EP4689670A1 - Method for identifying reactive oxygen species - Google Patents

Abstract

The invention relates to a method for in vitro quantifying the reactive oxygen species, or ROS, appearance rate in a biological sample, the method comprising - quantifying the amount of enzyme of mitochondrial complex I and III, and - calculating from the above amount the ROS, appearance rate.

Description

Description Title of the invention: Method for identifying reactive oxygen species Technical field The invention relates to a method for identifying reactive oxygen species, in particular involved in oxidative stress. State of the art ATP is an organic compound that provides energy to drive many processes in living cells, like nerve impulse propagation, condensate dissolution, and muscle contraction, all of that thanks to its two phosphoanhydride bonds. The energy stored in ATP is released with the hydrolysis of the terminal anhydride that transforms ATP into ADP (Adenosine Diphosphate). The mitochondria is considered to be a major actor in cellular metabolism because it contains the oxidative phosphorylation system which makes it possible to produce most of the ATP used by the cell, as well as the Krebs cycle and the betaoxidation of fatty acids which regenerate the substrates used by enzymes of oxidative phosphorylation. The electron transport chain (ETC) is a series of protein complexes located in the mitochondria that transfers electrons from electron donors to electron acceptors via redox reactions (both reduction and oxidation occurring simultaneously) and couples this electron transfer with the transfer of protons (H+ ions) across a membrane. This transfer of protons leads to an electrochemical proton gradient that drives the synthesis of ATP. The complex I and complex III of the respiratory chain are able to generate superoxide anion O₂ ^•−, which is formed by the reaction of oxygen with one electron. Reactive oxygen species (ROS) are molecules derived from the superoxide anion. For instance, hydrogen peroxide H₂O₂ is derived from the superoxide anion with the reaction: 2 O₂ ^•− + 2 H⁺ ^ O₂ + H₂O₂ ROS which are not regulated by cellular antioxidant defenses can react with the components of the cells and disturb its functioning : this imbalance between ROS and antioxidant defenses has been termed “oxidative stress”. Correlations have been shown between misregulation of ROS and various diseases, including cancer, diabetes, heart disease, and neurodegenerative diseases. Many neurodegenerative diseases have been linked to a dysfunction of one or several respiratory complexes associated to an oxidative stress. Hydrogen peroxide has been a focus of research geared toward understanding ROS in health and disease because it is a relatively long-lived ROS; thus, it is able to travel through a cell or even across cell membranes before it reacts with a target biomolecule. Various efforts have been made to detect hydrogen peroxide. For example, fluorescent scaffolds with boronate detection groups have been synthesized for the examination of hydrogen peroxide in cellular systems. Chemiluminescent probes based on peroxalate nanoparticles or on luminol, have been developed. However, the known method does not allow, for example to detect and/or quantify the superoxide anions. Accordingly, there remains a need in the field for compounds and/or methods of detecting ROS, including superoxide anions. Autosomal dominant optic atrophy 1 (DOA-1) is a neuro-pathology mainly caused by mutations in the gene OPA1 which codes a protein involved in mitochondrial dynamics. The symptoms and ages of onset of the disease are variable. There is no clear correlation between genotypes and phenotypes which can explain this variability and to date, there is no established medical treatment for the disease. The hypothesis of an oxidative stress has been proposed to explain the variability of symptoms observed in patients. Indeed, the mitochondrial energetic metabolism is altered in biological models (cell cultures and animal models) of DOA- 1 and low levels of antioxidant defenses have been measured in cells from patients suffering from severe forms of the pathology. (86) Accordingly, there is a need in the art to improve the understanding of the physio- pathological mechanisms involved in this disease. Accordingly, there remains a need in the field for compounds and/or methods of detecting ROS, including superoxide anion. Description of the invention The invention relates to a method for in vitro quantifying the reactive oxygen species, or ROS, appearance rate in a biological sample, the method comprising: a) determining the amount of 7 components involved in the activity of mitochondrial respiratory chain Complex I, or CI, or involved in the activity of mitochondrial respiratory chain Complex III, or CIII, or in the activity of both the mitochondrial respiratory chain CI and CIII, said 7 components being - the reduced form of Nicotinamide adenine dinucleotide, or NADH, - the oxidized form of Nicotinamide adenine dinucleotide, or NAD+, - quinone, or Q - quinol, or QH2, - molecular oxygen, or O₂, - the reduced form of cytochrome C, or cytCred, and - the oxidized form of cytochrome C, or cytCox, to obtain a respective concentration value - [NADH], corresponding to the amount of NADH measured in said biological sample, - [NAD+], corresponding to the amount of NAD+ measured in said biological sample, - [Q], corresponding to the amount of quinone measured in said biological sample, - [QH₂], corresponding to the amount of quinol measured in said biological sample, - [O₂], corresponding to the amount of molecular oxygen measured in said biological sample, - [CytC_Red] or [CytC_red], corresponding to the amount of reduced form of cytochrome C measured in said biological sample, - [CytC_ox], corresponding to the amount of oxidized form of cytochrome C measured in said biological sample, b) calculating a first ROS appearance rate V_{1 Ros} of ROS in said sample wherein V⁺ = 1727.2 mmol/min/mg ; KM,0^NADH = 10,6 K1 = 174.1, K2= 681.2, KM^NAD+ = 1028.6 µM, KM^Q= 11.6 µM, Kis^Q= 719128.5 µM, KM^QH2 = 16.3 µM, Kis^QH2 = 36.2 µM, Ki^NAD+ = 26.4 KO2 = 4871.0 and ^= 5. c) calculating a second Ros appearance rate V2 Ros of ROS in said sample wherein V2⁺ = 3579.29 mmol/min/mg ; K₁ = 23.05, K₂= 472.12, K_M,0 ^QH20 =184.77 µM, K_M ^QH2i = 397,14, K_M ^Qo= 43.08 µM, K_M ^Qi = 225.21µM, K_M ^cytCox = 8.11 µM, K_M ^cytCred= 419.16µM, K’_O2 = 914.51, ^’= 22.02 and β=173.54, d) obtaining the Ros appearance rate such that VRos = V1Ros + V2 Ros. The invention is based on the unexpected observation made by the inventors that an optimized model for measuring the activity of both mitochondrial Complex I and Complex III allows to simply assess the amount of Ros that can be produced in a cell, and then in an individual. The invention allows then to measure ROS production, not by measuring Ros themselve, but by measuring the activity of the enzymes and enzyme complexes from which the ROS are produced. Reactive oxygen species (hereafter referred as ROS or Ros) are highly reactive chemicals formed from diatomic oxygen (O₂). Examples of ROS include the following species: hydroxyl radical (HO^•), hydroxide ion (HO⁻), triplet oxygen (O₂ ^2•) superoxide anion (O₂ ^•−), peroxide ion (O₂ ⁻²), hydrogen peroxide (H₂O₂), and nitric oxide (NO^•). ROS are known to be produced by mitochondria, in particular buy Complexes I and III of the respiratory chain. Complex I of mitochondria is the first enzyme of the respiratory chain. This complex, i. e. the enzymes contained in the complex, oxidizes NADH, which is generated through the Krebs cycle in the mitochondrial matrix, and uses the two electrons to reduce ubiquinone to ubiquinol. Ubiquinol is reoxidized by the cytochrome bc1 complex and transfers electrons to reduce molecular oxygen to water at complex IV. The redox energy released during this process is used to transfer protons from the mitochondrial matrix to the periplasmic space that generates proton-motive force across the inner mitochondrial membrane at complex I, III, and IV. Complex V uses this proton-motive force to produce ATP from ADP and inorganic phosphate. This entire process constitutes OXPHOS. Because complex I is the major entry point for electrons to the respiratory chain and is suggested as the rate-limiting step in overall respiration, it plays a central role in energy metabolism. Complex I is the largest and most complicated component of the respiratory chain. Using the bovine heart as the model system, previous work has characterized all the complex I subunits and cloned the encoding genes. However, the functions of the individual subunits are largely unclear. Complex I is the only component of the mammalian respiratory chain whose three-dimensional structure is available only at a low resolution. Mitochondria are also major factors in modulating calcium signaling [4,5], which is a universal secondary messenger. As we will discuss further, complex I has been implicated in the regulation of reactive oxygen species (ROS), which are important molecules in various signaling pathways, including apoptosis. Mitochondrial complex III generates superoxide during the ubiquinone (Q)-cycle. The Q- cycle involves the transfer of two electrons to ubiquinone from complex I and complex II resulting in the reduction of ubiquinone to ubiquinol (QH2). The subsequent oxidation of ubiquinol at complex III requires the donation of those two electrons to the single electron carrier cytochrome c. The first electron transfer at complex III is to the Reiske iron-sulfur center protein (RISP). This electron is then transferred to cytochrome c1 and subsequently to cytochrome c. This one electron transfer from ubiquinol results in the unstable radical ubisemiquinone (Q^•-) which can donate its unpaired electron to oxygen to generate superoxide within the Q-cycle. Under most circumstances however, the unpaired electron of ubisemiquinone is transferred to the two heme groups of cytochrome b (Heme bL and Heme bH). The two hemes have different electron affinities because they are located in different polypeptide environments. Heme bL is located closer to the intermembrane space and has a lower affinity for electrons than Heme bH which is located closer to their matrix side. Ubisemiquinone transfers its electron to bL to form ubiquinone. Heme bL in turn donates an electron to Heme bH, which subsequently reduces another molecule of ubiquinone, forming ubisemiquinone. The Q-cycle at this stage is only half complete as only one electron from ubiquinol has been transferred to cytochrome c. After a second round of the Q-cycle, two molecules of ubiquinol have been oxidized on the intermembrane side of the inner membrane, two molecules of cytochrome c have been reduced, and one molecule of ubiquinone has been reduced on the matrix side of the inner membrane. Based on the data of the literature, the inventors were able to propose an optimized model for both mitochondrial complexes I and III. By simply measuring the concentration of specific substrate/products of these two complexes, and applying the optimized model, the inventors can easily and rapidly, but also efficiently quantify the ROS amount in a cell, a biological sample, and by extension to a patient. In the method defined above the concentration of - the reduced form of Nicotinamide adenine dinucleotide, or NADH, - the oxidized form of Nicotinamide adenine dinucleotide, or NAD+, - quinone, or Q - quinol, or QH₂, - molecular oxygen, or O₂, - the reduced form of cytochrome C, or cytCred or cytC_Red, and - the oxidized form of cytochrome C, or cytCox, is measured. In the present, the amount or concentration for example of the reduced form of Nicotinamide adenine dinucleotide, or NADH, the oxidized form of Nicotinamide adenine dinucleotide, or NAD+, quinone, or Q, - quinol, or QH2, molecular oxygen, or O2, the reduced form of cytochrome C, or cytCred or cytCRed, and/or the oxidized form of cytochrome C, or cytCox, is(are) mentioned in molar (M or mol.L^-1). Intracellular NAD+ and NADH levels can be measured with an NAD+/NADH Assay kit (Abcam) according to the manufacturer's instructions. Other similar tests are also usable. Quinone reduced and oxidized levels can be performed as described in Galinier et al., 2006 (58). The reduced form of cytochrome C, or cytCred, and the oxidized form of cytochrome C, or cytCox can be measured as described in Abramczyk, H.; Brozek-Pluska, B.; Kopec, M.; Surmacki, J.; Blaszczyk, M.; Radek, M. Redox Imbalance and Biochemical Changes in Cancer by Probing Redox-Sensitive Mitochondrial Cytochromes in Label-Free Visible Resonance Raman Imaging. Cancers 2021, 13, 960 (86). and Abramczyk, H. et al Scientific reports 2022 (87). Other methods known in the art can be used. In the present, "patient" or "subject" or “individual” means any individual, for example a mammal, for example a human being of any sex and/or age. For example, a human, such as an infant, a baby, a child, a teenager and adult. For example, a human aged between 7 days and 99 years. It may be, for example, an infant with an age ranging from 7 days to 14 months, a child with an age ranging from 14 months to 12 years, an adolescent with an age ranging from 12 to 17 years, an adult with an age ranging from 17 to 99 years. In the present, by "biological sample" means any biological liquid, for example, it may be a blood, plasma, serum, urine or synovial fluid sample, any biological tissue and/or any cells. It may be a serum or blood sample. It may be an eukaryotic cell, for example a muscle cell, a bone cell, a cancerous cell, epithelial cell. For example, when the biological sample is a eukaryotic cell it may be an isolated eukaryotic cell. In the present, the biological sample may be a biological sample previously taken from said patient or subject. It may be for example a sample taken from 5 minutes to 48 hours, for example from 1 hour to 24 hours, for example from 1 hour to 6 hours prior to its use in the process according to the invention. According to the invention, the volume of the biological sample can be from 10µl to 10 ml, for example from 12µl to 5 ml, for example from 100 to 200 µl, the volume of the biological sample can be from 10µl to 200 µl, for example equal to 15µl. The biological sample may be, for example, but not limited to, fresh, frozen, For example, when the sample is frozen, it may be advantageously frozen by flash freeze, for example with liquid nitrogen and then stored, for example at -80°C until analysis. In the present, reference sample means a biological sample being of the same nature as the tested sample. It may be for example a sample of the same nature as the tested sample obtained from a healthy reference "patient" or "subject" or “individual”. The term "healthy reference patient or subject or individual is used here to mean any individual, for example a mammal, for example a human being of any sex and/or age without any disease and/or without “oxidative stress” and/or without misregulation of ROS. For example, a human, such as an infant, a baby, a child, a teenager and adult. For example, a human aged between 7 days and 99 years. It may be, for example, an infant with an age ranging from 7 days to 14 months, a child with an age ranging from 14 months to 12 years, an adolescent with an age ranging from 12 to 17 years, an adult with an age ranging from 17 to 99 years. Then, the optimized equations as defined above are applied for each of the complexes I and III, to obtain the respective ROS appearance rate for each complex. To determine the global ROS appearance rate by adding the result for each complex. In other words, the invention relates to a method for quantifying, preferably in vitro, the reactive oxygen species, or ROS, appearance rate in a biological sample, the method comprising: a) determining the amount of 7 components involved in the activity of mitochondrial respiratory chain Complex I, or CI, or involved in the activity of mitochondrial respiratory chain Complex III, or CIII, or in the activity of both the mitochondrial respiratory chain CI and CIII, said 7 components being - the reduced form of Nicotinamide adenine dinucleotide, or NADH, - the oxidized form of Nicotinamide adenine dinucleotide, or NAD+, - quinone, or Q - quinol, or QH₂, - molecular oxygen, or O2, - the reduced form of cytochrome C, or cytCred, and - the oxidized form of cytochrome C, or cytCox, to obtain a respective concentration value - [NADH], corresponding to the amount of NADH measured in said biological sample, - [NAD+], corresponding to the amount of NAD+ measured in said biological sample, - [Q], corresponding to the amount of quinone measured in said biological sample, - [QH₂], corresponding to the amount of quinol measured in said biological sample, - [O₂], corresponding to the amount of molecular oxygen measured in said biological sample, - [CytC_Red], corresponding to the amount of reduced form of cytochrome C measured in said biological sample, - [CytCox], corresponding to the amount of oxidized form of cytochrome C measured in said biological sample, b) calculating a first ROS appearance rate V1 Ros of ROS in said sample ^_{^ ^^^} = 1727.2 ∗ ^_^^^^ ∗ ^_^^ wherein [^^^^] ^ ^{^}^^^^ ^_^^^ ^([^^^^ , ^[^^^^{^}]) _{= ^} [^^^^] _^^^^ ^{^} _^ in which ^^ ^_{^ ^^^^} ^{^} ^^^^.^ [^] ^_^ ^([ _^ ^] _, ^[ _^^^ ^]) ₌ ^^.^ [^] [^^ ] in which ^^ ^^.^^{∗^} _^ ^,^,^^_^ ^{^} ^^.^ ^{∗^}^,^,^^_^ [_^^] c) calculating a second ROS appearance rate V2 Ros of ROS in said sample ^ _^^^ = 3579.29 wherein [^^_^ ^] ^ ^{^^} _^^ ^ ([^^_^], [^]) = ^^ ^{[ ] [} _^ ^{] ^^} _^ ^ ^ _^ ^^.^^ ^ d) obtaining the ROS appearance rate such that VRos = V1Ros + V2 Ros. In other words, the invention relates to a method for quantifying, preferably in vitro, the reactive oxygen species, or ROS, appearance rate in a biological sample, the method comprising: a) determining the amount of 7 components involved in the activity of mitochondrial respiratory chain Complex I, or CI, or involved in the activity of mitochondrial respiratory chain Complex III, or CIII, or in the activity of both the mitochondrial respiratory chain CI and CIII, said 7 components being - the reduced form of Nicotinamide adenine dinucleotide, or NADH, - the oxidized form of Nicotinamide adenine dinucleotide, or NAD+, - quinone, or Q - quinol, or QH2, - molecular oxygen, or O2, - the reduced form of cytochrome C, or cytCred, and - the oxidized form of cytochrome C, or cytCox, to obtain a respective concentration value - [NADH], corresponding to the amount of NADH measured in said biological sample, - [NAD+], corresponding to the amount of NAD+ measured in said biological sample, - [Q], corresponding to the amount of quinone measured in said biological sample, - [QH2], corresponding to the amount of quinol measured in said biological sample, - [O₂], corresponding to the amount of molecular oxygen measured in said biological sample, - [CytC_Red], corresponding to the amount of reduced form of cytochrome C measured in said biological sample, - [CytC_ox], corresponding to the amount of oxidized form of cytochrome C measured in said biological sample, b) calculating a first ROS appearance rate V_{1 Ros} of ROS in said sample ^ _^^^ = 1727.2 wherein ^[^^^^^] 10.6 ^_^^^^^.^ 1 + ^^{^} ^^^.^ [_^] ^_^ ^([ _^ ^] _, ^[ _^^^ ^]) ₌ ∗ ^1 + 719128.5 + and [_^^] c) calculating a second ROS appearance rate V2 Ros of ROS in said sample ^_{^ ^^^} = 3579.29 wherein [_^^^ ^] 184,77 and [^_^] ^_^^([^_^]) = 914.51 1 + 22.02^_^^ + 173.54^_^^^^^^ d) obtaining the ROS appearance rate such that VRos = V_1Ros + V_{2 Ros.} Advantageously, the invention relates to the method as defined above, wherein the method comprising: b) calculating a first ROS appearance rate V_{1 Ros} of ROS in said sample wherein V2⁺ = 3909.06 mmol/min/mg ; K1 = 19.96, K2= 341.15, K_M,0 ^QH20 = 157.61 µM, K_M ^QH2i = 478.63, K_M ^Qo= 50.49 µM, K_M ^Qi = 280.26µM, K_M ^cytCox = 10.98 µM, K_M ^cytCred= 248.18µM, K’O2 = 760.78, ^’= 38.66 and β = 212.03 d) obtaining the ROS appearance rate such that VRos = V1Ros + V2 Ros. In other words, advantageously, the invention relates to the method as defined above, wherein the method comprising: a) determining the amount of 7 components involved in the activity of mitochondrial respiratory chain Complex I, or CI, or involved in the activity of mitochondrial respiratory chain Complex III, or CIII, or in the activity of both the mitochondrial respiratory chain CI and CIII, said 7 components being - the reduced form of Nicotinamide adenine dinucleotide, or NADH, - the oxidized form of Nicotinamide adenine dinucleotide, or NAD+, - quinone, or Q - quinol, or QH₂, - molecular oxygen, or O₂, - the reduced form of cytochrome C, or cytCred, and - the oxidized form of cytochrome C, or cytCox, to obtain a respective concentration value - [NADH], corresponding to the amount of NADH measured in said biological sample, - [NAD+], corresponding to the amount of NAD+ measured in said biological sample, - [Q], corresponding to the amount of quinone measured in said biological sample, - [QH₂], corresponding to the amount of quinol measured in said biological sample, - [O2], corresponding to the amount of molecular oxygen measured in said biological sample, - [CytCRed], corresponding to the amount of reduced form of cytochrome C measured in said biological sample, - [CytCox], corresponding to the amount of oxidized form of cytochrome C measured in said biological sample, b) calculating a first ROS appearance rate V1 Ros of ROS in said sample wherein ^[ _^^^^ ^] ^^^^ ^_^^^^ ^([^^^^^], ^[^^^^{^}]) ₌ ^{^} _^ [^^^^] in w ^^ _^^^^ ^{^} _^ hich ^_{^ ^,^ ^^^^} ^{∗^^,^^^^^ ∗^} _^^^ ^_{^ ^^^} ^{^} wherein V2⁺ = 3909.06 mmol/min/mg ; K1 = 19.96, K2= 341.15, KM,0^QH20 = 157.61 µM, KM^QH2i = 478.63, KM^Qo= 50.49 µM, KM^Qi = 280.26µM, KM^cytCox = 10.98 µM, KM^cytCred= 248.18µM, K’_O2 = 760.78, ^’= 38.66 and β = 212.03 d) obtaining the ROS appearance rate such that VRos = V1Ros + V2 Ros. In this advantageous embodiment, the inventors obtain better results when the formula for obtaining the ROS apparition rate is ameliorated by using the above-mentioned formulas. Here for model 3 of complex III, the inventors have an optional input, the % of saturation of Antimycin. More advantageously, the invention relates to the method as defined above, wherein the activity of CI is determined by the following formula: In addition to the apparition of ROS, the method proposed by the inventors allows to evaluate the activity of the Complex I. By determining the activity of the complex, it is possible to assess a possible dysfunction in the complex and thus possibly propose a therapy or cure for correcting the default. More advantageously, the invention relates to the method as defined above, wherein the activity of CIII is determined by the following formula: Similarly, in addition to the apparition of ROS, the method proposed by the inventors allows to evaluate the activity of the Complex III. By determining the activity of the complex, it is possible to assess a possible dysfunction in the complex and thus possibly propose a therapy or cure for correcting the default. The Invention also relates to a computer program comprising instructions which, when the program is executed by a computer, causes the computer to carry out steps b) to d) of the method as defined above. The computer program according to the invention is able, when implemented on a computer to calculate the ROS appearance rate and the activities of both complexes, by implementing the above mentioned formulas, based on the data relating to the concentrations of - [NADH], corresponding to the amount of NADH measured in said biological sample, - [NAD+], corresponding to the amount of NAD+ measured in said biological sample, - [Q], corresponding to the amount of quinone measured in said biological sample, - [QH2], corresponding to the amount of quinol measured in said biological sample, and - [O2], corresponding to the amount of molecular oxygen measured in said biological sample. Every support can be used such as personal computer, portable computer, tablet smartphone, or any dice available for computing the data according to the above-mentioned formulas. The invention also relates to a method, for determining, preferably in vitro, ROS detoxifying enzyme activity in a sample of an individual, the method comprising: - quantifying the ROS appearance rate in the biological sample by carrying out the method as defined above, to obtain a calculated rate, - comparing the calculated rate with a reference ROS appearance rate, said reference ROS appearance rate being obtained from a reference sample, the reference sample being of the same nature as the sample, thus obtaining a ratio R between the ROS appearance rate and the reference ROS appearance rate, and - concluding that * if R is lower than 0.85, then the sample contains efficient ROS detoxifying enzyme activity, to detoxify the sample during an oxidative stress, * otherwise the sample does not contain efficient ROS detoxifying enzyme activity (that detoxify the sample during the process of the production of ROS). With the method as defined above, it is possible to evaluate the detoxifying activity of an individual, and therefore to predict if, when submitted to an oxidative stress, the individual will be able to efficiently detoxify cells, or if an accumulation of ROS is expected, with the drawbacks thereof. In other words, the invention also relates to a method, for determining, preferably in vitro, ROS detoxifying enzyme activity in a sample of an individual, the method comprising: - quantifying the ROS appearance rate in the biological sample by carrying out the method as defined above, to obtain a calculated rate, i.e. a) determining the amount of 7 components involved in the activity of mitochondrial respiratory chain Complex I, or CI, or involved in the activity of mitochondrial respiratory chain Complex III, or CIII, or in the activity of both the mitochondrial respiratory chain CI and CIII, said 7 components being - the reduced form of Nicotinamide adenine dinucleotide, or NADH, - the oxidized form of Nicotinamide adenine dinucleotide, or NAD+, - quinone, or Q - quinol, or QH2, - molecular oxygen, or O2, - the reduced form of cytochrome C, or cytCred, and - the oxidized form of cytochrome C, or cytCox, to obtain a respective concentration value - [NADH], corresponding to the amount of NADH measured in said biological sample, - [NAD+], corresponding to the amount of NAD+ measured in said biological sample, - [Q], corresponding to the amount of quinone measured in said biological sample, - [QH2], corresponding to the amount of quinol measured in said biological sample, - [O₂], corresponding to the amount of molecular oxygen measured in said biological sample, - [CytC_Red], corresponding to the amount of reduced form of cytochrome C measured in said biological sample, - [CytC_ox], corresponding to the amount of oxidized form of cytochrome C measured in said biological sample, b) calculating a first ROS appearance rate V_{1 Ros} of ROS in said sample wherein V⁺ = 1727.2 mmol/min/mg ; KM,0^NADH = 10,6 K1 = 174.1, K2= 681.2, KM^NAD+ = 1028.6 µM, KM^Q= 11.6 µM, Kis^Q= 719128.5 µM, KM^QH2 = 16.3 µM, Kis^QH2 = 36.2 µM, Ki^NAD+ = 26.4 KO2 = 4871.0 and ^= 5. c) calculating a second ROS appearance rate V2 Ros of ROS in said sample wherein [^^_^ ^] ^ ^{^^} _^^ ^{^^} _^^ ^_^^^^([^^_^], [^]) = ^ ^{^^^^ ^} _^,^ ^^ ^{[^^} _^ ^] [^] in which ^_^ = ^{^^^^^ ^^} _^ ^^{^} ^ ^{^} ^ _^^ ^ _^^ ^ ^ _^ ^ ^^^ ^{^} _^ wherein V2⁺ = 3579.29 mmol/min/mg ; K1 = 23.05, K2= 472.12, KM,0^QH20 =184.77 µM, KM^QH2i = 397,14, KM^Qo= 43.08 µM, KM^Qi = 225.21µM, KM^cytCox = 8.11 µM, KM^cytCred= 419.16µM, K’O2 = 914.51, ^’= 22.02 and β=173.54 d) obtaining the ROS appearance rate such that VRos = V1Ros + V2 Ros, - comparing the calculated rate with a reference ROS appearance rate, said reference ROS appearance rate being obtained from a reference sample, the reference sample being of the same nature as the sample, to obtain a ratio R between the ROS appearance rate and the reference ROS appearance rate, and - concluding that * if R is lower than 0.85, then the sample contains efficient ROS detoxifying enzyme activity, (that detoxify the sample during the process of the production of ROS), , * otherwise the sample does not contain efficient ROS detoxifying enzyme activity that detoxify the sample during an oxidative stress. Advantageously, the invention relates the above-mentioned method, the method comprising: - quantifying the ROS appearance rate in the biological sample by carrying out the method as defined above, to obtain a calculated rate, - comparing the calculated rate with a reference ROS appearance rate, said reference ROS appearance rate being obtained from a reference sample, the reference sample being of the same nature as the sample, to obtain a ratio R between the ROS appearance rate and the reference ROS appearance rate, and - concluding that * if R is lower than 0.85, then the sample contains efficient ROS detoxifying enzyme activity, (that detoxify the sample during the process of the production of ROS), , * otherwise the sample does not contain efficient ROS detoxifying enzyme activity, that detoxify the sample during an oxidative stress. With the method as defined above, it is possible to evaluate the detoxifying activity of an individual, and therefore to predict if, when submitted to an oxidative stress, the individual will be able to efficiently detoxify cells, or if an accumulation of ROS is expected, with the drawbacks thereof. In other words, the invention also relates to a method, for determining, preferably in vitro, ROS detoxifying enzyme activity in a sample of an individual, the method comprising: - quantifying the ROS appearance rate in the biological sample by carrying out the method as defined above, to obtain a calculated rate, i.e. a) determining the amount of 7 components involved in the activity of mitochondrial respiratory chain Complex I, or CI, or involved in the activity of mitochondrial respiratory chain Complex III, or CIII, or in the activity of both the mitochondrial respiratory chain CI and CIII, said 7 components being - the reduced form of Nicotinamide adenine dinucleotide, or NADH, - the oxidized form of Nicotinamide adenine dinucleotide, or NAD+, - quinone, or Q - quinol, or QH2, - molecular oxygen, or O2, - the reduced form of cytochrome C, or cytCred, and - the oxidized form of cytochrome C, or cytCox, to obtain a respective concentration value - [NADH], corresponding to the amount of NADH measured in said biological sample, - [NAD+], corresponding to the amount of NAD+ measured in said biological sample, - [Q], corresponding to the amount of quinone measured in said biological sample, - [QH2], corresponding to the amount of quinol measured in said biological sample, - [O2], corresponding to the amount of molecular oxygen measured in said biological sample, - [CytC_Red], corresponding to the amount of reduced form of cytochrome C measured in said biological sample, - [CytC_ox], corresponding to the amount of oxidized form of cytochrome C measured in said biological sample, b) calculating a first ROS appearance rate V_{1 Ros} of ROS in said sample wherein V2⁺ = 3909.06 mmol/min/mg ; K₁ = 19.96, K₂= 341.15, K_M,0 ^QH20 = 157.61 µM, K_M ^QH2i = 478.63, K_M ^Qo= 50.49 µM, K_M ^Qi = 280.26µM, K_M ^cytCox = 10.98 µM, K_M ^cytCred= 248.18µM, K’O2 = 760.78, ^’= 38.66 and β = 212.03 d) obtaining the ROS appearance rate such that VRos = V1Ros + V2Ros, - comparing the calculated rate with a reference ROS appearance rate, said reference ROS appearance rate being obtained from a reference sample, the reference sample being of the same nature as the sample, to obtain a ratio R between the ROS appearance rate and the reference ROS appearance rate, and - concluding that * if R is lower than 0.85, then the sample contains efficient ROS detoxifying enzyme activity, (that detoxify the sample during the process of the production of ROS), * otherwise the sample does not contain efficient ROS detoxifying enzyme activity, (that detoxify the sample during the process of the production of ROS). More advantageously, the invention relates to the above-described method, wherein * if R is lower than 0,8, then the sample contains efficient ROS detoxifying enzyme activity, (that detoxify the sample during the process of the production of ROS), * otherwise the sample does not contain efficient ROS detoxifying enzyme activity, (that detoxify the sample during the process of the production of ROS). Advantageously, the invention also relates to a method, for determining, preferably in vitro, ROS detoxifying enzyme activity in a sample of an individual, the method comprising: - quantifying the ROS appearance rate in the biological sample by carrying out the method as defined above, to obtain a calculated rate, i.e. a) determining the amount of 7 components involved in the activity of mitochondrial respiratory chain Complex I, or CI, or involved in the activity of mitochondrial respiratory chain Complex III, or CIII, or in the activity of both the mitochondrial respiratory chain CI and CIII, said 7 components being - the reduced form of Nicotinamide adenine dinucleotide, or NADH, - the oxidized form of Nicotinamide adenine dinucleotide, or NAD+, - quinone, or Q - quinol, or QH2, - molecular oxygen, or O2, - the reduced form of cytochrome C, or cytCred, and - the oxidized form of cytochrome C, or cytCox, to obtain a respective concentration value - [NADH], corresponding to the amount of NADH measured in said biological sample, - [NAD+], corresponding to the amount of NAD+ measured in said biological sample, - [Q], corresponding to the amount of quinone measured in said biological sample, - [QH₂], corresponding to the amount of quinol measured in said biological sample, - [O₂], corresponding to the amount of molecular oxygen measured in said biological sample, - [CytC_Red], corresponding to the amount of reduced form of cytochrome C measured in said biological sample, - [CytC_ox], corresponding to the amount of oxidized form of cytochrome C measured in said biological sample, b) calculating a first ROS appearance rate V_{1 Ros} of ROS in said sample wherein V2⁺ = 3909.06 mmol/min/mg ; K₁ = 19.96, K₂= 341.15, K_M,0 ^QH20 = 157.61 µM, K_M ^QH2i = 478.63, K_M ^Qo= 50.49 µM, K_M ^Qi = 280.26µM, K_M ^cytCox = 10.98 µM, K_M ^cytCred= 248.18µM, K’_O2 = 760.78, ^’= 38.66 and β = 212.03 d) obtaining the ROS appearance rate such that VRos = V_{1 Ros} + V_{2 Ros,} - comparing the calculated rate with a reference ROS appearance rate, said reference ROS appearance rate being obtained from a reference sample, the reference sample being of the same nature as the sample, to obtain a ratio R between the ROS appearance rate and the reference ROS appearance rate, and - concluding that * if R is lower than 0.8, then the sample contains efficient ROS detoxifying enzyme activity, (that detoxify the sample during the process of the production of ROS), * otherwise the sample does not contain efficient ROS detoxifying enzyme activity, (that detoxify the sample during the process of the production of ROS). In one other aspect, the invention relates to a method for determining in vitro the response of a patient to a compound liable to reduce ROS amount in a biological sample, the method comprising: - quantifying the ROS appearance rate in the biological sample, after the administration of the compound to the patient, by carrying out the method as defined above, to obtain a final ROS rate, - comparing the initial ROS rate and the final ROS rate to obtain a ratio R between the final ROS and the initial ROS rate, - concluding that * if R is lower than 0.85, then the compound is able to reduce ROS amount in the sample, * otherwise the sample is not able to reduce ROS amount in the sample. With the above mentioned method, it is possible to evaluate the effect of a compound in the modulation of ROS production in a cell or a sample of an individual. In the present, by “compound liable to reduce ROS amount” refers to any molecule that is wished to be tested. These may be chemical and/or biological molecules, for example. It may be, for example, a therapeutic molecule that can be used to treat pathologies, or a drug, for example any substance or compound known to the person skilled in the art that has curative and/or preventive properties with regard to pathologies, lesions, traumas or human or animal diseases. It may, for example, be a pharmaceutical product for human and/or veterinary use. By measuring the ROS production by implementing the method according to the invention at a determined time (before any treatment) and after a period of time during which a treatment is administered, it is possible to evaluate if said treatment is efficient or not on modulating ROS production, preferably ROS reduction. Any difference of about 15% or more compared to the initial value will be considered as significantly different and associated with an effect of the treatment. By contrast, if the difference between the initial value of ROS amount and the final value of the ROS amount (after treatment) is lower than 15%, it is considered that he treatment has no effect on ROS production. Advantageously, the invention relates to a method for determining in vitro the response of a patient to a compound liable to reduce ROS amount in a biological sample, the method comprising: - quantifying the ROS appearance rate in the biological sample, before the administration of the compound to the patient, by carrying out the method as defined above, to obtain an initial ROS rate, the method comprising: - quantifying the ROS appearance rate in the biological sample by carrying out the method as defined above, to obtain a calculated rate, i.e. a) determining the amount of 7 components involved in the activity of mitochondrial respiratory chain Complex I, or CI, or involved in the activity of mitochondrial respiratory chain Complex III, or CIII, or in the activity of both the mitochondrial respiratory chain CI and CIII, said 7 components being - the reduced form of Nicotinamide adenine dinucleotide, or NADH, - the oxidized form of Nicotinamide adenine dinucleotide, or NAD+, - quinone, or Q - quinol, or QH2, - molecular oxygen, or O₂, - the reduced form of cytochrome C, or cytCred, and - the oxidized form of cytochrome C, or cytCox, to obtain a respective concentration value - [NADH], corresponding to the amount of NADH measured in said biological sample, - [NAD+], corresponding to the amount of NAD+ measured in said biological sample, - [Q], corresponding to the amount of quinone measured in said biological sample, - [QH₂], corresponding to the amount of quinol measured in said biological sample, - [O₂], corresponding to the amount of molecular oxygen measured in said biological sample, - [CytC_Red], corresponding to the amount of reduced form of cytochrome C measured in said biological sample, - [CytC_ox], corresponding to the amount of oxidized form of cytochrome C measured in said biological sample, b) calculating a first ROS appearance rate V_{1 Ros} of ROS in said sample wherein wherein V2⁺ = 3909.06 mmol/min/mg ; K1 = 19.96, K2= 341.15, KM,0^QH20 = 157.61 µM, KM^QH2i = 478.63, KM^Qo= 50.49 µM, KM^Qi = 280.26µM, KM^cytCox = 10.98 µM, KM^cytCred= 248.18µM, K’O2 = 760.78, ^’= 38.66 and β = 212.03 d) obtaining the ROS appearance rate such that VRos = V1 Ros + V2 Ros, - quantifying the ROS appearance rate in the biological sample, after the administration of the compound to the patient, by carrying out the method as defined above, to obtain a final ROS rate, - comparing the initial ROS rate and the final ROS rate to obtain a ratio R between the final ROS and the initial ROS rate, - concluding that * if R is lower than 0.85, then the compound is able to reduce ROS amount in the sample, * otherwise the sample is not able to reduce ROS amount in the sample. More advantageously, the invention relates to the above-described method, wherein * if R is lower than 0.8, then the compound is able to reduce ROS amount in the sample, * otherwise the sample is not able to reduce ROS amount in the sample. Advantageously, the invention relates to a method for determining in vitro the response of a patient to a compound liable to reduce ROS amount in a biological sample, the method comprising: - quantifying the ROS appearance rate in the biological sample, before the administration of the compound to the patient, by carrying out the method as defined above, to obtain an initial ROS rate, the method comprising: - quantifying the ROS appearance rate in the biological sample by carrying out the method as defined above, to obtain a calculated rate, i.e. a) determining the amount of 7 components involved in the activity of mitochondrial respiratory chain Complex I, or CI, or involved in the activity of mitochondrial respiratory chain Complex III, or CIII, or in the activity of both the mitochondrial respiratory chain CI and CIII, said 7 components being - the reduced form of Nicotinamide adenine dinucleotide, or NADH, - the oxidized form of Nicotinamide adenine dinucleotide, or NAD+, - quinone, or Q - quinol, or QH₂, - molecular oxygen, or O₂, - the reduced form of cytochrome C, or cytCred, and - the oxidized form of cytochrome C, or cytCox, to obtain a respective concentration value - [NADH], corresponding to the amount of NADH measured in said biological sample, - [NAD+], corresponding to the amount of NAD+ measured in said biological sample, - [Q], corresponding to the amount of quinone measured in said biological sample, - [QH2], corresponding to the amount of quinol measured in said biological sample, - [O2], corresponding to the amount of molecular oxygen measured in said biological sample, - [CytCRed], corresponding to the amount of reduced form of cytochrome C measured in said biological sample, - [CytCox], corresponding to the amount of oxidized form of cytochrome C measured in said biological sample, wherein V⁺ = 2121,1 mmol/min/mg ; KM,0 ^NADH = 8,1 µM, K1 = 185,2, K2= 1021,2, KM^NAD+ = 625,8 µM, KM^Q= 15,7 µM, KM^QH2 = 26 µM, Kis^Q= 1066907,0 µM, Kis^QH2 = 48,6 µM, Kis^NADH = 165076,9 µM K_i ^NAD+ = 34,3 K_O2 = 9551,4 et ^= 3,3. c) calculating a second ROS appearance rate V2 Ros of ROS in said sample wherein wherein V2⁺ = 3909.06 mmol/min/mg ; K1 = 19.96, K2= 341.15, KM,0^QH20 = 157.61 µM, KM^QH2i = 478.63, KM^Qo= 50.49 µM, KM^Qi = 280.26µM, KM^cytCox = 10.98 µM, KM^cytCred= 248.18µM, K’O2 = 760.78, ^’= 38.66 and β = 212.03 d) obtaining the ROS appearance rate such that VRos = V1 Ros + V2 Ros, - quantifying the ROS appearance rate in the biological sample, after the administration of the compound to the patient, by carrying out the method as defined above, to obtain a final ROS rate, - comparing the initial ROS rate and the final Ros rate to obtain a ratio R between the final ROS and the initial ROS rate, - concluding that * if R is lower than 0.8, then the compound is able to reduce ROS amount in the sample, * otherwise the sample is not able to reduce ROS amount in the sample. In one other aspect, the invention relates to a method for determining in vitro in a biological sample if a compound is a pro- or an anti-oxidant compound, the method comprising - quantifying the Ros appearance rate in the biological sample by carrying out the method as defined above, to obtain a calculated rate, - comparing the calculated rate with a reference Ros appearance rate, said reference Ros appearance rate being obtained from a reference sample, the reference sample being of the same nature as the sample, to obtain a ratio R between the Ros appearance rate and the reference Ros appearance rate, and - concluding that * if R is lower than 0.85, then the compound is an anti-oxidant compound, * if R is higher than 1.15, then the compound is a pro-oxidant compound, and * if R is comprised from 0.85 to 1.15, then the compound has neither anti- nor pro-oxidant properties. Advantageously the invention relates to the method defined above, the method comprising - quantifying the Ros appearance rate in the biological sample by carrying out the method as defined above, to obtain a calculated rate, - comparing the calculated rate with a reference Ros appearance rate, said reference Ros appearance rate being obtained from a reference sample, the reference sample being of the same nature as the sample, to obtain a ratio R between the Ros appearance rate and the reference Ros appearance rate, and - concluding that * if R is lower than 0.8, then the compound is an anti-oxidant compound, * if R is higher than 1.2, then the compound is a pro-oxidant compound, and * if R is comprised from 0.8 to 1.2, then the compound has neither anti- nor pro-oxidant properties. Brief description of the drawings The invention will be better understood in light of the following examples and the following figures: Figure 1: Oxygen consumption rates (OCR) were measured (ordinate) in siOPA1- and siCtrl-transfected HeLa cells, according time (abscissa). Spontaneous mitochondrial respiration was significantly lower in siOPA-transfected cells (0.33 ± 0.02 pMoles/min/mg protein) than control cells (0.53 ± 0.04 pMoles/min/mg protein). After oligomycin (1 µM) injection at 30 min, cell respiration was also significantly lower in siOPA1-transfected cells (0.11 ± 0.01 pMoles/min/mg protein) than control cells (0.17 ± 0.02 pMoles/min/mg protein). After FCCP (1 µM) injection at 50 min, maximal respiratory was significantly lower in siOPA- transfected cells (0.30 ± 0.04 pMoles/min/mg protein) than control cells (0.67 ± 0.10 pMoles/min/mg protein). Finally, rotenone (1 µM) plus antimycin A (1 µM) injections at 80 min inhibited mitochondrial respiration. Results are expressed as the mean ± SEM (n = 3). P values were determined by Student’s unpaired t-test p < 0.05* and p < 0.01**. Figure 2: Total ATP concentration (ordinate) was measured in µM/mg proteins for siOPA1 Hela cells (grey bar) and siCtrl-treated (white bar) cells. The ATP concentration was unchanged in siOPA1 Hela cells (grey bar) relative to siCtrl-treated (white bar) cells. Results are expressed as the mean ± SEM (n = 3). P values were determined by Student’s-paired t- test. Figure 3: Extracellular acidification rates (ECAR) (ordinate) was measured in mpH/min/mg proteins) for siOPA1 Hela cells (grey bar) and siCtrl-treated (white bar) cells. Data on the left hand bar were obtained at a basal level. Right hand bars were obtained after oligomycin injection. ECAR were unchanged in OPA1-treated cells (basal: 37.93 ± 1.33 pMoles/min/mg protein, oligomycin: 56.42 ± 3.08 pMoles/min/mg protein) relative to siCtrl-treated cells (basal: 46.40 ± 3.93 pMoles/min/mg protein, oligomycin: 69.14 ± 6.72 pMoles/min/mg protein) under basal conditions and after 1 µM oligomycin injection. As expected, ECAR increased in both siCtrl- and siOPA1-transfected cells after oligomycin injection. Results are expressed as the mean ± SEM (n = 3). P values were determined by Student’s unpaired t-test p < 0.05* p < 0.001*** Figure 4: Extracellular lactate (ordinate) was measured by colorimetric analysis in HeLa cells with (grey bar) or without (white bar) siOPA1 (Mean ± SEM, n = 2). P values were determined by Student’s-paired t-test. Figure 5: Representative immunoblots showing the effect of OPA1 downregulation in HeLa cells or Control cells (abscissa) on the levels of two subunits of the MRC complexes (complex I-IV) and three subunits of ATP synthase (complex V) relative to actin (ordinate). Histograms show representative quantities (ordinate) of NADH:Ubiquinone Oxidoreductase Subunit A9 (NDUFA9) or NADH:Ubiquinone Oxidoreductase Subunit B4 (NDUFB4) for complex I, succinate dehydrogenase complex, subunit A (SDHA) or succinate dehydrogenase complex, subunit B (SDHB) for complex II, Core 1 or Core 2 for complex III, COX I or COX IV for Complex IV, alpha subunit (F1), gamma subunit (F1) or subunit d (F0) for complex V, in HeLa cells with (grey bar) or without (white bar) siOPA1. The quantity of NDUFB4 (n = 10) was significantly lower in siOPA1-transfected HeLa cells (0.47 ± 0.07) than siCtrl-transfected cells (0.85 ± 0.09). The quantity of SDHB (n =10) was significantly lower in siOPA1-treated cells (0.68 ± 0.16) than control cells (0.96 ± 0.16). The level of Core 2 (n = 10) was significantly lower in siOPA1- treated cells (0.75 ± 0.05) than control cells (1.238 ± 0.10). The level of COX I (n = 10) was lower in siOPA1-treated cells (0.37 ± 0.05) than control cells (0.69 ± 0.06). P values were determined by Student’s-paired t-test (p < 0.05*, p < 0.01**, and p < 0.001***). Figure 6: Activity (ordinate) of complex I (n = 3), II (n = 16), III (n = 3), and IV (n = 16) measured in vitro in both siOPA1- (grey bar) and siCtrl-treated (white bar) HeLa cells. Succinate dehydrogenase (complex II) activity was lower in siOPA1-treated cells (6.438 ± 0.701) than control cells (8.688 ± 0.7229). Results are expressed as the mean ± SEM. P values were determined by Student’s paired t-test (p < 0.05*, p < 0.01**, and p < 0.001***). Figure 7: Total ROS fluorescence (ordinate) was measured for siOPA1- (grey bar) and siCtrl-treated (white bar) HeLa cells. Total ROS measured by the H₂-DCFDA probe in siOPA1- transfected HeLa cells (72.63 ± 4.6) was lower than in siCtrl-treated cells (95.48 ± 9.1). Figure 8: Aconitase activity (ordinate) was measured for siOPA1- (grey bar) and siCtrl- treated (white bar) HeLa cells. Aconitase activity was lower in siOPA1-treated cells (3.185 ± 1.41) than siCtrl-treated cells (4.811 ± 1.361). Figure 9: Representative immunoblots showing siOPA1- and siCtrl-treated HeLa cells (abscissa) proteins according to their molecular size (ordinate). Histograms represents aconitase relative quantity (ordinate) in siOPA1- (grey bar) and siCtrl-treated (white bar) HeLa cells. These results show that OPA1 downregulation in HeLa cells has no effect on the expression of aconitase relative to actin. Figure 10: Mitochondrial superoxide production was measured using MitoSOX Red (mitochondria-targeted superoxide indication) with fluorescent microscopy (X40). MitoSOX (ordinate) measurement was performed after 72h siCtrl (white) or siOPA1 (grey) transfection. Representative histogram of quantitative fluorescence intensity was analysed by ImageJ software. The ROS level was higher in siOPA1-tranfected cells (1,9444 ± 0,2278) than control cells (1,386 ± 0,08754). Representative micrographs of mitochondrial MitoSOX immunocytofluorescence and DNA Hoechst staining in siCtrl- or siOPA1-treated HeLa cells 72h after transfection. Scale bar represents 10 µm. Results are expressed as the mean ± SEM with n = 8 (A), n = 6 (B), and n = 8 (C) and n=3 (D) with more than 200 cells. Statistical analysis was performed using a nonparametric test (Mann-Whitney) for A and D and Student’s paired t-test for B (p < 0.05*). Figure 11: Representative micrographs of NRF2 immunocytofluorescence and DNA Hoechst staining or Merge in siOPA1- or siCtrl-treated HeLa cells 72 h after transfection. Figure 12: Representative histogram showing the percentage of cells with NRF2 nuclear translocation (ordinate) according to time 66, 67, 68, 69, 70, and 72 h (abscissa) after siOPA1 (grey) or siCtrl (white) transfection. Nuclear immunostaining of NRF2 was observed in 15.18 ± 1.86% of control HeLa cells and 51.68 ± 5.57% of siOPA1-treated cells 72 h after transfection. Results are expressed as the mean ± SEM with n =4 to 14 (400 cells per condition). P values were determined by a nonparametric test (Mann-Whitney test), p < 0.001***. Scale bar: 10 µm. Figure 13: Representative immunoblots and relative quantities of SOD1, SOD2, Catalase, NQO1, GSTP1, FHC, and FLC protein in both siCtrl- and siOPA1-treated HeLa cells. The quantity of SOD1 was higher in siOPA1-treated cells (1.05 ± 0.03) than control cells (0.79 ± 0.05). The quantity of GSTP1 was higher in siOPA1-treated cells (1.07 ± 0.06) than control cells (0.82 ± 0.11). Figure 14: Total SOD activity (SOD1 and SOD2) (ordinate) was measured for siOPA1- (grey bar) and siCtrl-treated (white bar) HeLa cells. Total SOD activity was higher in siOPA1-treated cells (1.01 ± 0.12) than control cells (0.73 ± 0.08). Figure 15: Catalase activity (ordinate) was measured for siOPA1- (grey bar) and siCtrl- treated (white bar) HeLa cells. Catalase activity was the same in siOPA1- and siCtrl-treated cells. Results are expressed as the mean ± SEM with n = 8 (A), n = 5 (B) n =7 (C) n=5 (D) and n=7. Statistical significance was determined by Student’s-paired t-test, p<0.01**. Figure 16: Simulations of Complex I activity (ordinate) with the mathematical model in a context of SiOPA1 (grey bar) or SiCtrl-treated (white bar) cells. Figure 17: Simulations of ROS production by complex I (ordinate) with the mathematical model in a context of SiOPA1 (grey bar) or SiCtrl-treated (white bar) cells. Figure 18: Representative immunoblots and histograms showing protein levels of OPA1 (inner membrane), citrate synthase (matrix), HSP60 (matrix), VDAC (outer membrane) and TOM20 (outer membrane) relative to actin (ordinate), in OPA1 down-regulated cells and control cells (abscissa). OPA1 protein level is drastically decreased in siOPA1-transfected HeLa cells (92%). In OPA1 down-regulated HeLa cells, there are no differences of all protein quantities compared to control cells. Figure 19: Citrate synthase activity (ordinate), a TCA cycle enzyme, in siOPA1-transfected (grey bar) or siCtrl-transfected (white bar) HeLa cells. Results are expressed as mean +/- SEM with n=8 (A), n=16 (B). P values were determined by Student’s-paired t-test p<0.001***. Figure 20: The mitochondrial network in siOPA1-transfected HeLa cells is punctuated and filamentous in siCtr-transfected HeLa cells (abscissa). Nuclei are stained with DAPI and the mitochondrial network is coloured with mitotracker (ordinate). Scale bar represents 10 µm. Figure 21: Ratio of NAD⁺/NADH, H⁺ and total intracellular levels of NAD⁺ and NADH, H⁺ (ordinate) were measured in HeLa cells transfected with siOPA1 (grey bar) or siCtrl (white bar). Ratio of NAD⁺/NADH, H⁺ and total intracellular levels of NAD⁺ and NADH, H⁺ were unchanged in HeLa cells transfected with siOPA1, when compared to siCtrl cells. Results are expressed as mean +/- SEM n=5. P values were determined by Student’s-paired t-test. Figure 22: Reduced (GSH) and oxidized (GSSG) glutathione levels (ordinate) are measured in HeLa cells transfected with siOPA1 (grey bar), compared to control cells (white bar). Figure 23: Oxidized and reduced quinones are evaluated. Quinone redox state is the ratio of oxidized form of quinone on total forms (ordinate). Quinone redox state were measured in HeLa cells transfected with siOPA1 (grey bar) or siCtrl (white bar). Results are expressed as mean +/- SEM n=4 (A), n=8 (B). P values were determined by Student’s-paired t-test. Figure 24: Representative micrographs of NRF2immunocytofluorescence and DNA Hoechst staining (ordinate) in siOPA1- or siCtrl-treated HeLa cells (abscissa) 72 h after transfection. (The scale bar represents 5 ^m) Figure 25: Graphical Abstract. In OPA1 depleted cells, some subunits of the first four complexes of the Mitochondrial Respiratory Chain are decreased without altered total ATP production. The cells fall in a pro-oxidative state as underlined by the decreased aconitase activity, the NRF2 transcription factor which is translocated into the nucleus and the GSTP1 and SOD1 protein quantities that are significantly increased and in turn decrease the intra- cellular ROS levels. Figure 26: Complex I activity: simulation of Catalytic speed of NAD⁺ (ordinate) in several configurations indicated on the graph with the different models from 1 to 5 with biological data from Heiske et al. (20) (* represents biological data from Heiske et al.(20) named literature ; lines with different colors are the results of simulation from model 1 to 5). A – I : For different concentrations of NADH, and with constant concentration of quinone Q (from 1.1 to 70 µM). J – R : For different concentrations of quinone Q and with constant concentration of NADH (from 1 to 47.5 µM). S – U : For different concentrations of quinol QH₂ or a ratio of QH₂/Q_tot with constant concentration of NADH and quinone Q. V – X : For different concentration of NAD⁺ and with constant concentration of NADH and quinone Q. Figure 27: Complex I ROS production: Catalytic speed of O₂ ^.− (ordinate) in several configurations indicated on the graph with the different models from 1 to 5 with biological data from Kussmaul and Hirst (26) and and Grivennikova and Vinogradov(2006) (16) (* represents biological data from Kussmaul and Hirst (26) and Grivennikova and Vinogradov (16) named literature ; solid line or with dashes are the results of simulation from model 1 to 5) A : For different concentrations of NADH B : For different concentrations of NAD⁺ C : For a concentration of NADH = 30 µM and Q = 100 µM Figure 28: Complex III activity: simulation of Catalytic speed of NAD⁺ (ordinate) in several configurations indicated on the graph with the different models from 1 to 3 with biological data from Heiske et al. (19) (* represents biological data from Heiske et al.(19) named literature ; solid line or with dashes are the results of simulation from model 1 to 3) A – C : For different concentrations of CytoC_ox and with constant concentration of quinol QH₂ (from 70 to 210 µM). D – F : For different concentrations of quinole QH2, and with constant concentration of CytoCox (from 20 to 80 µM). G – J : For different concentrations of quinone Q, and with constant concentration of quinol QH2 and of CytoCox (from 20 to 80 pM) K – N : For different concentrations and percentage of CytoCred and with constant concentration of quinol QH2 (210 µM). Figure 29: Complex I ROS production: Catalytic speed of O2^.− (ordinate) for different percentages of quinone Q (abscissa) indicated on the graph with the different models from 1 to 3 with biological data from Dröse and Brandt (11) (* represents biological data from (11) named literature; solid line or with dashes are the results of simulation from model 1 to 3) Examples Example 1 - Dominant optic atrophy (DOA) is characterized by moderate to severe loss of visual acuity with insidious onset in early childhood (Amati-Bonneau et al., 2009 (45); Lenaers et al. (67), 2012; Yu-Wai-Man et al., 2011 (83)). This disease primarily affects retinal ganglion cells (RGCs), of which the axons forming the optic nerve degenerate. The estimated prevalence is between 1:10,000 in Denmark and 1:50,000 worldwide. There is considerable inter- and intra- familial variability and penetrance may be as low as 40%. Different studies have shown certain OPA1 mutations to be associated with a severe multi-systemic disorder (DOA+ syndrome) (Amati-Bonneau et al., 2008 (46); Cohn et al., 2007 (53); Yu-Wai-Man et al., 2010 (82); Zeviani, 2008 (85)). DOA+ patients present additional neurological complications, such as ataxia, sensorineural deafness, sensory-motor neuropathy, progressive external ophthalmoplegia, and parkinsonism, as well as myopathy. Altogether, these findings highlight the widespread deleterious consequences of OPA1 mutations, not only for RGCs but also for other cell populations (Barboni et al., 2013 (47); Chao de la Barca et al., 2016 (51); Mackey and Trounce, 2010 (70); Spinazzi et al., 2008 (78); Zeviani, 2008 (85)). There is currently no effective treatment for this complex disease. Most DOA patients (~75%) harbour mutations in the OPA1 gene, which encodes a mitochondrial GTPase (Delettre et al., 2000 (57)) localized in the inter-membrane space (IMS) and anchored to the mitochondrial inner membrane (Griparic et al., 2004 (62); Ishihara et al., 2006 (63); Olichon et al., 2002 (73); Satoh et al., 2003 (75)). Most OPA1 mutations result in premature termination, with the ensuing OPA1 haploinsufficiency as a major pathogenic mechanism (Amati-Bonneau et al., 2009 (45)). OPA1 protein has been shown to be involved in fusion of the mitochondrial inner membrane and the structure of the cristae in various cell lines. Fusion and fission of mitochondria control mitochondrial morphology and regulate the major mitochondrial functions (Bertholet et al., 2016 (48)). These processes also contribute to the quality control of the organelle. Through its role in organizing the structure of the cristae, OPA1 sequesters cytochrome c inside the cristae and thus exerts an anti-apoptotic function. Data on skin fibroblasts, muscle, and lymphoblasts from DOA patients globally show altered mitochondrial morphology and energetics, as well as increased sensitivity to apoptosis (Alavi et al., 2009 (44); Chevrollier et al., 2008 (52); Olichon et al., 2006 (74); Spinazzi et al., 2008 (78); Yu-Wai-Man et al., 2011 (83); Zanna et al., 2008 (84)). However, there are numerous contradictory reports concerning the existence and nature of energetic defects in DOA patients and thus, the extent to which these processes contribute to the pathogenesis of DOA is still unknown. Furthermore, two invertebrate DOA models and, more recently, a mammalian DOA model have linked the critical generation of ROS to OPA1 dysfunction (Millet A. et al., 2017 (71); Millet et al., 2016 (72); Shahrestani et al., 2009 (77); Tang et al., 2009 (79)). The inventors previously developed a deterministic mathematical model able to predict The ROS production of Complex I of the MRC as well as The complex I catalytic activity. The method used is based on in vitro data referring to the activity (Heiske et al., 2014 (20)) and production of ROS (Grivennikova and Vinogradov, 2006 (16); Kussmaul and Hirst, 2006 (26)) in several operating configurations of Complex I. These data are introduced in algorithms with normalization to be able to score the future sets of parameter. The molecular behavior is transcribed with the Michaelis and Menten equations of enzymatic kinetics of Complex I reactions. Several models are created with different levels of accuracy, thus increasing the number of parameters to be optimized. After having randomly generated a population of parameters, each solution is passed in a differential evolution algorithm to generate an optimized solution population. The models are connected through a cascade structure and are called successively from the simplest model to the most accurate. According to the algorithm, the selected set of parameters can simulate the activity of Complex I and the production of ROS at the same time with results qualitatively and quantitatively close to the input data. The inventors thus addressed the question of the involvement of OPA1 in oxidative metabolism. As haploinsufficiency is primarily responsible for DOA and the effects of OPA1 inactivation are not restricted to RGCs, the inventors assessed the general impact of OPA1 inactivation on oxidative phosphorylation and the redox state by downregulating OPA1 in HeLa cells using an RNA interference strategy. The inventors found cellular respiration to be diminished when OPA1 levels were decreased. This was accompanied by an increase in mitochondrial ROS production, which was buffered by the activation of antioxidant defences, leading to a pro-oxidative state. The inventors’ algorithm of Complex I is able to simulate its activity that fits with the inventors’ in vitro data and refine hypothesis for the ROS production. 2. Results 2.1. OPA1 downregulation affects cellular oxygen consumption and the quantity and activity of mitochondrial respiratory chain complexes. The inventors evaluated the effect of OPA1 downregulation on respiration and glycolysis in HeLa cells transfected with siRNA against OPA1 (siOPA1) using siRNA against Luciferase (siCtrl) as a control. Seventy-two hours after transfection, the quantity of OPA1 was 92% lower in siOPA1 treated cells than siCrtl cells but actin levels were not altered (Figure 18). The inventors assessed oxygen consumption rates (OCR), a direct measure of oxidative phosphorylation activity, and extracellular acidification rates (ECAR), representative of glycolysis, since it accounts for approximately 80% of acidification (Wu et al., 2007 (89), Xie et al., 2009 (90)), using the Seahorse XF24 flux analyser (Seahorse Bioscience Inc, North Billerica, MA, USA). Basal respiration was 38% lower in siOPA1-transfected HeLa cells than siCtrl-treated cells (Figure 1). Furthermore, ATP-linked respiration measured in the presence of oligomycin, which inhibits ATP synthase, was reduced by 40%. The maximal OCR measured in the presence of the protonophore FCCP, which uncouples oxidation and phosphorylation, was reduced by 58%. The maximal OCR in siOPA1-treated cells was not significantly different from the basal OCR, in contrast to siCtrl-transfected cells. Rotenone (complex I inhibitor) and antimycin (complex III inhibitor) considerably decreased the OCR in both siOPA1- and siCtrl-transfected Hela cells, showing that more than 95% of oxygen consumption was due to mitochondrial respiration (Figure 1). Of note, the decrease of mitochondrial respiration in siOPA1-treated cells did not correlate with a diminution in mitochondrial biomass. Indeed, citrate synthase activity and quantity and HSP60, VDAC, and TOM20 levels were unchanged (Figures 18 and 19). The inventors examined the mitochondrial network of SiCtrl- and SiOPA1-treated Hela cells; the SiOPA1 treated cells showed a fragmented mitochondrial network, in contrast to the filamentous mitochondrial network observed in SiCtrl-treated cells (Figure 20). Although OPA1 downregulation induced a decrease in mitochondrial respiration, total intracellular ATP levels remained unchanged (Figure 2). Furthermore, the decrease in OPA1 levels did not induce a shift towards glycolysis in HeLa cells, as measured by ECAR (Figure 3) and extracellular lactate levels in the culture media (Figure 4). Similarly, oligomycin treatment increased acidification rates in both siCtrl- and siOPA1-treated HeLa cells (Figure 3). The effect of OPA1 downregulation on cellular respiration could be explained by a decrease in the level or activity of mitochondrial respiratory chain (MRC) complexes. The levels of the subunits NDUFB4 (Complex I), SDHB (Complex II), Core2 (Complex III), and COX I (Complex IV) were 44, 29, 39, and 46% lower, respectively, in siOPA1-treated HeLa cells than siCtrl treated cells (Figure 5). However, the levels of other subunits, such as NDUFA9 (Complex I), SDHA (Complex II), Core1 (Complex III), and COX IV (Complex IV), were unchanged (Figure 5). The levels of three subunits of ATP synthase (alpha and gamma for F1 complex and d for F0 complex) were unaffected by OPA1 silencing in HeLa cells (Figure 5). The inventors then assessed the in vitro activities of complexes I to IV by spectrophotometry. Although the activity of complexes I, III, and IV was not affected in OPA1-downregulated HeLa cells, complex II activity was reduced by approximately 25% (Figure 6). Succinate dehydrogenase is also a tricarboxylic acid (TCA) cycle enzyme. The inventors thus assessed the level of NADH, H+, the major TCA cycle product, in HeLa cells. The total intracellular level of NADH, H+/NAD+ was unchanged in siOPA1-treated HeLa cells (Figure 21). Accordingly, the levels of two other TCA cycle enzyme activities, fumarase and malate dehydrogenase, were also unchanged (data not shown). 2.2. OPA1 downregulation results in an imbalanced intracellular redox state The loss of OPA1 results in impaired mitochondrial respiratory chain function, without disruption of the supply of NADH, H+. This can lead to an increase in electron leaks and thus increased reactive oxygen species production. OPA1 downregulation could thus lead to an imbalance of intracellular redox homeostasis in HeLa cells. The inventors investigated this possibility by first measuring total ROS content using an H2- DCFDA probe. Surprisingly, ROS levels decreased by 23% in OPA1 downregulated HeLa cells 72 h after transfection (Figure 7). The inventors then investigated aconitase activity, which has been shown to be highly sensitive to oxidation due to a damaged FeS core. Inhibition of its activity is thus widely used as a signature of increased mitochondrial ROS production (Gardner et al., 1994 (60); Kelly et al., 2010 (65); Vincent et al., 2005 (80)). Aconitase activity was significantly lower (by 34%) in siOPA1-treated HeLa cells than control cells (Figure 8). This decrease could not be attributed to differences in protein quantity, which was unchanged (Figure 9). 2.3. OPA1 downregulation activates a major antioxidant pathway: the NRF2 transcription factor Although the total intracellular level of ROS was reduced, the inhibition of aconitase activity provided evidence of an increase in intra-mitochondrial ROS production. This suggests that intracellular antioxidant responses were activated upon OPA1 downregulation. The inventors thus first analysed the levels of two redox state markers, glutathione and quinone. The ratio between reduced (GSH) and oxidized (GSSG) glutathione in siOPA1-treated HeLa cells increased by 78%, but this increase was not significant (Figure 22). The quinone redox state was not altered by the loss of OPA1 (Figure 23). The inventors then investigated the nuclear factor (erythroid-derived 2)-like 2 (NRF2) pathway, which accounts for a large part of antioxidative responses. The inventors assessed the intracellular localisation of NRF2, which indicates its activation, by immunocytofluorescence in HeLa cells from 66 to 72 h post-transfection (Figures 11 and 12). The kinetics of NRF2 immunostaining showed significant NRF2 nuclear relocalisation starting 67 h after OPA1 downregulation (Figure 12). The inventors already knew that the level of OPA1 protein was reduced by 50% 48 h and 90% 72 h post-transfection (data not shown). The inventors thus hypothesized that NRF2 translocation would occur between 48 and 72 h post- transfection. Seventy-two hours post-transfection, 51% of siOPA1-treated HeLa cells showed NRF2 nuclear localisation, whereas only 15% of siCtrl-treated cells showed NRF2 in the nucleus (Figures 11, 12 and 25). The inventors thus assessed the levels of several NRF2 target proteins, such as superoxide dismutases 1 and 2 (SOD1 and SOD2), catalase, NQO1, GSTP1, and ferritin heavy (FHC) and light (FLC) chains. The quantity of SOD1 protein was significantly higher (33%) in siOPA1-treated HeLa cells than control cells (Figure 13). This increase correlated with a 37% increase in total SOD (SOD1 and SOD2) activity (Figure 14). There was also an increase of 30% in the quantity of GSTP1 protein in siOPA1-treated HeLa cells (Figure 13). Neither the quantity nor activity of catalase were altered upon downregulation of OPA1 (Figures 13 and 15). Furthermore, there was no change in NQO1, FHC, or FLC protein levels in siOPA1-transfected HeLa cells. The data are summarized in Table 1. OPA1 deficiency H2DCFDA= total cellular ROS Decrease Aconitase activity Decrease Mitosox= mitochondrial ROS Increase Nuclear translocation of NRF2 Increase SOD1 Increase GSTP1 Increase Total SODs activity Increase Table 1: Summary table of performed tests of oxidative metabolism for OPA1 deficiency in HeLa cells. Altogether, these results are showing that OPA1 down regulation increases mitochondrial ROS levels, activating the nuclear translocation of NRF2 which in turn up- regulates some antioxidant defenses decreasing the total cellular ROS. This generates a pro- oxidative stress sensitizing cells to subsequent stress. 2.4. A deterministic mathematical model of complex I is able to predict the results obtained with OPA1 down regulation. The data presented in Figure 16 show that the inventors’ algorithm fed with data from the inventors’ analysis didn’t find any difference in complex I activity while OPA1 is down-regulated or not as well as the inventors presented in vitro activities in Figure 6. the algorithm doesn’t express the results in the same units as the in vitro results of Figure 6 (micromole per min per milligram of proteins instead of micromole per min per million of cells). The inventors verified the stability of the protein contents in OPA1 down regulated and WT cells (SiLuc treated cells 99,23 +/- 22,56 n=6 and SiOPA1 treated cells 77,38 +/- 16,20 p value= 0,4516 with an unpaired T test) as they are strictly identical the inventors can compare the two types of results. Moreover, the inventors’ algorithm presents no differences in ROS production by Complex I (Figure 17). 3. Discussion Mitochondrial respiration was impaired upon OPA1 downregulation in HeLa cells, as previously shown in several cell types (Millet et al., 2016 (72)). This, however, did not trigger a shift towards glycolysis. Overall, the mitochondrial biomass did not change, as estimated both by measurement of the levels of several mitochondrial proteins and citrate synthase activity. However, the level of specific subunits of the first four complexes of the mitochondrial respiratory chain decreased. Therefore, the decrease in mitochondrial respiration measured in siOPA1-treated cells did not correlate with a decrease in the quantity of mitochondria but may be the consequence of impaired activity of the MRC complexes. Only the activity of complex II was decreased. 3.1. Cellular respiration is impaired in OPA1 downregulated cells Oxygen consumption rates (OCR) were significantly decreased in OPA1 downregulated cells. This phenomenon, also analysed in vitro in OPA1 downregulated neurons, was not accompanied by a shift towards glycolysis (Millet et al., 2016 (72)). The decrease in OCR shown in siOPA1-treated cells could not be linked to a decrease in mitochondrial biomass, as the quantities of citrate synthase, HSP60, VDAC, and TOM 20 and the activity of citrate synthase remained unchanged in both siCtrl and siOPA1 treated cells. This phenomenon is similar to that observed in our previous study on OPA1 downregulated rat neurons in primary culture (Millet et al., 2016 (72)). In addition, the level of subunits of the first four complexes of the MRC were found to be similar in both OPA1 downregulated HeLa cells and rat neurons in vitro, with a marked decrease in the level of certain subunits. However, the quantity of complex V (ATP synthase) was unchanged upon OPA1 inactivation in both cellular models (Bertholet et al., 2013 (49); Millet et al., 2016 (72)). Such a similar mitochondrial metabolism pattern in two very different cell types suggests that OPA1 downregulation triggers mitochondrial dysfunction possibly involved in both neuronal and extra-neuronal degeneration, as seen in DOA+ patients. 3.2. The redox state is imbalanced in OPA1-downregulated cells A marked decrease in the quantity of MRC subunits and/or dysfunction of the activities of the complexes, while the level of NADH remains stable, results in increased mitochondrial ROS production. The fact that the NADH, H+, NAD+, and NAD+/NADH ratio were unchanged in the siCtrl and siOPA1 treated HeLa cells implies that the amounts of substrates provided to the MRC were also unchanged in both populations. As already demonstrated in siOPA1- treated rat neurons in primary culture, total intracellular ROS levels decreased. However, aconitase activity also decreased, suggesting an increase in mitochondrial ROS levels. Hence, the increase in intra-mitochondrial ROS production, which did not correlate with the total intracellular level of ROS, suggests the activation of antioxidant responses upon OPA1 downregulation. We verified this possibility by first analysing two redox state markers, glutathione and quinone. The ratio between reduced (GSH) and oxidized (GSSG) glutathione in siOPA1-treated HeLa cells increased by 121% relative to control cells, but was not statistically different (Figure 22), and by 78.1% in siOPA1-transfected neurons (p < 0.05), as previously described (Millet et al., 2016 (72)). The quinone redox state (oxidized quinones/(oxidized + reduced quinones)) was not altered by the loss of OPA1 (Figure 23). Altogether, these results suggest that OPA1 downregulation activates antioxidant responses by increasing the reduced form of glutathione, the major non-enzymatic antioxidant compound in the cells. Quinones appear not to be involved in mitochondrial ROS detoxification under our conditions. Once again, the similarities between the data obtained with neurons and HeLa cells is striking. These data suggest activation of the antioxidant signaling pathway to buffer the increase in mitochondrial ROS production. 3.3. Antioxidant defenses are activated in OPA1 downregulated cells. The intra-nuclear level of NRF2, a major transcription factor involved in antioxidant defenses (Ma, 2013 (69)) was analyzed, in a time-course experiment to assess its activation upon OPA1 downregulation. There was a significant increase in the nuclear translocation of NRF2 in siOPA1-transfected cells from 67 to 72 h post-transfection. Of note, the level of OPA1 decreased by 50% and 90% 48 and 72 h post-siOPA1 transfection, respectively. This correlation between the decrease in OPA1 content and the nuclear translocation of NRF2 is particularly intriguing, as it underscores the link between OPA1 and the redox state. The levels of various NRF2 targets in the two conditions; the quantity of SOD1 and GSTP1 increased upon OPA downregulation, as well as SOD activity were measured. Similarly, both the level and activity of catalase, another NRF2 target, were elevated in siOPA1-treated neurons (Millet et al., 2016 (72)). The detoxification of the superoxide anion was clearly activated in both cellular types, underlying the importance of the phenomenon and the universal involvement of OPA1 in redox signaling. This study is clearly in accordance with the literature linking OPA1 with the redox state in invertebrates (Kanazawa et al., 2008 (64); Yarosh et al., 2008 (81)) and also confirms our results in neurons and transgenic mice cortices (Daloyau et al., 2018 (56); Millet et al., 2016 (72)), with better identification of the actors. Moreover, this paper underlines the ubiquitous effect of OPA1 deficiency on oxidative metabolism in different cell types. A recent study analysing the expression of OPA1 during H₂O₂ treatment showed a large decrease in the long form of OPA1 (Garcia et al., 2018 (59)). The destabilization of the long form of OPA1 could, in turn, disorganize the inner membrane, emphasizing the effect of the reorganisation of the MRC and increasing ROS production. In other words, this phenomenon may represent a vicious circle (Figure 25). 3.4. Replicative cells as biological material to predict and treat OPA1 gene mutation-related disorders The high convergence of the results based on two biological models of DOA that are so strikingly different paves the way towards novel ways of analysing, predicting, and possibly treating OPA1 dysfunctions. Moreover, the use of a mathematical model for the complex I activity and ROS production is interesting. In this work the algorithm simulates perfectly the complex I activity whether there is or not OPA1 expression. The activities are stable in both cases with in silico simulations (Figure 16) and in vivo analysis (Figure 6). In term of ROS production, the simulations suggest that Complex I wouldn’t be the principal ROS producer. In this context, Complex III would be the most probable actor of the increase in mitochondrial ROS production. In silico simulations with our mathematical model of Complex I activity combined with ROS production will become a powerful tool to integrate different biological data and the predict the evolution of the described processes. This is a real proof of concept for translational medicine in the area of neurodegenerative diseases. We plan to analyse the oxidative metabolism of DOA and DOA+ patients using fibroblasts from patient biopsies (Millet et al., 2016 (72)) and epithelial cells to predict the evolution of the disease, which is currently impossible to control. Experimental and mathematical models be combined to enhance the power of wet-lab investigations and more accurately predict the evolution of the disease by considering mitochondrial ROS production and the detoxifying pathways of ROS (Merabet et al., 2017 (29); Millet A. et al., 2017 (71)). Mitochondria have long been proposed to play a key role in aging (Green et al., 2011 (61)). As a consequence of their central role in ATP formation via the MRC, mitochondria are the major source of ROS and are thus highly involved in oxidative stress processes (Brookes et al., 2004 (50)). However, mitochondria are also targets of these molecules (Brookes et al., 2004 (50)). Under physiological conditions, approximately 1 to 3% of molecular oxygen is incompletely reduced during redox reactions in the MRC, leading to the production of ROS superoxide anion (O2^.−) by-products. In this scenario, complex interactions in antioxidant defence systems repress oxidative stress within mitochondria (Kienhofer et al., 2009 (66)). Cellular systems that protect against oxidants involve antioxidant defence enzymes (superoxide dismutase [SOD], glutathione peroxidase [GPx], and catalase) (Kienhofer et al., 2009 (66)), oxidant scavengers (vitamin E, vitamin C, carotenoids, uric acid, and polyphenols), and mechanisms that repair oxidant-induced damage to lipids, proteins, or DNA. Despite these protective mechanisms, the uncontrolled generation of ROS can overwhelm the capacity of antioxidant protection, causing mitochondrial dysfunction (for example Parkinson disease directly involves Complex I dysfunction). In vivo studies in transgenic mice have shown that the overexpression of catalase targeted to mitochondria reduces age-associated diseases and increases their lifespan (Lopez-Armada et al., 2013 (68); Schriner et al., 2005 (76)). Based on these observations, our entire body of work clearly suggests that the downregulation of OPA1 in cellular and animal models induces an imbalance of the redox state, which could lead to premature cellular ageing. 4. Materials and Methods 4.1. Cell culture HeLa cells from the American Type Culture Collection (Manassas, VA) were cultured in Dulbecco’s Modified Eagle’s Medium with 4.5 g/l glucose (DMEM, Invitrogen), supplemented with 10% FCS, penicillin (100 units/ml) and streptomycin (100 mg/ml), in an incubator at 37°C and 5% CO2. HeLa cells were electroporated using the Cell Line Kit R (Amaxa, Lonza) with 1.5 µg control siRNA (D-001210-02, Dharmacon Research) or human OPA1 siRNA (D- 005273-03, target sequence AAAGAAGGCUGUACCGUUA (SEQ ID NO: 1), Dharmacon Research) per 10⁶ cells. 4.2. Measurement of oxygen consumption rates, extracellular acidification rates and ATP levels Oxygen consumption rates (OCR) and extracellular acidification rates (ECAR) were measured using the XF24 Extracellular Flux Analyser (Seahorse Bioscience, North Billerica, MA). HeLa cells (15.103) transfected with siCtrl or siOPA1 were plated on XF24 microplates three days before OCR measurements. Dual-analyte sensor cartridges were soaked in XF Calibrant Solution (Seahorse Biosciences) in 24-well cell-culture microplates overnight at 37°C to hydrate. Approximately one hour prior to experimentation, injection ports on the sensor cartridge were filled with oligomycin (1 µM), Carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP) (1 µM), and rotenone (1 µM) plus antimycin A (1 µM). Plates were then loaded into the XF24 instrument for calibration. For measuring oxygen consumption, the DMEM growth media of HeLa cells was replaced by DMEM supplemented with NaCl (143 mM), Phenol Red (3 mg/ml), glucose (10 mM), glutamine (2 mM), and pyruvate (2 mM) at pH 7.4, and the plates maintained at 37°C 1 h prior to experimentation. Plates were then loaded into the Seahorse XF24 analyser following the manufacturer’s instructions. ATP measurements in HeLa cells were determined using the ATP Colorimetric/Fluorometric Assay kit (Abcam). Intracellular ATP levels were determined using the colorimetric assay, following the manufacturer’s instructions, on 1.106 HeLa cells transfected with control siRNA or OPA1 siRNA. ATP content was measured in duplicate (570 nm) and calculated per microgram of protein. 4.3. In vitro activities of respiratory chain complexes The activity of respiratory complexes II and IV and citrate synthase was measured as previously described (Agier et al., 2012 (88)). The activity of respiratory complexes I and III was measured as previously described (Spinazzi et al., 2011 (78)). 4.4. Extracellular lactate levels Extracellular lactate levels were measured using a colorimetric assay (BioMérieux). Lactate was measured in media supernatants (1/10 dilution) at 505 nm 72 h after transfection of HeLa cells with siCtrl or siOPA1, following the manufacturer’s instructions. 4.5. Immunoblot analysis Transfected HeLa cells were lysed for 30 min in a RIPA buffer containing 50 mM Tris-HCL pH 7.5, 250 mM NaCl, 5 mM EDTA, 5 mM EGTA, 1 mM Dithiothreitol, 0.1% Triton X-100, 0.1% SDS, 1% Deoxycholate, 1% NP40, and protease inhibitors (« Complete » protease inhibitor mixture, Roche Applied Science). Cell lysates were then centrifuged at 14,000 g at 4°C for 10 min. The total protein concentration was determined in the supernatant using the Bradford Protein-assay (Bio-Rad). Proteins (100-200 μg) were separated by SDS-PAGE (8-15%) and transferred onto nitrocellulose membranes (Whatman, Protran). Free binding sites were blocked with 5% non- fat dry milk in 1X Tris Buffered Saline pH 7.6 containing 0.2% Tween 20 (blocking buffer). The membranes were probed with various primary antibodies (anti-OPA1 (1/300, BD-Biosciences), anti-actin (1/25,000, Chemicon), anti-HSP60 (1/8,000, Sigma), anti-OXPHOS (1/200, Mitosciences), anti-NDUFB4 (1/500, Mitosciences), anti-NDUFA9 (1/100, Mitosciences), anti- SDHA (1/1,000, Abcam), anti-Core 1 (1/500, Invotrogen), anti-COXIV (1/250, Cell Signaling Technology), anti-ATP5C1 (1/500, Abgent), anti-ATP5H (1/5,000, Abcam), anti-aconitase (1/500, Abcam), anti-SOD1 and anti-SOD2 (1/2,000, Epitomics), anti-catalase (1/3,000, Abcam), anti-NQO1 (1/3,000, Abcam), anti-GSTP1 (1/8,000, Oxford Biochemical Research), anti-ferritin heavy chain (1/500, Abcam), and anti-ferritin light chain (1/4000, Abcam) and incubated overnight at 4°C in blocking buffer. After chemiluminescent detection of horseradish peroxidase-conjugated secondary antibody (1/50,000, Abcam), scanned photographic films were analysed using ImageJ software. 4.6. Immunocytochemistry or mitotracker staining HeLa cells were fixed for 20 min with PBS containing 3.7% formaldehyde and permeabilized for 5 min in 1X PBS, 0.25 % TritonTM X-100 and incubated for 10 min at -20°C with methanol prior to nuclear NRF2 detection. Non-specific binding sites were blocked with 3% BSA in 1X PBS for 15 to 30 min at room temperature. Cells were immunostained with rabbit polyclonal anti-NRF2 antibody (1/50, Santa Cruz Biotechnology) for 1 h at 37°C. HeLa cells were then incubated with Alexa fluor 488-conjugated secondary antibodies (1/300, Molecular Probes), labelled with 0.25 µg/ml Hoechst in 1X PBS for 5 min, and mounted in Mowiol. Immunolabelling of HeLa cells 66, 67, 68, 69, 70, and 72 h after siCtrl or siOPA1 transfection was visualized under a fluorescence microscope (Nikon Eclipse 80i) and the images acquired using an NIS- Element (Nikon Digital Sight DUS2 camera). HeLa cells showing accumulation of NRF2 staining in the nucleus were counted by stack with Hoechst labelling of the nucleus using ImageJ software. Mitochondrial network was stained with Mitotracker Red (Invitrogen molecular probes FM Invitrogen M22425) in culture medium during 15 min at 37°C and 5% CO2. Then cells were fixed for 20 min with PBS containing 3.7% formaldehyde and permeabilized for 5 min in 1X PBS, 0.25 % TritonTM X-100 and the mounted in DAPI-mounted medium. 4.7. Measurement of reactive oxygen species levels and aconitase activity ROS levels in HeLa cells were measured using the fluorescent dye 2',7'- dichlorodihydrofluorescein diacetate (CM-H2-DCFDA, Molecular Probes) at 4 µM for 30 min at 37°C. Fluorescence intensities were measured at 493 nm in a WALLAC VICTOR 1480 Multilabel Counter. Mitochondrial superoxide production was measured using MitoSOX Red (mitochondria- targeted superoxide indication) 5 µm upon the manufacturer’s protocol with fluorescent microscopy (X40). MitoSOX measurement was performed after 72h siCtrl or siOPA1 transfection. Quantification of fluorescence intensity was analysed by ImageJ software. Aconitase activity in HeLa cells was measured at 525 nm (UVIKON Spectrophotometer 922) using a protocol already described in (Colombani et al., 2009 (54)). 4.8. Enzymatic antioxidant activities Superoxide dismutase (SOD) activities (Mn SOD and Cu/Zn SOD) were assayed by inhibition of pyrogallol auto-oxidation in HeLa cell extracts, as performed for aconitase activity. One enzymatic unit of SOD activity was defined as the amount of enzyme that inhibited pyrogallol auto-oxidation by 50% (Galinier et al., 2006 (58)). Catalase activity was determined by measuring the decomposition of H2O2 at 240 nm (Galinier et al., 2006 (58)). 4.9. Statistical analysis Most of the experiments were statistically treated using Student’s paired t-test because of the systematic comparison between siCtrl and siOPA1-treated HeLa cells. Oxygen consumption rates between siCtrl and siOPA1-treated HeLa cells were investigated using a nonparametric test (Mann-Whitney). P values: p < 0.05*, p < 0.01**, p < 0.001***. 4.10. Immunoblot analysis The membranes were probed with various primary antibody (anti-OPA1 (1/300, BD- Biosciences), anti-actin (1/25000, Chemicon), anti-HSP60 (1/8000, Sigma), anti-citrate synthase (1/3000, Abcam), anti-VDAC (1/1000, Abcam) and anti-TOM20 (1/3000, Abcam)) and incubated overnight at 4°C in blocking buffer. After chemiluminescent detection of horseradish peroxidase-conjugated secondary antibody (1/50000, Abcam), scanned photographic films were analysed using ImageJ software. 4.11. Citrate synthase activity Citrate synthase activity was evaluated in HeLa cells transfected with siCtrl or siOPA1 lysates. Citrate synthase activity was determined by measurement of TNB produced by the reaction of CoA-SH and 5’5 dithiobis 2 nitrobenzoic acid (DTNB).930 µl of reaction media (100 µM DTNB, 100 mM TrisHCL pH 8.0, 300 µM acetyl-CoA, 500 µM oxaloacetate, 0,1 % Triton X-100) and 40 µg of proteins were measured at 412 nm (37°C). Reactions start after adding 50 µl of 10 mM oxaloacetic acid diluted in 100 mM Tris HCl pH 8.1. 4.12. NAD⁺ and NADH, H⁺ levels Intracellular NAD⁺ and NADH levels in HeLa cells were measured with an NAD⁺/NADH Assay kit (Abcam) according to the manufacturer's instructions. Briefly, 2.10⁶ HeLa cells were transfected with small interfering RNA control or against OPA1. Cells were washed with cold PBS and extracted with NADH/NAD extraction buffer by two freeze/thaw cycles (20 min on dry ice and then 10 min at room temperature). Total NAD (NADt) and NADH levels were detected in a 96-well plate, and color was developed and read at 450 nm. NAD+/NADH ratio was calculated as (NADt − NADH)/NADH. 4.13. Glutathione and quinone levels HeLa cells were lysed with 200 µl of 5 % metaphosphoric acid and then centrifuged 1,500 g at 4°C during 10 min. Final supernatant was used for glutathione assay (reduced GSH and oxidized GSSG measurements) performed by reverse-phase high-perforance liquid chromatography (HPLC) as previously described (Galinier et al., 2006 (58)). Quinone reduced and oxidized levels were performed as described (Galinier et al., 2006 (58)). Example 2 - Multi objective optimization of mitochondrial process model: a novel tool for precision medicine 1. INTRODUCTION In the context of precision medicine adapted to different pathologies (neurodegenerated diseases, metabolic diseases or inflammation processes) traditional biological approaches are still not sufficient. A system biology methodology associated to engineering techniques are necessary. This procedure is used to build a knowledge model of biological mechanisms. In a cellular system, mitochondria arc the main provider of energy: Adenosine Tri Phosphate (ATP) via the oxidative phosphorylation. The oxidative phosphorylation system consists of five enzyme complexes anchored in the inner membrane of the mitochondria. Four of these complexes (complexes I to IV) make up the respiratory chain. By moving protons (H+), complexes I, VI and IV establish an electrochemical gradient. This process is powered by the redox energy released during the successive electron transfers from the Nicotine of electrons from reduced Nicotinamide adenine dinucleotide (NADH) and succinate to oxygen. The fifth complex uses this electrochemical gradient created by complexes I, III and IV to catalyse the phosphorylation of adenosine diphosphate (ADP) to ATP (Mitchell, 1961 (30); Mitchell, 1966; (31)). During a normal function of the MRC, Reactive Oxygen Species (ROS) are basically produced essentially by the first and third complexes. ROS arc derivatives of dioxygen with sur numerar electrons. However, complex 1 and complex III dysfunctions, caused by different factors (genetic, environmental or pathologies) result in a dramatic increase of their production of ROS. This increase, if it is not balanced by antioxidant defences, induces lipids, proteins and DNA destruction 1. This phenomenon finally ends in cell death most of the time associated with inflammation processes. We previously developed a stochastic mechanical model of complex I able to simulate complex I activity and its ROS production (Merabet, 2017 (29)). In the present example, the inventors focused on a deterministic model of complex I and complex III. 2. BIOLOGICAL MECHANISM OF MITOCHONDRIAL RESPIRATORY CHAIN Complex I is an electron entry point in the respiratory chain 2. It catalyzes the oxidation of NADH to NAD⁺ and the reduction of ubiquinone (Q) into ubiquinol (QH2}. This reaction allows to move four protons from the matrix to the intermembrane space. ”x” refers to the mitochondrial matrix and ”is” refers to the intermembrane space. NADH is oxidized by a non-covalently bound flavin mononucleotide (FMN). The hilly reduced flavin FMNH- and the semi-reduced flavin FMNH transfer these electrons one by one to a chain of eight ironsulfur (FrS) clusters constituting the redox centers of the Complex I. The last cluster of the chain is the electron donor to the Q-binding site. It is well accepted that biological electron transfer is a process of tunnelling (Moser et al.,1992 (32)). Complex III oxidizes ubiquinol and reduces cytochrome c. In mammals, complex III is a dimer with each monomer consisting of 11 subunits as described in Iwata et al., 1998 (23). Among these 11 subunits, three are conserved among species, from bacteria to mammals, and contain four groups involved in the redox function of complex TH: the bH and bL hemes of cytochrome b, cytochrome c1, and the [2Fe-2S] center of the Rieske protein (ISP). The mechanism of the reaction catalyzed by complex III is called cycle Q. It was initially proposed by Mitchell (1975) (91) and subsequently modified (Hunte et al., 2003 (22); Berry et al.,2000 (4)). This mechanism is based on the existence of two binding sites for the Q/QH2 redox couple called site Qo site and Qi site. The oxidation of ubiquinol at the Qo site induces the release of two protons and the transfer of two electrons to complex III. The first electron is transferred to the Fb-S group of the PSI before being transferred to cytochrome cl and filially reducing cytochrome c (high potential chain). The other electron enters the low potential chain with the reduction of heme bL and is then transferred to heme bH and filially to the ubiquinone of the Qi site, forming a stabilized semiquinone (Hunte et al., 2003 (22); Berry et al.,2000 (4)) that can be detected and released from the positive side of the membrane and a second cytochrome c is reduced ma the high potential chain while a second electron is transferred to the Qi site via the low potential chain, reducing the semiquinone to ubiquinol. This second phase is accompanied by the use of two protons (Berry et al., 2000 (4)). Reactive oxygen species (ROS) and more precisely superoxide anion the first reactive oxygen species is mainly produced in mitochondria by complexes I and III of the MRC. Complexes I and III of the respiratory chain are considered to be the main producers of superoxide anion and its derivatives within the mitochondria (Bleier and Drose, 2013 (5); Wong et al., 2017 (43); Pryde and Hirst, 2011 (36); Murphy, 2009 (92)). Superoxide production by complex I has been shown using isolated complexes (Kussmaul and Hirst, 2006 (26); Esterlıâzy et al., 2008 (93)), submitochondrial particles (Vinogradov and Grivennikova, 2005 (42); Pryde and Hirst, 2011 (36); Genova et al., 2001 (15); Fato et al.,2009 (13); Grivennikova and Vinogradov, 2013 (17); Maranzana et al., 2013 (28); Kim et al.,2018 (24)) and intact mitochondria from multiple sources. Two modes of superoxide production by complex I have been identified: a direct production mode in the presence of NADH and a reverse production mode in the presence of succinate. The production of ROS in direct mode is maximal when the NADH/NAD+ ratio is high (strongly reduced NAD pool) and, in mitochondria and submitochondrial particles, when complex I is inhibited by a Q-site inhibitor (e. g. rotenone) (Kussmaul and Hirst, 2006 (26) Lambert and Brand, 2004 (27)). The second mode of production was observed with coupled submitochondrial particles and intact mitochondria. This mode of production relies on the reverse transport of electrons in complex I: this reverse transport requires the presence of a highly reduced quinone pool and a sufficiently high electrochemical gradient. Measured production rates in reverse mode can reach three times those in direct mode, measured under optimal conditions of KOS production by the direct mode (fully reduced NAD pool and presence of Q-site presence of complex I Q-site inhibitors such as rotenone). The reverse mode of production of complex I has been known since the 1960s as described in Hinkle et al., 1967 (21) but has long been considered as a phenomenon without physiological relevance. However, several studies suggest, that this process, which does not require inhibition or dysfunction of complex 1, may have physiological validity. Indeed, this process could play an important role in redox signalling mechanisms in basal location and in pathological oxidative mechanisms, especially in the case of pathological mechanisms (Chouchani et al.,2016 (8); Pell et al.,2018 (35); Robb et al,2018 (38)). Complex III is capable of producing large amounts of superoxide when inhibited by antimycin A at the Qi site. This production takes place at the Qo site and the superoxide produced is released oil both sides of the inner membrane of the mitochondria. In the absence of antimycin A the production of O2^{. -} by complex III is low to undetectable (T^irrens et al., 1985 (41); Muller et al,2002 (33); Muller et al.,2003 (34); Drose and Brandt, 2008 (11); Borek et al., 2008 (6); Chen et al., 2003 (7); Ksenzenko et al., 1983 (25)). But, in the presence of a physiological membrane potential, purified yeast complex III recoustituted iii phospholipid vesicles, also produce non-negligible amounts of ROS (Rottenberg et al.,2009 (39)). The rate of produced ROS would increase exponentially as a function of membrane potential and could reach values similar to those obtained lining antimycin A. Moreover, the absence of production by complex 111 under basal conditions does not mean that this production could not increase under pathological conditions (Bleier and Drose, 2013 (5)). This oxygen electron donor would be a semiquinone produced at the Qo site (Drose and Brandt, 2008 (11); Fisher et al., 2016 (14)). 3. MODELLING OF ENZYME KINETICS 3.1 Complex I Complex I is considered as a system that can transfer electrons from a donor (NADH) to several recipients (Q, O2) depending on the circumstances: The inventors use the Michaelis and Menten equation to relate observations to formulas. At the 2 active sites, the substrates need to bind to complex I to allow electron transfer (donor or recipient). In addition, once the products are formed, they access the active site, blocking the following substrates. The products become competitive inhibitions for their own substrate. The functions of active sites ^^⁄ ^ take the following form ⁽^, ^⁾ ^ _^^ ^^^^⁄ ^_^^^^^⁄ ^_^^ . The inventors identify x and y which are equivalent to molecule concentrations and kMx and kMy which are the Michaelis constants and are to be determined by all the inventor’s algorithms. For fNADH And for fQ There is also a phenomenon of inhibition by substrates. These ones could physically prevent the products from leaving the active site, slowing down the resulting activity. Complex I has the ability to accumulate a limited number of electrons within itself. Thus, the more electrons the complex stores, the more the bottleneck effect will be felt as the reaction rate of electron donation from NADH to the complex is reduced. Thus, if the capacity of the quinone Q to accept electrons is low, the complex empties slowly, the bottleneck is consequent and the NADH can donate its electrons less quickly, thus ^_^ ^{^^^^}decreases. The production of the superoxide anion ^_^ ^{.^} is proportionally linked to the quantity of oxygen but is also inhibited by two phenomena: • NAD⁺ blocks access to oxygen when donating an electron to the complex, this effect is modelled by competitive inhibition; • the complex functions properly when the availability of the quinone Q is sufficiently high. The inventors add the empirical parameter alpha to control this phenomenon. As the two types of inhibition do not operate at the same sites, they are multiplied in the following formula: By using the mechanisms of Complex I in direct mode, the inventors can transcribe its activity for each electronic pathway. When all the substrates are present at their site, a NADH molecule will donate 2 electrons, both of them for quinone site or directly for the oxygen reduction. The inventors use the constant V⁺ to model the Complex I capacity to accept the electron transfer. The Inventors can separate the different pathways to have the normal pathway with quinone reduction. And the ROS production A second operating mode can be added to the direct mode: the reverse mode. In reverse mode, quinol QH₂ becomes the electron donor and NAD⁺ or O₂ the recipients. The ^_^^^ ^{^}and ^_^^^ functions are then used with a mirror construction of their direct mode equivalent. For the ^_^ ^{.^} production, the inventors assume that the electron donor is flavin as before but the presence or absence of quinone inhibiting ^_^ ^{.^} production in direct mode has no impact in reverse mode, so wc remove it from the equation 4. Once all these equations have been established, we need to find the values that the constants , and V⁺ should take. For this purpose, the concentrations NADH, NAD⁺, QH2, Q arid O₂ are considered to be known arid are taken from the literature. The inventors use a total of 5 direct models of complex I. The first one (numbered 1) is the simplest of all and uses the presented equations before 2, 3, 4, 6 and 7. The model N*2 uses a sigmoid function to represent one of the Michaelis constants. Models 3 and 4 take into account the inhibition of ^_{^^ 2} by ^_^ and NAD⁺ respectively. The 5th and last model implement the steric inhibition of NADH. Each model improves the previous model, so model 5 takes into account, all the additions mentioned and is the most complete. 3.2 Complex III Complex III is considered as a system that can transfer electrons from a donor (QH2) to several recipients (Q, Cytc, O2) depending on the circumstances: • In the absence of quinone al the Qi site, cytochrome C will be the recipient; • In the presence of antimycin, cytochrome C and oxygen will receive the electrons. The inventor’s use the Michaelis and Menten equations to relate observations to formulas. At the 3 active sites, the substrates need to bind to complex III to allow electron transfer (donor or recipient), in addition, once the products are formed, they access the active site, blocking the following substrates. These products become competitive inhibitions for their own substrate. The functions of active sites take following form ⁽^, ^⁾ ^ ^{^⁄ ^} _^^ ^^{^^⁄ ^} _^^ ^{^^⁄ ^} _^^ : For ^_^^^^ , for ^_^^^^^^ And for fQ The effects of antimycin on complex III and the QH2 oxidation rate allow the inventors to speculate that a dependence between the QH2 oxidation rate and the ability to receive electrons from cytochrome C exists. If the capacity of cytochrome C to accept electrons is high then QH2 can donate them faster, (n) therefore ^ ^{^^} ^ ^{^} increases; and vice versa. ^ ^{^^} ^ ^{^} can therefore be modeled as a function of ^_^^^^^^ as a sigmoid to enrich the model. The electrons evolving in complex III will be accepted in priority by the 2 substrates Q and cytochrome C, so The inventors can modify the functions of^_^^^^^^, ^_^ and ^_^^ to include antimycin inhibition. By using the mechanisms of complex III, the inventors can transcribe its activity in each configuration. When all the substrates are present at their site, a quinole molecule will donate 2 electrons, 1 for each site. Cytochrome C will be reduced and quinone will be partially reduced, giving: The inventors use the constant V⁺ to model the complex III capacity to accept the electron transfer. When there is no quinone, the 2 electrons in the quinole reduce 2 cytochromes. The presence of quinone is considered to be an inhibition here. In the presence of antimycin, the electrons of the quinole will partition between tlie cytochrome and the heme bL. A quinone molecule at the Q_o site collects the electron from the heme bL by reverse transfer and gives it to oxygen. The QH2, Q, cytcox, cytcred, O2 and antimycin concentrations are input data from literature. The inventors have to find the value of the other constant ^ ^{^^} ^ ^{^^}, ^ ^{^} ^^{^}, ^ ^{^^^^^} ^ ^{^^}, ^ ^{^^^^^} ^ ^{^^^}, ^ ^{^} _{^ ^} use the complex III model. The inventors use a total of 3 models of complex III. The first one (numbered 1) is the simplest and uses the presented equations. Model No 2 uses a sigmoid function to represent one of the Michaelis constants. The 3rd and last model implements the inhibition of complex III by antimycin. Each model improves the previous model, so model 3 takes into account all the additions mentioned and is the most complete. 4. MULTIOBJECTIVE OPTIMIZATION Optimization function The inventors use experimental data from articles: • 3 for the complex I, one for the activity (Heiske et al., 2014 (20)) and another for the ROS production (Kussmaul and Hirst, 2006 (26); Grivennikova and Vinogradov, 2006 (16)) • 2 for the complex III, one for the activity (Heiske et. al., 2017 (19)) and another for the ROS production (Dröse et Brandt, 2008 (11)). The inventors search to minimize the maximal difference between model evaluation and the experimental data: the optimization is ensured via the constant, values. fi is the i-th objective function, ^_^^^,^ is a vector of experimental catalytic speed of the i-th objective and ^_^^^,^ is the vector of catalytic speed evaluated with the model. The magnitude order between the catalytic speed and the ROS production speed being too large, we use the Grodzevich et Romanko, 2006 (18) method. To optimize the parameters, the inventors establish objective functions from experimental data from the literature and from the modeling of these data by our model. Thus the inventors seek to reduce as much as possible the difference between the experimental data and the inventors’ model. The optimization domain is bounded by 2 points: • the Utopia point: this is the ideal objective vector which minimizes each objective function individually, • the Nadir point: this is the vector referencing the worst optimization of each objective function with the solutions found for the Utopia point. Normalization allows us to give a score between 0 and 1 to each objective function. A score close to 0 means being close to the Utopia point, and therefore a good multi-objective solution. Conversely, a score close to 1 will result in a solution to be ruled out. Dominance These scores make it possible to sort the most interesting solutions according to their dominance. A solution a dominates a solution b if each of its scores less than or equal to the respective score of b AND if there is at least one score of a lower than respective b score. k is number of objective function of an optimization. The solutions are arranged by fronts: front 1 contains all the solutions which are not dominated, front 2 contains the solutions dominated only once, and so on until having all the fronts. The crowding distance (Deb, 2002 (10)) allows solutions to be ranked within the same dominance front. For each objective, the solutions with the highest and lowest scores will have an infinite distance. The intermediate solutions will have a crowding distance calculated by the normalized absolute difference of the solutions of the objective functions adjacent to solution i (i+1 and i-1). In other words, for each objective function fj, with j ^ { 1, ..., k}, the solutions are sorted by increasing score and the intermediate solutions Xi, i ^ { 2, ..., NP-1} are calculated via : with the assumption ^_^ ⁽^_^^ ⁾ ≠ ^_^ ⁽^_^ ⁾. A total crowding distance can be calculated by adding the distances of each objective for a given solution Xi. Thus, the set of solutions is prioritized first by dominance ranks and then within a rank, the solution with the largest clutter distance will be the best solution. Initial population The initial candidate population is usually created randomly to cover a wider field of the study area. However, the experimental data and the objective functions allows up to know a priori the relevant intervals for the different parameters. Nevertheless, this method can make converge towards local minima and skew the continuation of procedures. Therefore, Afi’s method was used which generates a population of size NP according to an uniform random distribution while being bounded by the user, a population of opposite candidates is built in parallel to enrich the total population. A candidate X = ⁽x_^, x_^ , … , x_^ ⁾ of dimension n is constructed for all i ∈ [1, 2, … , n] to have x_^ ∈ [l_^, u_^] with x_^ = l_^ + ⁽u_^ − l_^ ⁾ ∗ U⁽0,1⁾. The opposite candidate X^{^} = ⁽x^{^} _^, x^ ^, … , x^ ^⁾ is defined by x^ ^ = l_^ + u_^ − x_^~ All these candidates make up a population of 2NP candidates. The inventors use the dominance sorting method explained above for a population of the NP best candidates. Evolutionary algorithm Once the population is formed, each ^_^,^ candidate (p-th candidate of the population generation G) passes through a differential evolution algorithm having 3 operators: mutation, crossing and selection. Mutation: this operator creates a new candidate from 3 candidates from the current population. For a candidate ^_^,^ 3 random candidates are drawn ^^_^^,^ , ^_^^,^ , ^_^^,^^ with p,p1,p2,p3 ∈ {1,2, ...,NP} and p ^ ^ p1 ^ p2 ^ p3. The mutated candidate ^_^,^ is defined by: F is a control parameter between 0 and 1. Crossing : the mutated candidate ^_^,^ = ^{^}^_^,^,^, … , ^_^,^,^ ^{^} is crossed with the initial candidates ^_^,^ = ^^_^,^,^, … , ^_^,^,^^ create a temporary candidate ^_^,^ = ^^_^,^,^, … , ^_^,^,^^ with p ∈ {1,2,…, NP}, i ∈ {l,2,…,n}, r a random value between 0 and 1,and rr a random value generated only one time for each p, and Cr ∈ [0; 1] the crossing rate. Selection : Usually, the selection is between the initial candidate and the crossed candidate, the best one is selected to incorporate the initial population of the next generation, the other candidate is simply discarded. Here, the unselected candidate is placed in a pool, once the whole population has been run through the evolutionary algorithm, this pool is challenged with the previously selected candidates to keep only the best NPs for the next generation’s population. It is possible to stop the evolutionary algorithm prematurely once a certain population convergence has been reached. This convergence is measured with the stability of the density of the non-dominated solutions. In other words, the stopping criterion is based on the evolution of the crowding of solutions: convergence is observed when the crowding distance no longer evolves significantly over several generations, so the search for solutions is stopped. The inventors note d ^ the maximum clutter distance at generation ^. The inventors can measure the stability of the clutter over L generations by calculating the difference : ^_^ = ^ m^ℓa^x^ ^_ℓ − ^ m^ℓi^n^ ^_ℓ with the condition on the stopping criterion: ^_^ < ^_^^^ At this stage, a difficulty may arise since d ^ may undergo oscillations even after convergence. In this case, with a too large Δlim criterion, the oscillations will not be taken into account and the search for a solution will stop too soon. On the contrary, if Δlim is too small, the stop will be too fast. In this respect, (ROUDENKO, 2004 (40)) uses the standard deviation of the dl instead of the stability of the clutter: wherein^{^} ^^{^} _^ ^{^} is the mean of ^_ℓ during L generations. 5. RESULTS AND DISCUSSION The inventors have constructed a model of the catalytic activity of complex I and its production of superoxide anion in direct and reverse mode. To build this model classical enzymatic kinetic equations were modified to fit the specificities of complex I. The inventors optimized the parameters of the direct model with a multi-objective algorithm. The model of complex I is able to simulate the catalytic activity - Figure 26 - and production of ROS - Figure 27 - in direct mode for different configurations and concentration of substrates and products. For different concentrations of NADH, and with constant concentration of quinone Q (from 1.1 to 70 µM) our model No 5 is the most accurate comparing to the experimental data. The result from model 1 to 5 is shown Figure 26 A - I. For different concentrations of quinone Q and with constant concentration of NADH (from 1 to 47.5 µM) our model No 5 is the most of the time better comparing to the experimental data. The result from model 1 to 5 is shown Figure 26 J - R. For different concentrations of quinol QH2 or a ratio of QH2/Qtot with constant concentration of NADH and quinone Q inventors’ model No 5 is the most accurate comparing to the experimental data. The result from model 1 to 5 is shown Figure 26 S - U. Finally, for different concentrations of NAD⁺ and with constant concentration of NADH and quinone Q inventors’ model No 5 is the most accurate comparing to the experimental data. The result from model 1 to 5 is shown Figure 26 V - X. For different concentrations of NAD⁺ the model No 5 is the best for low concentrations of NADH but it is divergent, for high concentration of NADH. The result from model 1 to 5 is shown Figure 27 A. For different concentrations of NAD⁺ the model No 5 is the best. The result from model 1 to 5 to shown Figure 27 B. For a concentration of NADH = 30µM and Q = 100 µM, all models are acceptable The result from model 1 to 5 to shown Figure 27 C. The inventors can observe a real improvement of the simulations through the iterative process from model 1 to 5. For different concentrations of CytoC_ox and with constant concentration of quinol QH₂ (from 70 to 210 µM) inventors’ model No 3 is the most accurate comparing to the experimental data. The result from model 1 to 3 is shown Figure 28 A - C. For different concentrations of quinole QH2, and with constant concentration of CytoCox (from 20 to 80 µM) model No 3 is the most accurate comparing to the experimental data. The result, from model 1 to 3 is shown Figure 28 D - F. For different concentrations of quinone Q, and with constant concentration of quinol QH2 and of CytoCox (from 20 to 80 pM) our model No 3 is the most accurate comparing to the experimental data. The result from model 1 to 3 is shown Figure 28 G - J. For different concentrations and percentage of CytoCred and with constant concentration of quinol QH₂ (210 µM) model No 3 is the most accurate comparing to the experimental data. The result from model 1 to 3 is shown Figure 28 K - N. For different percentages of quinone Q the model No 3 is the best. The result from model 1 to 3 is shown Figure 29. The inventors applied the modelling method used for complex I to complex III. The inventors created a model capable of simulating the production of ROS and the catalytic activity of complex III. The inventors also performed a sensitivity analysis in the same way as for complex I. Gauthier et al (Gauthier et al., 2013a) constructed a mathematical model of the production of ROS by complexes I and III to study the production of ROS in cardiac myocytes under different metabolic conditions. However, the model focuses only on the production of ROS and is not tested for the catalytic activity of the respiratory chain which is lacking. Another model for the production of ROS by complexes I and III has been proposed by Bazil el al. (2). This model includes the respiratory chain model developed by Beard et al. (3). The model of Beard et al. (3) models the activity of respiratory complexes by a law of mass action. In the Bazil et al. model (2), the model for complex I is the one previously proposed by Bazil et al. (1). In addition, a production of ROS with the semiquinone donor, in disagreement with the experimental data obtained on complex I, is assumed in the model Bazil et al. (1). Other models of complex III have also been proposed but focus solely on the mechanism of ROS production or quinol oxidation by complex III (Quinlan et al., 2011 (37); Crofts et al., 2006 (9)). 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Claims

Claims 1. A method for in vitro quantifying the reactive oxygen species, or ROS, appearance rate in a biological sample, the method comprising: a) determining the amount of 7 components involved in the activity of mitochondrial respiratory chain Complex I, or CI, or involved in the activity of mitochondrial respiratory chain Complex III, or CIII, or in the activity of both the mitochondrial respiratory chain CI and CIII, said 7 components being - the reduced form of Nicotinamide adenine dinucleotide, or NADH, - the oxidized form of Nicotinamide adenine dinucleotide, or NAD+, - quinone, or Q - quinol, or QH₂, - molecular oxygen, or O₂, - the reduced form of cytochrome C, or cytCred, and - the oxidized form of cytochrome C, or cytCox, to obtain a respective concentration value - [NADH], corresponding to the amount of NADH measured in said biological sample, - [NAD+], corresponding to the amount of NAD+ measured in said biological sample, - [Q], corresponding to the amount of quinone measured in said biological sample, - [QH₂], corresponding to the amount of quinol measured in said biological sample, - [O₂], corresponding to the amount of molecular oxygen measured in said biological sample, - [CytCRed], corresponding to the amount of reduced form of cytochrome C measured in said biological sample, - [CytCox], corresponding to the amount of oxidized form of cytochrome C measured in said biological sample, b) calculating a first ROS appearance rate V1 Ros of ROS in said sample wherein [^^^^] ^ (^[ ^^^^], ^[^^^ ^{^} _^^^^ ^ ^ ^{^}]) ₌ [^^^^] in wh ^^ _^^^^ ^{^} _^ ich ^_^^^^ ^{^ ^} _{^ ^^^} ^{^} [^] ^ ^ ^ ⁽[^], [^^_^]⁾ = [^] [^^ ] in which ^^ ^ _^ ^{∗^} _^ ^{^} _^,^,^^ ^ ^{∗^} _^ ^ ^ wherein V⁺ = 1727.2 mmol/min/mg ; KM,0^NADH = 10,6 K1 = 174.1, K2= 681.2, KM^NAD+ = 1028.6 µM, K_M ^Q= 11.6 µM, K_is ^Q= 719128.5 µM, K_M ^QH2 = 16.3 µM, K_is ^QH2 = 36.2 µM, Ki^NAD+ = 26.4 KO2 = 4871.0 and ^= 5. c) calculating a second Ros appearance rate V2 Ros of ROS in said sample wherein V2⁺ = 3579.29 mmol/min/mg ; K1 = 23.05, K2= 472.12, KM,0^QH20 =184.77 µM, KM^QH2i = 397,14, KM^Qo= 43.08 µM, KM^Qi = 225.21µM, KM^cytCox = 8.11 µM, KM^cytCred= 419.16µM, K’O2 = 914.51, ^’= 22.02 and β=173.54, d) obtaining the Ros appearance rate such that VRos = V_1Ros + V_{2 Ros.} 2. The method according to claim 1, wherein the method comprising: b) calculating a first ROS appearance rate V_{1 Ros} of ROS in said sample wherein V⁺ = 2121,1 mmol/min/mg ; K_M,0 ^NADH = 8,1 µM, K₁ = 185,2, K₂= 1021,2, K_M ^NAD+ = 625,8 µM, K_M ^Q= 15,7 µM, K_M ^QH2 = 26 µM, K_is ^Q= 1066907,0 µM, K_is ^QH2 = 48,6 µM, K_is ^NADH = 165076,9 µM Ki^NAD+ = 34,3 KO2 = 9551,4 et ^= 3,3, and c) calculating a second ROS appearance rate V_{2 Ros} of ROS in said sample wherein V2⁺ = 3909.06 mmol/min/mg ; K1 = 19.96, K2= 341.15, KM,0^QH20 = 157.61 µM, KM^QH2i = 478.63, KM^Qo= 50.49 µM, KM^Qi = 280.26µM, KM^cytCox = 10.98 µM, KM^cytCred= 248.18µM, K’O2 = 760.78, ^’= 38.66 and β = 212.03 d) obtaining the ROS appearance rate such that VRos = V1Ros + V2 Ros. 3. The method according to claim 1 or 2, wherein the activity of CI is determined by the following formula: 4. The method according to claim 1 or 2, wherein the activity of CIII is determined by the following formula: 5. A computer program comprising instructions which, when the program is executed by a computer, causes the computer to carry out steps b) to d) of the method as defined in anyone of claims claim 1 to 4. 6. A method, for determining in vitro Ros detoxifying enzyme activity in a sample of an individual, the method comprising: - quantifying the Ros appearance rate in the biological sample by carrying out the method as defined in anyone of claims 1 to 4, to obtain a calculated rate, - comparing the calculated rate with a reference Ros appearance rate, said reference Ros appearance rate being obtained from a reference sample, the reference sample being of the same nature as the sample, to obtain a ratio R between the Ros appearance rate and the reference Ros appearance rate, and - concluding that * if R is lower than 0.85, then the sample contains efficient Ros detoxifying enzyme activity, to detoxify the sample during an oxidative stress, * otherwise the sample does not contain efficient Ros detoxifying enzyme activity, (that detoxify the sample during an oxidative stress). 7. The method according to claim 6, wherein * if R lower than 0.8, then the sample contains efficient Ros detoxifying enzyme activity, to detoxify the sample during an oxidative stress, * otherwise the sample does not contain efficient Ros detoxifying enzyme activity, to detoxify the sample during an oxidative stress. 8. A method for determining in vitro the response of a patient to a compound liable to reduce Ros amount in a biological sample, the method comprising - quantifying the Ros appearance rate in the biological sample, before the administration of the compound to the patient, by carrying out the method as defined in anyone of claims 1 to 4, to obtain an initial Ros rate, - quantifying the Ros appearance rate in the biological sample, after the administration of the compound to the patient, by carrying out the method as defined in anyone of claims 1 to 4, to obtain a final Ros rate, - comparing the initial Ros rate and the final Ros rate to obtain a ratio R between the final Ros and the initial Ros rate, - concluding that * if R lower than 0,85, then the compound is able to reduce Ros amount in the sample, * otherwise the sample is not able to reduce Ros amount in the sample. 9. The method according to claim 8, wherein * if R is lower than to 0.8, then the compound is able to reduce Ros amount in the sample, * otherwise the sample is not able to reduce Ros amount in the sample. 10. A method for determining in vitro in a biological sample if a compound is a pro- or an anti- oxidant compound, the method comprising: - quantifying the Ros appearance rate in the biological sample by carrying out the method as defined in anyone of claims 1 to 4, to obtain a calculated rate, - comparing the calculated rate with a reference Ros appearance rate, said reference Ros appearance rate being obtained from a reference sample, the reference sample being of the same nature as the sample, to obtain a ratio R between the Ros appearance rate and the reference Ros appearance rate, and - concluding that * if R is lower than 0.85, then the compound is an anti-oxidant compound, * if R is higher than 1.15, then the compound is a pro-oxidant compound, and * if R is comprised from 0.85 to 1.15, then the compound has neither anti- nor pro-oxidant properties.