Dr Etienne Aux

Dr Ludovic Lefai

Pr Michel Pelosi

Pr Jean-Marc Rigoulet

Lobaccaro

Fréderic, Stéphanie, Mathilde Merci Isabelle

Merci Professeur Julien

Maria Gallego

José Yelamos

Anaïs, Guy, Zulimar Merci Eriel

Layal… Gracias

Jean Rostand

a ti…)

Keywords: Mitochondrial DNA, DNA repair, BER, microarrays, Poly(ADP-ribosyl)ation, PARP, PARG ADN mitochondrial, Réparation, BER, puces ADN, Poly(ADP-ribosyl)ation, PARP, PARG

Thank you Reiner for your moral support what was my energy

Beaucoup d'entre nous qui espèrent devenir docteur, ignorons son origine Ceux qui luttaient à mort dans les arènes du Colisée de Rome afin de survivre, malgré de graves blessures, devaient se rétablir rapidement pour affronter le prochain combat .Survivre à chaque combat grâce à l'expérience du précédent était la stratégie du gladiateur. La résistance physique et mentale étaient ses véritables armes, mais plus encore, celui qui réussissait à intégrer une équipe de combat était appelé Docteur.

Il est plus difficile, de réussir l'intégration d'une équipe, que la notre au sein d'une existante. S'intégrer est la mission de gladiateur, intégrer est le résultat d'un Docteur….

Remercier est un acte de gratitude en réponse aux résultats positifs obtenus grâce à la participation de tierces personnes. L'être humain, en tant qu'entité sociale, est constamment soumis à une grande interaction avec des personnes qui permettent le développement ou la frustration de ses projets.

Généralement celui qui réalise une thèse remercie ceux qui ont rendu possible celle-ci.

Néanmoins, Aujourd'hui je remercie ceux qui n'ont pas rendu possible la fin de l'étudiant.

… et durant toute cette nouvelle vie qui commence aujourd'hui, de par mes résultats positifs, je dirai merci à l'équipe d'accueil qui représenta l'arène ou j'ai livré tous mes combats ; ............................................................................. b) Is PARP protein involved in mtDNA repair in Drosophila melanogaster? ..................................... c) How is PARP regulated at a transcriptional level in different Drosophila mutants for key BER enzymes? ..................................................................................................................................................................... I.→.Animals ...........................................................................................................................................................................

Abreviations

INTRODUCTION

I. Mitochondria

According to the endosymbiosis theory, mitochondria origin is from -proteobacteria, which after the internalization by an anaerobic cell, (Figure 1), carried out an endosymbiotic relationship with them, based in exchanges of metabolites, energetic sources such as ATP and complex mechanism of signaling [START_REF] Kutschera | Endosymbiosis, Cell Evolution, and Speciation[END_REF].

The intermembrane space

The intermembrane space is the space between the outer and the inner membrane. Its nature is very similar to cytoplasm; it became clear very recently that the intermembrane space plays a pivotal role in the coordination of mitochondrial activities with other cellular processes. In human, these activities include the exchange of proteins, lipids, or metal ions between the matrix and the cytosol, the regulated initiation of apoptotic cascades, signalling pathways that regulate respiration and metabolic functions, the prevention of reactive oxygen species produced by the respiratory chain, or the control of mitochondrial morphogenesis 1 .

In Yeast mitochondria, several proteins were localized in the intermembrane space such as:

Ups1p, Ups2p, Cytochrome bz and Cytochrome c (Cyt c) and peroxidase [START_REF] Maccecchini | Import of Proteins into Mitochondria[END_REF][START_REF] Tamura | Role for Two Conserved Intermembrane Space Proteins, Ups1p and Ups2p, [corrected] in Intra-Mitochondrial Phospholipid Trafficking[END_REF].

In normal functioning of mitochondria, Cyt c remains attached (electrostatic interactions)

with proteins (complex II and IV) inserted in inner mitochondrial membrane. A disorder in electrostatic potential of inner membrane leads to the traffic of Cyt c to cytosol where it is involved in apoptosis pathway [START_REF] Liu | Induction of Apoptotic Program in Cell-Free Extracts: Requirement for dATP and Cytochrome c[END_REF].

I.1.2.2. The mitochondrial matrix

The mitochondrial matrix is the scenario of metabolic pathways implicated in ATP production, such as: fatty acid oxidation, amino acid catabolism and Krebs cycle (also named the citric acid cycle CAC). (Figure 3) The machinery enzymes of the CAC is represented in different sub-compartments (metabolic canalization) in the mitochondrial matrix, with the exception of succinate dehydrogenase, which is bound to the inner mitochondrial membrane as part of Complex II.

The mitochondrial matrix can integrate cytosolic and mitochondrial pathways where metabolites such as, amino acids, fatty acid and glucose become in ATP. Recently, thanks to microscopy and mass spectrometry techniques, 495 proteins inside the human mitochondrial matrix were identified. The method was based on a genetically targetable peroxidase enzyme that biotinylated nearby proteins, which were subsequently purified and identified by mass spectrometry. Also it was identified 31 proteins not previously involved in mitochondria [START_REF] Rhee | Proteomic Mapping of Mitochondria in Living Cells via Spatially Restricted Enzymatic Tagging[END_REF].

I.1.3. The Outer mitochondrial Membrane

The mitochondrial outer membrane (OMM) separates the intermembrane space from the cytosol, which encloses totally the organelle. OMM lipid composition is near 40% of phospholipids permitting the whole exchange of metabolites between mitochondrial internal compartments and the cytosol in eukaryote cell. OMM creates a diffusion barrier for small molecules such as adenine nucleotides, creatine phosphate and creatine, causing ratedependent concentration gradients as a prerequisite for the action of ADP shuttles via creatine kinases or adenylate kinases. If the OMM becomes leaky, Cyt c and apoptosis-inducing factor can be released [START_REF] Gellerich | Function of the Mitochondrial Outer Membrane as a Diffusion Barrier in Health and Diseases[END_REF]Horvath SE. et Daum G., 2013).

The protein voltage-dependent anion-selective channel (VDAC) [START_REF] Rostovtseva | VDAC Structure, Function, and Regulation of Mitochondrial and Cellular Metabolism[END_REF] constitutes the main pathway for ATP, ADP, and other mitochondrial metabolic substrates in OMM. Recent findings suggest that regulation of VDAC by cytosolic proteins, such as kinases, and cytoskeletal proteins, dynamically and globally controls mitochondrial metabolism in normal cells and might play a role in aerobic glycolysis in cancer cells (the Warburg effect). Bigger molecules can only cross the OMM by active transport through mitochondrial membrane transport proteins, and mitochondrial pre-proteins are imported through specialized translocase complexes (Figure 4) as translocase outer membrane (TOM).

The OMM also contains enzymes involved in: fatty acids elongation, hormones oxidation and tryptophan catabolism. In Eukaryotes, the IMM presents different receptors involved in diverse pathways such as:

Ca 2+ internalization [START_REF] Hajnóczky | Old Players in a New Role: Mitochondria-Associated Membranes, VDAC, and Ryanodine Receptors as Contributors to Calcium Signal Propagation from Endoplasmic Reticulum to the Mitochondria[END_REF] or in mtDNA maintenance [START_REF] Rossi | Mitochondrial Localization of PARP-1 Requires Interaction with Mitofilin and Is Involved in the Maintenance of Mitochondrial DNA Integrity[END_REF].

Other mitochondrial membrane lipids such as phosphatidylcholine, phosphatidylserine, phosphatidylinositol, sterols and sphingolipids have to be imported [START_REF] Becker | Role of phosphatidylethanolamine in the biogenesis of mitochondrial outer membrane proteins[END_REF].

The more important phospholipid from IMM is the cardiolipin (CL), which provides essential structural (Figure 5) and functional support to several proteins involved in mitochondrial bioenergetics. Thus, CL leads to IMM freely permeable only to oxygen, carbon dioxide, and water, but slightly permeable to the passage of ions and molecules across the membrane without carriers. CL mediates the cooperativity between cytochrome-c-binding sites in the dimeric enzyme complex [START_REF] Arnold | Cell Respiration Is Controlled by ATP, an Allosteric Inhibitor of Cytochrome-c Oxidase[END_REF].

A) B)

I.1.5.2. mtDNA replication

The Figure 8 shows different replication modes of mammalian mtDNA. Replication of leading strand (H-strand) initiates at OH and proceeds unidirectionally (black arrow), displaying the parental H-strand as ssDNA. When OL (or OLalt) is exposed, the lagging strand (L-strand) synthesis initiates in the opposite direction (gray arrow).

In the ribonucleotide incorporation throughout the lagging strand (RITOLS) model, replication of leading strand (H-strand) initiates at a discrete origin (OR) and proceeds unidirectionally (black arrow), displaying the parental H-strand. The lagging strand (L-strand) is initially laid down as RNA (red line) using the displayed H-strand as template. The RNA is subsequently replaced by DNA (gray arrow), which probably starts as soon as OL is exposed by a yet unclear mechanism. In the strand-coupled mode (right pathway) initiation of both leading-(H-strand) and lagging strand (L-strand) synthesis occurs bidirectionally from multiple origins across a broad zone downstream of OH. This is followed by progression of both forks until the forks arrest at OH [START_REF] Antoshechkin | The HMG-Box Mitochondrial Transcription Factor Xl-mtTFA Binds DNA as a Tetramer to Activate Bidirectional Transcription[END_REF].

I.1.5.3. mtDNA transcription

In human cells, each strand contains promoter for transcriptional initiation, one on the light-strand promoter (LSP) and two on the heavy-strand promoter (HSP), (Figure 7).

Transcription from the mitochondrial promoters produces polycistronic precursor RNA encompassing all the genetic information encoded in each of the specific strands, and the primary transcripts are processed to produce the individual tRNA and mRNA molecules [START_REF] Clayton | Replication and Transcription of Vertebrate Mitochondrial DNA[END_REF]. T he transcription of mtDNA operates in each promoter independently and it is carrying out oriented in opposite direction. The heavy strand has two sites of transcription initiation such as HSP1 and HSP2 Besides, the strand-coupled and the RITOLS models coexisting as interconnected events leading as multiple priming sites of the new lagging strand for the DNA synthesis.

More recently, Reyes and collaborators observed that long tracts of RNA are associated with replicating molecules of mtDNA and they suggested that the mitochondrial genome of mammals is copied by a non classical mechanism (synthesis in parallel of each strand with 5'-3' orientation)

Finally using DNA synthesis in organelle, they demonstrated that isolated mitochondria incorporate radiolabeled RNA precursors, as well as DNA precursors, into replicating DNA molecules. They indicate that RITOLS is a physiological mechanism of mtDNA replication, and that it involves a 'bootlace' mechanism, illustrated in (Figure 9) in which processed transcripts are successively hybridized to the lagging-strand template, as the replication fork advances [START_REF] Reyes | Mitochondrial DNA Replication Proceeds via a "Bootlace" Mechanism Involving the Incorporation of Processed Transcripts[END_REF]. (PAC) transgenic mice, it was reported that also mammalian TFAM may serve as a key regulator of mtDNA copy number. This results that the human TFAM protein is a poor activator of mouse mtDNA transcription, despites its high capacity for unspecific DNA binding [START_REF] Campbell | Mitochondrial Transcription Factor A Regulates Mitochondrial Transcription Initiation, DNA Packaging, and Genome Copy Number[END_REF]. Now, therefore it would be possible to dissociate the role of TFAM in mtDNA copy number regulation from mtDNA expression and mitochondrial biogenesis in mammals in vivo.

The yeast POLRMT contains a unique amino-terminal extension, which is absent in homologous bacteriophage RNA polymerases. The function of the amino-terminal extension is separable from the known RNA polymerization activity of the enzyme (Wang Y et Shadel

GS., 1999)

In support of this notion, the domain interacts specifically with Nam1, a protein originally identified as a high copy suppressor of mtDNA point mutations that affect splicing of introns in budding yeast. The N-terminal domain may therefore provide the means to couple factors involved in additional aspects of RNA metabolism directly to the transcription machinery [START_REF] Rodeheffer | Nam1p, a Protein Involved in RNA Processing and Translation, Is Coupled to Transcription through an Interaction with Yeast Mitochondrial RNA Polymerase[END_REF].

According with S. cerevisiae transcription model, it will be of interest to analyze human mitochondrial transcription. It was demonstrated in human that the levels of TFAM directly regulate the activity of both TFB1M/POLRMT -and TFB2M/POLRMT -dependent mtDNA transcription both in vivo and in vitro (Falkenberg M.et al., 2002).

The TFAM protein contains two tandem HMG box domains separated by a 27-amino-acid residue linker region and followed by a 25-residue carboxy-terminal tail. Mutational analysis of TFAM has revealed that the tail region is important for specific DNA recognition and essential for transcriptional activation (Shadel GS, et Clayton., 1995).

TFAM can bind, unwind and bend DNA without sequence specificity, similar to other proteins of the HMG domain family [START_REF] Fisher | DNA Wrapping and Bending by a Mitochondrial High Mobility Group-like Transcriptional Activator Protein[END_REF].

The sequence-specific binding of a TFAM tetramer [START_REF] Antoshechkin | The HMG-Box Mitochondrial Transcription Factor Xl-mtTFA Binds DNA as a Tetramer to Activate Bidirectional Transcription[END_REF], upstream of HSP and LSP, may allow the protein to introduce these structural alterations at a precise position in the promoter region and perhaps partially unwind the start site for transcription.

This model may explain why the exact distance between the TFAM binding site and the start site for LSP transcription is of critical importance (Dairaghi DJ, et al., 1995).

The POLRMT (with proof-reading ability) can collaborate with Twinkle protein (with 5' to 3' DNA helicase) in DNA replication (McKinney EA, Oliveira MT 2013).

Also, the Figure 10 shows POL , POLand POLRMT action The POLRMT was initially identified in Saccharomyces cerevisiae. This enzyme is a unique mitochondrial RNA polymerase that is distinct from the nuclear RNA polymerases [START_REF] Greenleaf | Yeast RPO41 Gene Product Is Required for Transcription and Maintenance of the Mitochondrial Genome[END_REF][START_REF] Kelly | Isolation of the Nuclear Gene Encoding a Subunit of the Yeast Mitochondrial RNA Polymerase[END_REF].

In human cells POLRMT was later identified and sequence analysis demonstrated that it was structurally related to the bacteriophage T7 RNA polymerase [START_REF] Tiranti | Identification of the Gene Encoding the Human Mitochondrial RNA Polymerase (h-mtRPOL) by Cyberscreening of the Expressed Sequence Tags Database[END_REF].

The POLRMT as the T7 homologue cannot initiate transcription on its own [START_REF] Fuchs | In Vitro Synthesis of T3 AND T7 RNA Polymerase at Low Magnesium Concentration[END_REF]), but requires the simultaneous presence of a high mobility group-box protein (mitochondrial transcription factor A; TFAM), and either transcription factor B1 (TFB1M) or B2 (TFB2M) [START_REF] Fernández-Silva | Replication and Transcription of Mammalian Mitochondrial DNA[END_REF].

Both TFB1M and TFB2M can form a heterodimeric complex with POLRMT. The four proteins of the basal mitochondrial transcription machinery have been purified in recombinant form and used to reconstitute transcription in vitro with a promoter-containing DNA fragment [START_REF] Falkenberg | Mitochondrial Transcription Factors B1 and B2 Activate Transcription of Human mtDNA[END_REF].

The POLRMT is not only required for transcription, besides, at the origin of heavy strand Abbreviations: HSP, heavy-strand promoter; LSP, light-strand promoter; OH, origin of H-strand; F, tRNA phenylalanine; P, tRNA prolin (Wanrooij S. et al., 2008).

The Figure 12 shows in three steps the different proteins involved in mtDNA transcription. The POLRMT has the capacity to specifically initiate RNA primer synthesis at the OL stem-loop structure. At this point, POL replaces POLRMT and lagging strand DNA synthesis is initiated. Interestingly, unlike in H-strand replication, the primer hand-off between POLRMT and POL could be reconstituted in vitro, suggesting that this event in OL dependent initiation is not a regulated event (Wanrooij S. et al., 2008).

The proposed model is mainly based on in vitro data, but receives support from twodimensional gel electrophoresis (2D-PAGE) analysis in combination with siRNA knockdown of POLRMT. In human cells, depletion of POLRMT generates replication intermediates with delayed lagging strand synthesis, thereby providing evidence for POLRMT acting as a lagging-strand primase also in vivo [START_REF] Fusté | Mitochondrial RNA Polymerase Is Needed for Activation of the Origin of Light-Strand DNA Replication[END_REF]. Recently, it has been suggested that transcription in mitochondria is also coordinated with other crucial processes such as DNA replication and translation, indicating the importance of studies of molecular mechanisms of mitochondrial gene expression in these organelles [START_REF] Miu | Transcription and its regulation in mammalian and human mitochondria[END_REF].

It was reported different levels among the steady-state of the promoter-proximal transcripts and steady-state of promoter-distal from mRNA transcripts [START_REF] Gelfand | Synthesis and Turnover of Mitochondrial Ribonucleic Acid in HeLa Cells: The Mature Ribosomal and Messenger Ribonucleic Acid Species Are Metabolically Unstable[END_REF].

The second heavy-strand promoter dedicated to rRNA gene transcription justifies this difference in transcript levels [START_REF] Montoya | Identification of Initiation Sites for Heavy-Strand and Light-Strand Transcription in Human Mitochondrial DNA[END_REF][START_REF] Montoya | The Pattern of Transcription of the Human Mitochondrial rRNA Genes Reveals Two Overlapping Transcription Units[END_REF].

Besides, the existence of a tridecamer template sequence downstream of the 16S rRNA 3′-end, which serves as a binding site for the mitochondrial transcription termination factor mTERF [START_REF] Christianson | A Tridecamer DNA Sequence Supports Human Mitochondrial RNA 3'-End Formation in Vitro[END_REF][START_REF] Kruse | Termination of Transcription in Human Mitochondria: Identification and Purification of a DNA Binding Protein Factor That Promotes Termination[END_REF][START_REF] Fernandez-Silva | The Human Mitochondrial Transcription Termination Factor (mTERF) Is a Multizipper Protein but Binds to DNA as a Monomer, with Evidence Pointing to Intramolecular Leucine Zipper Interactions[END_REF].

Although mTERF binding to the termination sequence blocks transcription bidirectionally in a partially purified human mitochondrial system, it only blocks heterologous RNA polymerases in the direction opposite that of mitochondrial rRNA synthesis [START_REF] Shang | Human Mitochondrial Transcription Termination Exhibits RNA Polymerase Independence and Biased Bipolarity in Vitro[END_REF].

The number of mitochondrial proteins is estimated 10 % of the total cellular proteome. But only a few polypeptides of OXPHOS are translated in mitochondria (due to mtDNA coding limitation).

Certainly, the mitochondrial translation system is very similar to the bacterial system. It is inhibited by chloramphenicol and initiation is carried out by a formyl-methionine. Another feature of this system is the use of a genetic code that differs from the universal genetic code for several codons [START_REF] Osawa | Recent Evidence for Evolution of the Genetic Code[END_REF].

The Table 1 shows mitochondrial genetic code use. Thus, mitochondria can replicate its genome without mitochondrial division or mitochondrial fission takes place without mtDNA replication. The Figure 14 shows the mitochondria of ONH astrocytes were stained with MitoTracker Red. Control ONH astrocytes exposed to vehicle contained classic elongated tubular mitochondria. In contrast, ONH astrocytes exposed to H 2 O 2 contained small rounded mitochondria.

However, ONH astrocytes pretreated with CoQ10 showed a partial preservation of mitochondrial morphology compared with the ONH astrocytes exposed to H 2 O 2 .

Furthermore, the mitochondria appearance as network-like regrouped organelles with the same functionally is supports by fission and fusion mitochondrial cycles. Also these organelles can be morphologically and functionally independent within cells [START_REF] Noh | Inhibition of oxidative stress by coenzyme Q10 increases mitochondrial mass and improves bioenergetic function in optic nerve head astrocytes[END_REF].

I.3. Mitochondrial fusion and fission

Mitochondrial fusion and fission cycles are opposing processes involved in the division and the morphology of the mitochondrial population dynamic [START_REF] Chen | Mitochondrial Dynamics in Mammals[END_REF].

Mitochondrial fusion is regulated by proteins localized in OMM named mitofusin (Mfn)

family [START_REF] Mozdy | A Fuzzy Mitochondrial Fusion Apparatus Comes into Focus[END_REF]. Mitofusin 2 is involved in ER and mitochondria exchange of Ca2 + . Mitochondria are in constant remodeling in the cell with half-life between 6 to 10 days and this variation is because of fusion and fission regulation [START_REF] Jakobs | High Resolution Imaging of Live Mitochondria[END_REF].

Fusion and fission events were originally studied in the yeast Saccharomyces cerevisiae and allowed the identification of many protein actors as MMM (Mitochondrial Morphology and Maintenance). Most of the MMM genes are highly conserved through of species evolution and highly implicated in the mitochondrial morphology changes by coordinated fusion and fission.

These results were confirmed in human mitochondria where it has been shown that fusion and fission reactions are involved in matrix exchanges [START_REF] Legros | Mitochondrial Fusion in Human Cells Is Efficient, Requires the Inner Membrane Potential, and Is Mediated by Mitofusins[END_REF][START_REF] Santel | Mitofusin-1 Protein Is a Generally Expressed Mediator of Mitochondrial Fusion in Mammalian Cells[END_REF] . These studies confirm that alterations in mitochondrial energy generation, ROS production, and apoptosis can all contribute to the pathophysiology of mitochondrial disease (Wallace DC., 2002).

Studies in the yeast Saccharomyces cerevisiae have shown that in two mitochondria synchronized in fusion and fission events, the fission between external membranes leads to the very slow exchange of mtDNA and very rapid exchange of mitochondrial matrixes, several cycles of outer and inner membrane fusion and fission are required for the exchange of mtDNA damaged [START_REF] Okamoto | Mitochondrial Morphology and Dynamics in Yeast and Multicellular Eukaryotes[END_REF] (Figure 16).

Recently, it has been demonstrated that mitochondrial network mobility is associated with the cytoskeleton proteins as actin and involved in cytosqueleton dynamic (Kremneva K et al., 2013). Nevertheless, proteins involved in DNA repair present in matrix can be exchanged very fast [START_REF] Kaufman | In Organello Formaldehyde Crosslinking of Proteins to mtDNA: Identification of Bifunctional Proteins[END_REF][START_REF] Nunnari | Mitochondrial Transmission during Mating in Saccharomyces Cerevisiae Is Determined by Mitochondrial Fusion and Fission and the Intramitochondrial Segregation of Mitochondrial DNA[END_REF][START_REF] Otera | New Insights into the Function and Regulation of Mitochondrial Fission[END_REF].

Mutations in MMM lead to modify the mitochondrial profile of subcellular localization and mitochondrial morphology.

The Figure 17 shows different interactions of MMM complex with proteins that plays important role in the cell [START_REF] Millau | A Microarray to Measure Repair of Damaged Plasmids by Cell Lysates[END_REF].

I.4. Mitochondrial Functions

Figure 18: The CAC has integrative functions between mitochondrial matrix and cytosolic pathways in ATP production. The 3 main sources for ATP production are: carbohydrates, lipids and proteins (Krauss S., 2001).

In the eukaryotic cell mitochondria is involved in several mechanisms such as: Ca 2+ storage [START_REF] Leanza | Intracellular Ion Channels and Cancer[END_REF], thermogenesis [START_REF] Tine | Genomic Characterization of the European Sea Bass Dicentrarchus Labrax Reveals the Presence of a Novel Uncoupling Protein (UCP) Gene Family Member in the Teleost Fish Lineage[END_REF]) and ATP production.

The more important mitochondrial function in eukaryotic cell is the ATP production. The

Figure 18 shows the different pathways for ATP synthesis from various sources such as: amino acids, fatty acids and glucose. The protein catabolism is involved directly with CAC in the formation of oxaloacetate and 2-oxoglutarate. On the other hand, glucose and fatty acids became in acetyl-CoA by glycolysis and ß-oxidation respectively.

Finally, the ATP production depends of OXPHOS capacity. The ATP production (Figure 19) is generated from a gradient of protons (H + ) which is used by the ATP synthase to convert ADP and phosphate ions in ATP.

This gradient of protons is the result of several oxidation-reduction reactions and depends of three important coenzymes such as: nicotinamide adenine dinucleotide (NAD); nicotinamide adenine dinucleotide phosphate (NADP); and flavin adenine dinucleotide (FAD). OXPHOS is carried out in IMM involving the electrons traffic through respiratory chains complexes.

During cellular respiration, ATP production is controlled by product excess, ATP being an allosteric inhibitor of Cyt-C oxidase (Arnold S. et Kadenbach B., 1997).

I.5. Mitochondrial diseases

The Table 2

I.5.1. Mendelian: associated with genes of the nuclear genome

When DNA disorders concern OXPHOS system, hereditary mitochondrial diseases usually affect tissues with high energy needs, like neuromuscular or central nervous system.

Frequently the deleterious mitochondrial mutations are heteroplasmic, thus wild type and mutated forms of mtDNA coexist in the same cell. The mitochondria functions start to shut down when a threshold of mtDNA mutation was exceeded. This threshold is variable and differs from mutation to mutation: for example 60 to 70% in chronic progressive external ophthalmoplegia (CPEO) and probably 95% in the syndromes of mitochondrial encephalopathy, myopathy, lactic acidosis, and stroke-like episodes (MELAS), and myoclonic epilepsy with ragged red fibers (MERRF) [START_REF] Tonin | Characterization of Chemically Modified Oligonucleotides Targeting a Pathogenic Mutation in Human Mitochondrial DNA[END_REF]. The threshold effect may explain the tissue-specific patterns of clinical expression (Nonaka I., 1992).

Consequently, a shift in the proportion between mutant and wild type molecules could restore mitochondrial functions [START_REF] Tonin | Characterization of Chemically Modified Oligonucleotides Targeting a Pathogenic Mutation in Human Mitochondrial DNA[END_REF].

Mutations in mtDNA that encoded OXPHOS system are related to damage effect in respiratory function increasing electron leak and ROS production and therefore mtDNA damage. This result guides to vicious cycle that leads to the differential accumulation of mutation in mtDNA during aging (Wei YH. et al., 1998). It was show the relationship among damages in mtDNA and illnesses apparition such as: MELAS, MERRF, KSS, LHON and NARP (Thorburn DR et Rahman S., 1993), where this last one is the consequence of ATPase 6 mutation.

I.5.2. not Mendelian: associated with maternal inheritance

The strictly maternal inheritance of mitochondria is the result of the removal of sperm tail containing mitochondria prior to egg entry, and the destruction of the sperm mitochondria in very little time after fertilization [START_REF] Sutovsky | Ubiquitinated Sperm Mitochondria, Selective Proteolysis, and the Regulation of Mitochondrial Inheritance in Mammalian Embryos[END_REF]. Thus, this mechanism proposed to promote the strictly inheritance of mtDNA in humans and animals [START_REF] Thompson | Ubiquitination of Prohibitin in Mammalian Sperm Mitochondria: Possible Roles in the Regulation of Mitochondrial Inheritance and Sperm Quality Control[END_REF]. As well, pedigree schematized by

II.2. DNA repair mechanisms

II.2.1. Base Excision Repair (BER)

The BER system is a multi-step process that repair damage to modified bases following oxidation, methylation, deamination, or spontaneous loss of the DNA base itself (A

Memisoglu A et Samson L., 2000).
The Table 3 shows the more studied human DNA glycosylases Endogenous DNA damages were largely as a consequence of unavoidable hydrolysis and oxidation. The endogenous ROS production is one main source of damage to DNA present in eukaryotes, particularly in aerobic organisms responsible of certain human disease.

Human

Most of the single-base damages are repaired through the base excision repair (BER) BER initiated by a DNA glycosylase leaves an abasic site. Depending on the numbers of nucleotides eliminated (short or long excision), BER will progress either by short-patch repair or long-patch repair using different proteins in each mechanism [START_REF] Krokan | Base Excision Repair[END_REF].

After glycosylase intervention, the protein AP endonuclease (APE1) cleaves the AP site to yield a 3' hydroxyl adjacent to a 5' deoxyribosephosphate. This excision can be carried out by bifunctional glycosylases like OGG1.

In order to facilitate ligation 3'-5' ends, the empty DNA zone is completed by the DNA Polymerase. The Pol β is the main human polymerase involved in short and long-patch BER.

In short-patch pathway, BER ligation is permitted by DNA ligase III that catalyzes, in presence of two partners XRCC1 and Poly ADP ribose polymerase 1 (PARP1), the nicksealing step.

For the long-patch pathway, DNA ligase III is replaced by DNA ligase I (Rice PA., 1999 ;

II.2.1.1. BER in mitochondria

In

II.3.2. Single or double strand breaks repair

In mammalian cells single strand break can be repaired by BER mechanism and DSB by Non-Homologous End Joining (NHEJ), and Homologous Recombination (HR), (Figure 28)

which is only available S and G2 phases when a sister chromatid can be used as a template for the repair reaction (Lopez-Contreras AJ, et Fernandez-Capetillo O., 2012).

II.3.2.2. Homologous recombination (HR)

Homologous recombination is a universal process, conserved from bacteriophage to human, which is important for the repair of double-strand DNA breaks. The general process is as follow : the free ends of a DSB are first processed by an exonuclease. The ATM kinase is recruited to DSB via an interaction with the MRN (Mre11-Rad50-Nbs1) complex. Once at the break, ATM that becomes activated, phosphorylating multiple substrates. In a reaction that depends in multiple endo and exonuclease activities. The detection method may influence the type of lesion processing, either damage reversal, if a specific enzyme such as APTX or TDP1 is able to resolve the lesion, or damage excision, a more generic pathway that excises the DNA damage using the long patch-BER pathway.

a). Proteins involved in mtDNA strands breaks repair

The Table 4 illustrates some proteins involved in mtDNA recombination pathways in yeast, mammalian and plant. In 2008, our group demonstrated that bleomycin-induced DSB were efficiently repaired in Drosophila mitochondria using S2 cells, with a comparable rate to that was observed for nuclear repair [START_REF] Morel | Bleomycin-Induced Double-Strand Breaks in Mitochondrial DNA of Drosophila Cells Are Repaired[END_REF]. (Chen., 2013) .

Normally, the Yeast Mgm101 protein (Figure 32) is involved in single-strand annealing but in response to oxidative stress or to other signals which generate dsDNA breaks, Mgm101 can be involved in dsDNA breaks repair pathways in mitochondria (Chen., 2013). The Figure 33 shows that Mgm101 can be activated by ROS and guide mitochondria to repair mtDNA by pathways such as HR and NHEJ (Chen., 2013). The Figure 34 shows the gene duplication that occurred before the divergence of archaea and eukaryotes giving rise to two lineages, RADandRADrecA has remained as a singlecopy gene. In eukaryotes, both RADand RADgenes experienced several duplication events, but, in archaea, they remained as single-copy genes.

Eukaryotic recA genes originated from proteobacteria (recAmt) and cyanobacteria (recAcp) recA genes after two separate endosymbiotic events. recAmts were subsequently lost in the ancestors of animals and fungi [START_REF] Lin | Origins and evolution of the recA/RAD51 gene family: evidence for ancient gene duplication and endosymbiotic gene transfer[END_REF]. Finally a complex including ligase 4 and XRCC4 factor ligates the ends together.

Although XLF binding to XRCC4 was known, its exact function needs to be elucidated From the 18 member of ARTs described (Figure 36 The automodification domain contains a BRCT, D and E motifs. BRCT motif guides to PARP-1 interaction with similar motif in proteins such as: XRCC1, BRCA-1 and BRCA-2.

The catalytic domain with motif F is the smallest PARP-1 fragment retaining catalytic activity. The NAD + acceptor sites is named PARP signature motif and it is a sequence of 49 amino acids highly conserved through the evolution [START_REF] Elkoreh | Type 1 Inositol-1,4,5-Trisphosphate Receptor Is a Late Substrate of Caspases during Apoptosis[END_REF]. PARP-1, with the N-terminal two first zinc finger motives, makes genome screening until DNA-nicks detection [START_REF] Eustermann | The DNA-Binding Domain of Human PARP-1 Interacts with DNA Single-Strand Breaks as a Monomer through Its Second Zinc Finger[END_REF]. This results into auto-posttranslational modification (auto ADP-ribosilation) but also histones poly(ADP-ribosyl)ation.

III.2.2. PARP-1 in DNA repair

The The crystal structure of the murine PARP-2 is very similar to that of PARP-1, with the difference that PARP-2 hasnot Zinc-finger motifs, (Figure 43). This structural difference leads the acceptor site that reflects differences in terms of substrates [START_REF] Oliver | Crystal Structure of the Catalytic Fragment of Murine poly(ADP-Ribose) Polymerase-2[END_REF].

The catalytic domain has the strongest resemblance to that of PARP-1 with 69% similarity.

PARP-2 carries out multiple modifications in histone H2B while PARP-1 preferentially makes only one modification in H1 [START_REF] Poirier | Poly(ADP-Ribosyl)ation of Polynucleosomes Causes Relaxation of Chromatin Structure[END_REF].

III.3. Tankyrases

Tankyrases (ART-5 and 6 in Figure 36) contain large ankyrin domain repeats that mediate protein-protein interactions using a 33-residue sequence motif. This sequence was first identified in yeast and Drosophila and was named for the cytoskeletal protein ankyrin, which contains 24 copies of this repeat. Subsequently, ankyrin repeats have been found in many proteins spanning a wide range of functions.which facilitate target selection and activation [START_REF] Mosavi | The Ankyrin Repeat as Molecular Architecture for Protein Recognition[END_REF]).

Tankyrase1 (TRF1-interacting, ankyrin-related ADP-ribose polymerase), ART5 was identified as a partner of the human telomeric protein TRF1 in a two-hybrid screening [START_REF] Sbodio | Identification of a Tankyrase-Binding Motif Shared by IRAP, TAB182, and Human TRF1 but Not Mouse TRF1. NuMA Contains This RXXPDG Motif and Is a Novel Tankyrase Partner[END_REF]. Thus, its functions are involved in telomere length regulation in mitotic division [START_REF] Hsiao | Tankyrase Function at Telomeres, Spindle Poles, and beyond[END_REF]. Accordingly, Tankyrase-1 (ART-5) and

Tankyrase-2 (ART-6) show multiple subcellular localizations such as: telomeres, nuclear pores, pericentriolar material during mitosis, and Golgi complexes. It was hypothesized that tankyrases are involved in energy homeostasis in mammals, thus present in mitochondria (Yeh TY, et al., 2009).

III.4. Other PARPs

The

III.5. PARPs inhibitors

PARP-1 has been the target for design of PARP inhibitors. These have been claimed to have applications in the treatment of many disease including cancer, inflammations and retroviral infection. It must made some questions concerning to PARP-1 inhibitors before their use in treating of human diseases.

These questions are related to chemical properties of the inhibitors, specificity, including isoforme-selectivity. Another question concerns the fact that different diseases may require therapeutic PARP-1 inhibition to be either short-term or chronic. The answers to these questions could determine the future of disease therapy through inhibition of PARP (Woon

EC et al., 2005).

The Figure 44 shows different DNA repair pathways involving PARP inhibitor action.

The PARP inhibition can perturb highly the HR mechanism. Successful repair by each of these three different pathways leaves behind a unique repair product whose genetic signature can be traced in the progeny of the males. Unfortunately, BER system was not studied in order to demonstrate glycosylases actions after oxidative DNA damages.

Figure 47: A-B: PARP-B protein localizes to nucleoli in all Drosophila tissues, including polytene chromosomes of larval salivary glands (A) and diploid nuclei of larval brain (B). The dissected larval salivary glands and brains expressing PARP1-DsRed (red) were stained with the DNA binding dye Draq5 (green). Positions of nucleoli are indicated with arrows. SGlarval salivary gland; BRlarval brain. C-D. PARP-B deletion displaces nucleoli protein as detected by nucleoli-specific antibody AJ1 (red). AJ1 detects nucleoli in every cell of Drosophila mid-intestine in wild-type second-instar larvae (C), but only in a few cells in ParpCH1 mutants (D). DNA is detected with OliGreen dye (green). E-H. Deletion or disruption of PARP-B protein

MATERIAL AND METHODS

All chemicals were obtained from Sigma unless otherwise stated

I. .Animals

All flies used in this study were from D. melanogaster strain either generated in our laboratory (Wild type flies) or obtained from Bloomington Stock collections for mutants (University of Indiana, USA), (Table 5).

They were reared on axenic medium [START_REF] Alziari | Mitochondrial DNA Expression in Drosophila Melanogaster: Neosynthesized Polypeptides in Isolated Mitochondria[END_REF] at 19°C.

D. melanogaster Phenotype (eyes color ) Origin

Wild Type (WT) white Laboratory Only ogg1 -/xrcc1 -mutant strain was generated using classical genetic crossings illustrated in Figure 48.

II. .Sub-cellular fractionation

II.1. Sub-cellular fractions purification (nucleus and mitochondria)

Mitochondria were prepared according to protocols previously described in Alziari and collaborators briefly [START_REF] Alziari | Mitochondrial DNA Expression in Drosophila Melanogaster: Neosynthesized Polypeptides in Isolated Mitochondria[END_REF], 300-400 males fruitflies were used for each mitochondria preparation. They were immobilized on ice and ground into small aliquots (50 individuals) with a mortar and pestle in 2 mL of isolation buffer. The resulting suspension was filtered on Sefar Nitex membrane, and centrifuged (240g, 10 min.). The pellet was kept for further nuclear purification and the supernatant (lysate) was centrifuged (3000g, 10 min).

The resulting pellet was suspended in 2 mL of isolation buffer, and centrifuged twice for 10 min at 7000g. The final pellet was the "crude mitochondrial fraction".

The following steps were conducted according to Alziari and collaborators Preformed Percoll gradient was prepared by a 55,000g centrifugation of 55% Percoll solution for 1 h.

The crude mitochondrial fraction was gently layered onto this preformed Percoll gradient.

After further centrifugation (55,000g, 1 h), purified mitochondria could be detected as discrete bands in the middle part of the gradient. Bands were collected from the tube with a needle, pooled, and diluted volume to volume with isolation buffer. This solution was centrifuged twice for 10 min at 7000g. The resulting pellet was the "pure mitochondrial fraction".

The next step for mitochondria purification was mitoplast preparation. Pure mitochondrial fraction was incubated for 15 min at 4 °C with 0.1 mg of digitonin per mg protein with frequent stirring. After dilution with two volumes of isolation buffer, the solution was centrifuged (10,000g, 5 min). The pellet was washed twice (10,000g, 5 min), and finally suspended in 200-400 L of isolation buffer. This fraction was "purified mitoplasts".

The first pellet obtained after the first centrifugation of the crushed fruitfly suspension was suspended in isolation buffer, filtered on Sefar Nitex membrane, and centrifuged for 10 min at 240g. The pellet was suspended in 2 mL of isolation buffer and centrifuged for 10 min at 650g, giving the "crude nuclear fraction".

The purified nuclear fraction was obtained using a subsequent purification step on a sucrose cushion. Briefly, a 2 mL solution of 2M sucrose was prepared in 3.5 mM MgCl 2 , 3 mM DTT and loaded onto a polyallomer tube. The crude nuclear fraction was gently layered on the sucrose cushion top, and centrifuged at 124,000g for 1 h. The pellet was washed twice at 10,000g for 10 min in 1 mL of isolation buffer, and suspended in 500 L.

II.2. Mitochondrial fractions validation

II.2.1. Western blot analysis

Equivalent protein amounts (15 g) from mitochondrial or nuclear fractions were run on 10% or 15% SDS-PAGE, and blotted on nitrocellulose membrane (GeHealthcare). Western blot analyses were performed with the following dilutions using antibodies raised against lamin 1:750 (DSBH), histone H3 1:2,000, Rieske 1:13,500 (gift from Dr C. Godinot, Lyon), PDH 1:2,000 (Mitosciences). Various incubations and detection were as previously described by Dubessay and collaborators [START_REF] Dubessay | Aging Impact on Biochemical Activities and Gene Expression of Drosophila Melanogaster Mitochondria[END_REF].

II.2.2. Enzyme assays

All the enzymatic measurements were made with a heat-controlled spectrophotometer (Shimadzu) at 28 °C. Both activities were assayed using a previously described protocol.

a. Cytochrome oxidase assay

The activity of Cyt c oxidase was measured as the decrease in absorbance due to the oxidation of Cyt c at 550 nm (= 18,500 M -1 . cm -1 ). The addition of 10 g of protein from mitochondrial or nuclear fractions to the reaction mixture (50 mM KH 2 PO 4 , mM EDTA, 100

M cytochrome c, pH 7.4) initiated the reaction.

b. Citrate synthase assay

Citrate synthase activity was measured as the increase in absorbance due to the reduction of 5,5'-dithiobis-2-nitrobenzoic acid (DTNB) at 412 nm, coupled to the reduction of coenzyme A by citrate synthase in the presence of oxaloacetate (= 13,600 M -1 . cm -1 ). The assay was performed with 10 g of proteins added to the reaction mixture (100 mM Tris HCl, pH 8, 2.5 mM EDTA, 37 M acetyl-CoA, 75 M DTNB, 320 M oxaloacetate).

III. Comprehensive BER repair analysis through microarray technology

The excision reactions were conducted using the modified oligonucleotide arrays already described elsewhere [START_REF] Pons | Age-Associated Modifications of Base Excision Repair Activities in Human Skin Fibroblast Extracts[END_REF].

Briefly, each well of a 24-well slide was functionalized by a series of lesion-containing oligonucleotide duplexes (lesion ODNs) and a control duplex (control ODN, with no lesion).

The duplexes were tethered to the slide surface by one end, and labeled with a Cy3 at the other end. All the duplexes were attached in duplicate.

Eight lesion substrates were present: ethenoadenine (EthA) paired with T, hypoxanthine (Hx) paired with T, 8oxoguanine (8oxoG) paired with C, A paired with 8oxoG, thymine glycol (Tg) paired with A, tetrahydrofuran (THF), as abasic site (AP site) analog, paired with A, uracil (U) with G and U paired with A.

Excision reactions were conducted with 10 g of protein per well at room temperature ( 23°C) for 1 h in 80 L of excision buffer (10 mM Hepes/KOH pH 7.8, 80 mM KCl, 1 mM EGTA, 0.1 mM ZnCl 2 , 1 mM DTT, 0.5 mg/mL BSA). The slides were subsequently rinsed at room temperature for 3 x 5 min with 80 L of washing buffer (PBS containing 0.2 M NaCl and 0.1% Tween 20). On each slide, two wells were incubated with the excision buffer only (control wells) to serve as a reference, arbitrarily set at a fluorescence level of 100% for the calculation of the excision rates (Figure 49) The spot fluorescence was quantified using a GenePix 4200A scanner (Axon Instrument, Molecular Devices, Sunnyvale, CA, USA). Total spot fluorescence intensity was determined using the GenePix Pro5.1 software (Axon Instrument).

Each extract was tested twice. The results between replicates (four spots) were normalized using the NormalizeIt software as described by Millau and collaborators. A first operation calculated the excision rate of each of the lesion ODNs in the extract-containing wells compared with the lesion ODNs of the control wells. A correction was then applied that took into account the possible degradation of the control ODN (no lesion). Final results for each lesion ODN, expressed as percentage of cleavage, were calculated using the formula: (100-(1percentage of fluorescence of lesion ODN/percentage of fluorescence of control ODN)).

IV. →Molecular Analysis

IV.1. Expression analyses

These analyses were performed either with S2 cells lines from Drosophila melanogaster or with fruitfly stock IV.1.1. mRNA extraction 50 flies or S2 cells (10 6 cells) were disrupted in 1 ml TRIZOL. The total RNA was purified according to the manufacturer's protocol (Invitrogen). The samples were solubilised in 50 µl distilled water and immediately stored at -80°C.

IV.1.2. cDNA production

Reverse transcriptase reactions were made using random primers (2.5 mM) and reverse transcriptase enzyme RT M-MLV (1 U) (PROMEGA) at 37°C during 60 minutes. All Reverse transcriptase reactions were made including no-template, no-RT enzyme as controls of genomic DNA contamination. After cDNA amplification the PCR products were incubated with RNAse (4g/L) at 37°C during 30 min and quantified in Nanodrop1000 spectrophotometer. cDNA aliquots were stored at -80°C. Tables 8 and9 show the PARP-B-target construct and vector source. All DNA digests were made according to the manufacturer's protocol from PROMEGA.

IV.1.3. RT-q-PCR

IV.3. PARP-B-target construction

The DNA product purification was made using NucleoSpin Gel and PCR Clean-Up from MACHEREY NAGEL and the ligation reactions were performed with T4 ligase (2.5 U) from PROMEGA at 4°C overnight.

DNA

IV.4. DNA electrophoresis

The gels were made with different agarose percentage such as 0.8, 1.0 and 2.0%. The agarose was prepared in TAE1X buffer (4 mM Tris, 1 mM EDTA). Ethidium bromide was added 1 drop (50mg/ml) per 50 ml of gel solution at 40°C. Electrophoreses were performed in TAE1X buffer and run at constant voltage (110V).

IV.5.2. Transformation

DNA plasmid (80-100ng) was added to competent cells kept on ice-cold and incubated for 1 hour. A 1 min heat shock (42°C) was performed, followed by 2 min on ice. LB medium without ampicillin (500 L) were added and bacteria were incubated at 37°C with 200 cycles/min in a rotary shaker during 45 min. Bacteria were plated on agar containing 100 g/mL ampicillin.

V.2. Protein sequences homology

The protein Align Sequences analysis was made using BLAST:

http://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastp&PAGE_TYPE=BlastSearch&BLAST_SPEC=blast2 seq&LINK_LOC=blasttab&LAST_PAGE=blastn&BLAST_INIT=blast2seq

V.3. Caspase cleavage sites determination

The caspase cleavage site determination was carried using the program PeptideCutter

VI.2. Cells transfection

Cells transfection was carried out on confluent cells using Cellfectin Reagent (Invitrogen) Day 1: Cell preparation: 3 mL of S2 cells (2 x 10 6 cells/mL) were plated in a 35 mm well with complete Schneider's Drosophila Medium (SDM). In order to induce the similar cellular stress that in protein induction mechanism, S2 cells were incubated with Cu 2 SO 4 (0.7mM) during 24 hours.

VI.4.2. Oxidative stress

In order to induce a cellular stress and to promote DNA alterations, oxidative stress conditions were created using various [H 2 O 2 ] (Table 11).

VI.4.3. Copper /oxidative stress

The stress combination was performed as follow. Cells were incubated during 24h with 0.7mM CuSO 4 then 40 mM H 2 O 2 was added during 30 min.

VII. .Microscopy

Forty picomoles of Mitotracker CMXRos (Molecular Probes) were added to 5 ml of S2 cells suspension (collected at confluency) and incubated for 30 min at 24°C. The fluorochrome was eliminated by warm PBS 1X (24°C) with 3 quick washes.

Cells fixation was performed using paraformaldehyde (PFA) 4% at 4°C during 30 min. 50 l of cells lines (GFP tagged) were directly layered with coverslip.

Triton100X (0.2%) was added to J2 line on slides. Protein detection was made using different primaries antibodies such as anti-V5 or anti-PARP (Santa Cruz). Both were diluted 1/500 and 1/200 respectively and incubated for 1 hours at room temperature and washed with PBS1X, 3 times. After BSA (3%) blocking during 30 min at room temperature, 3 other washes with PBS1X were performed.

Both secondary antibodies, anti-rabbit alexa488 (for anti-V5) and anti-goat alexa488 (for anti-PARP) were diluted 1/320 and incubated for 1 hour. Cells were observed with Axioplan-2 microscope (Zeiss) about ten images per slide were captured using Axiovision software (Zeiss) at the following wavelengths: MitoTracker: the excitation Band was 550-700 nm; DAPI: Violet Excitation Band was 401-418nm and FITC: Blue Excitation Band was 480 -500nm.

VIII. .Proteins analyses from Drosophila

VIII.1. Protein extraction

After 24 hours induction with 0.7mM Cu 2 SO 4 , The S2 cells were washed 3 times with PBS1X. Washed cells were diluted in extraction buffer. Cell lysis was performed with ultrasound (Vibracell; BIOBLOCK SCIENTIFIC) on ice with 15 cycles of 30 sec pulse and 1 min.

VIII.2. Electrophoresis and blotting

Laemmli 1X (10% glycerol, 5% β-mercaptoethanol, 1% SDS, 0.02% bromophenol blue) was added to 20 g protein as determined by Bradford assay (BioRad) and heated at 95°C during 5 min. Proteins samples run was made in 10% SDS-PAGE (Table 12) at constant voltage (80V) during 3 hours.

Protein staining was directly performed in gel using 0.1% Coomassie Brilliant Blue (triphenylmethane) solution.

Protein blotting was performed after SDS-PAGE on nitrocellulose blotting membrane (Millipore) at 200mA during 1 hour. Proteins on membrane were stained with 2% ponceau red.

Gels

VIII.3. Parp-B protein detection

This was made using anti-V5 (PROMEGA) and anti-PARP (Santa Cruz) antibodies diluted at 1/200 and the respectively secondary antibodies anti-rabbit and anti-goat both diluted 1/10000. Parp-B-Hist 6 detection was made according to manufacturer's protocol using anti-Hist 6 diluted 1/20000 and anti-mouse diluted 1/10000 both antibodies.

Incubation times were: 2 hours for primary antibodies and 1 hour for secondary antibodies and all incubations were made at room temperature. Luminata TM crescendo kit (Millipore)

was used as a substrate for peroxidase labeled-antibodies.

VIII.4. Mass spectrometry

According to Dr. BONHOMME protocol (personal communication), bands were excised from gel using cutting blade and forceps. Each band cut were about into 1 x 1 mm pieces and place in 1.5mL LoBind tube (Eppendorf). All reagents were HPLC grade. The gel pieces were rinsed with 300μL water for 15 min at room temperature on shaker. Acetonitrile (300μL) was added and washed for 15 min at room temperature on shaker.

Supernatant was discarded. Gel pieces were washed with 100mM ammonium bicarbonate (300μL) for 15 min at room temperature on shaker. The supernatant was discarded. One hundred millimolar ammonium bicarbonate in 50% acetonitrile (300μL) was added and incubated at for 15 min at room temperature on shaker, supernatant was discarded and 100μL acetonitrile was added to the pellet for 5 min at room temperature on shaker. The supernatant was discarded.

The gel pieces were dried in a Speedvac for 5 min. Ten millimolar DTT (50μL) was added and incubated for 1 hour at 60°C. Supernatant was discarded. 50μL were added to 50mM IAA and incubated for 30 min in dark at room temperature. Supernatant was discarded. Washing gel pieces were with 300μL 100mM Ammonium Bicarbonate for 15 min at room temperature on shaker. Supernatant was discarded. Washing gel pieces were with 300μL 20mM Ammonium Bicarbonate in 50% Acetonitrile for 15 min at room temperature on shaker. Supernatant was discarded. It was adding 100μL HPLC Acetonitrile for 5 min at room temperature on shaker. Supernatant was discarded. Drying of gel pieces was in a Speedvac for 5 min. It was adding 20μL of Trypsin solution and incubated for 1 hour at room temperature.

Finally, enough Ammonium Bicarbonate at 40mM was added in 10% Acetonitrile to completely cover the gel pieces and incubate overnight at 37°C. It was added 150μL HPLC water to pieces for 10 minutes at room temperature on shaker. Removed supernatant and placed in a 0.5mL LoBind tube (Eppendorf). It was extract the gel pieces twice with 50μL of 50% Acetonitrile/5% TFA for 60 min each time at room temperature. It was pooled all extracts and dried in a Speedvac.

The purification and concentration of peptides for in silico analysis were carried out using ZipTips [START_REF] Tiss | Serum Peptide Profiling Using MALDI Mass Spectrometry: Avoiding the Pitfalls of Coated Magnetic Beads Using Well-Established ZipTip Technology[END_REF].

The

. Purity analysis

As a first step prior to any repair measurement in mitochondria, we sought to obtain purified functional mitochondrial fractions with no contaminants from cytosol or nuclear compartments, in order to ensure that tested repair activities came solely from the expected compartment.

Mitochondrial and nuclear purity was estimated using Western blot analyses with specific antibodies: anti-PDH (mitochondrial matrix), anti-Rieske (mitochondrial inner membrane, component of the respiratory complex III), anti-lamin (Dm0, nuclear envelope), and antihistone H3 (nucleoplasm).

The crude mitochondrial fraction obtained by differential centrifugation showed, by

Western blot analysis, a persistency of nuclear proteins such as lamin and histone H3, suggesting a partial nuclear contamination of this fraction, independent of fruitfly age (Figure 56, lane CM). The incubation of these fractions with low digitonin concentration in order to eliminate mitochondria outer membrane did not get rid of these contaminations (data not shown).

A subsequent purification step from crude mitochondrial fractions on Percoll gradient (Figure 56, lanes PM) not only preserved the fraction integrity as shown by the presence of mitochondrial matrix protein PDH, but was also sufficient to eliminate residual traces of nuclear components. An additional treatment of these fractions with digitonin removed all residual contaminants (Figure 56, lanes Mpl). All the results confirmed that these fractions (purified mitochondria (PM) and mitoplasts (Mpl)) could be further analyzed for mitochondrial BER activity assays.

In parallel, crude nuclear fractions were prepared and analyzed by Western blot. As observed for mitochondria, these fractions were still contaminated with other compartment After determining the fractions purity, mitochondrial functionality was assessed using enzyme assays. Two enzyme activities were chosen: cytochrome oxidase (complex IV of the respiratory chain, Figure 57A), and citrate synthase (a mitochondrial matrix protein from the Krebs cycle, Figure 57B).Change (2.6-fold) for citrate synthase activity between crude mitochondria and mitoplasts was significant (p < 0.05). By contrast, the increase observed for cytochrome oxidase between these two fractions (1.34-fold) with young fruitflies was not significant. All these results confirmed that mitochondrial fractions were functional for further studies.

I.1.1.2. Functionality analysis

I.1.2. Nuclear BER activities

Validation of microarray approach on Drosophila model Specific BER glycosylase and endonuclease activities were measured using modified oligonucleotide microarrays designed for the determination of BER capacities in nuclear extracts. As a first step, nuclear BER activities were analyzed to confirm the efficiency of the microarray with fruitfly extracts.

Most of the eight DNA lesions present on the microarray were detected and excised by these extracts, suggesting the presence of the corresponding specific glycosylases/endonuclease in these samples. However, four main activities were observed in nuclear extracts from young individuals (Figure 58A).

The endonuclease activity that cleaved THF paired with A with approximately 60%

excision rate was predominant. The cleavage rate of Hx, U and A, paired respectively with T, G and 8oxoG was also high (more than 25% excision). Other activities were lower than 10%.

During aging, only Hx-T and A-8oxoG were significantly decreased (68%, p < 0.05).

Hence, as also for various cell models, these modified oligonucleotide microarrays were efficient in determining BER activities in nuclear extracts from Drosophila melanogaster.

I.1.3. Mitochondrial BER activities

Mitochondrial fractions, both crude mitochondria (Figure 66B) and pure mitoplasts (Figure 66C) from young individuals, were tested in the same conditions. In crude mitochondria samples, four major excision activities were observed with the following efficiency rate order: THF-A (80% excision rate) > U-G>, Tg-A> Eth-A.Other activities were not detectable (Hx-T; 8oxoG-C; A-8oxoG and U-A). Pure mitoplast fraction was analyzed to compare with the crude mitochondria preparation. The global activity profile was quite similar to the one obtained with crude mitochondrial fraction, but provided additional information. The main changes were observed with the excision percentages, which were globally higher in mitoplasts than in crude mitochondria. In particular, excision of U-A was effective in mitoplast fraction whereas it was almost undetectable in crude mitochondria.

An interesting observation was made regarding the effect of aging (Figures 58B and58C).

Two tendencies observed with crude mitochondria were significant with the mitoplasts. A marked increase (2.4-fold, p < 0.05) of U-G excision was detected, whereas other activities declined. Aging-associated decrease was significant for THF (-18%). Even though endonuclease activity was high in both nuclear and mitochondrial extracts, these results show that BER activities in mitochondria were different from their nuclear counterpart, and that aging interferes with these capacities.

Most of the observed activities were a consequence of the presence of specific mitochondrial isoforms from various BER glycosylases. In silico analyses were performed to investigate the ability of candidates proteins to be involved in such activities. These proteins were described in several organisms mitochondria but were less known for Drosophila model.

II. Mitochondrial addressing predictions

Two different computer programs were used. In a first step, PsortII was chosen for its ability to give a mitochondrial addressing prediction, but also predictions for other compartments locations. MitoProtII was used as complement software to reinforce the previous results.

A group of 8 proteins from Drosophila and one from human was investigated. On one hand, molecules directly involved in BER process were tested such as one glycosylase (Ogg1-PA), two isoforms of a specific endonuclease (Rrp1 PA and PB), the Ligase III partner As shown in Table13, two populations of proteins were present. A first group with a high prediction index for nuclear addressing (≥ 75 %) including Rrp1-PA and PB, XRCC1-PA, PARP-PB and PC was observed and a second group (one protein) with a high prediction index for mitochondria (≥75%). Between, these two groups, some proteins were analyzed without any strong predictive index for any locations.

However, we must keep in mind that this predictive approach is based on the presence of consensus sequences in proteins such as NLS (nucleus targeting) or MTS (mitochondria targeting) and that many proteins have been shown to be present in mitochondria without any detectable MTS region.

This first analysis suggested that some of these proteins could be present in Drosophila mitochondria. The

III. .Base excision repair genes expression in Drosophila

III.1 Relative mRNA expression in wild-type context

Using the Flybase database (http://flybase.org/), few BER genes expression (identical to those illustrated in chapter II) was analyzed in young male flies (5 days old). Among the five candidates, Xrcc1 showed the higher level of expression, followed successively by Rrp1, then ParpB/C and PARG and finally Ogg1.

Levels of expression of the same BER candidates were evaluated in 5 days old male flies in order to get a representative pattern of their expression in basal conditions (Figure 58). All these values were estimated by RT-q-PCR using Rp49 (Ribosomal protein used as external control of DNA repair systems) as an internal reference. The same expression pattern than that obtained with Flybase data was observed In fact specific gene (glycosylase) displays a lower expression level than those encoding crossroads proteins such as Xrcc1 and Rrp1. The reality of the double knockout was confirmed by the lack of ogg1 and xrcc1 expression in the tested flies (Figure 62). Interestingly, the observed expression pattern was quite similar to that obtained with single xrcc1 knockout strain. A strong decrease in Rrp1 and PARP B/C expression was detected and an increase in PARG expression (+ 145%). Such a behavior suggests that the lack of XRCC1 protein remains a key element in the global regulation process of other BER partners.

IV. .PARP AS A CANDIDATE FOR MITOCHONDRIAL DNA REPAIR IN

DROSOPHILA?

IV.1 PARP isoforms relative expression

In Drosophila melanogaster, two different PARP isoforms have been described: PARP B and C. Both of them are transcribed from a single copy gene but PARP-C is a product of an alternative splicing process leading to the removal of exon 5 compared to PARP-B mRNA [START_REF] Kawamura | An Alternative Form of poly(ADP-Ribose) Polymerase in Drosophila Melanogaster and Its Ectopic Expression in Rat-1 Cells[END_REF].

Little information is known regarding either the exact roles for each of these forms or their eventual functional relationships. To help into the understanding of these two PARP isoforms, we started by analyzing the relative expression of both of them in various contexts.

IV.1.1 In whole Drosophila organism

In 5 days old wild type males (Figure 63), PARP C expression was 1.5 fold higher than that of PARP B. In the mean time, the expression of PARG mRNA appeared similar to that for PARP B. The same study was performed in rrp1 -(Flybase code 10213) mutant flies. This mutant context was chosen due to the fact that it was the only one displaying an increase in PARP B/C expression.

Our purpose was to investigate the role of PARP B/C or its effects on other BER molecules in Drosophila; this is why rrp1 mutant was chosen with the PARP B/C upregulation in this genetic background. Interestingly, PARP-C mRNA level remained unchanged compared to PARP-B that was increased by 1.5 fold in mutant flies. In the case of PARG, the observed increase was almost by 170 %.

IV.1.2. In S2 cells

The same kind of quantification was performed in Drosophila derived cell lines: S2 cells in order to get information on a model that could be used further under stress conditions.

After RT-q-PCR experiment on mRNA extracted from S2 cells in normal culture conditions, PARP-B and PARP-C level appeared almost identical. PARP-B/PARP-C Expression Ratio (ER) is about 1 (Figure 64) compared to ER about 0.7 in WT whole flies.

IV.2. PARP isoforms relative expression in stress conditions

PARP is known in several models to be a key actor during cell responses to various stresses playing roles in DNA repair, apoptosis, and gene transcription [START_REF] Shall | Poly(ADP-Ribose) Polymerase-1: What Have We Learned from the Deficient Mouse Model?[END_REF].

IV.2.1. Copper stress

As a first stress, copper was chosen for its ability to inhibit the repair of some oxidative DNA damages and to interfere with the process of poly-(ADP-ribosyl)ation [START_REF] Schwerdtle | Impact of Copper on the Induction and Repair of Oxidative DNA Damage, poly(ADP-Ribosyl)ation and PARP-1 Activity[END_REF].

IV.2.3 Combination of copper and oxidative stress

Some of the detrimental effects observed with copper are mediated through oxidative stress generation. In the following experiment, incubation was performed with a combination of copper/ H 2 O 2 .

There is a potentiating effect of both components, illustrated by a 30% increase for PARP-B mRNA levels and by 45% increase for PARP-C with high significance (Figure 66).

Nevertheless, PARP-B and PARP-C level were increased but their ER=0.9 was similar to that observed with S2 control (ER about 1).

IV.3. PARP isoforms relative expression after stress and recovery

After copper/H 2 O 2 stress followed by 30 minutes recovery in normal medium, a decrease was observed for PARP-B (-38%) and for PARP-C (-52%) mRNA levels. However, ER remains unchanged compared to S2 cells in control conditions (Figure 67).

IV.4. Subcellular PARP localization in Drosophila melanogaster model

In order to get a precise view of PARP sub-cellular localization in S2 cells, specific constructs were generated in pMT-puro expression vectors (Annexes 2). All the obtained constructs are illustrated in annexes. The cloning procedure was described in the Materials and Methods chapter.

IV.4.1. Expression vectors and transfection in S2 cells

Two constructs were generated in pMT-puro vector. The first one encodes a PARP fusion protein with a V5 tag (Figure 52) at the C-terminus end of the protein and will be used for immunodetection in Western blot analysis but also for ¨in situ¨ localization of PARP protein.

The second construct encodes a PARP-GFP (Figure 53

IV.4.2.2. PARP PB-V5 under copper induction

In a second set of experiments (Figure 69) , PARP was overexpressed in S2 cells and detected through the use of antibodies directed against the V5 tag of the fusion protein. With this procedure, a peripheral MitoTracker detection was observed, but also a colocalisation with ParPB-V5 suggesting at least, a partial mitochondrial presence of PARP. In the mean time, a nuclear signal was also observed for the recombinant protein.

IV.4.2.3. Parp PB-GFP under copper induction

Using the same strategy, but with a PARP-GFP fusion protein (Figure 70), a strong positive GFP signal was observed in the nucleus. However, a green but faint signal was also co-localized with MitoTracker in a diffuse manner in the cytosol, suggesting once again a potential mitochondrial localization. In order to create redox imbalance within the cells but also to induce DNA damage in the mean time, S2 cells were incubated with hydrogen peroxide. This experiment was performed to determine if oxidative stress induces PARP relocalisation within treated cells.

In basal S2 cells, a strong positive signal corresponding to endogenous PARP, was detected in the nuclear region, but also a green faint signal was observed partly co-localized with MitoTracker in a diffuse manner within the cytosol (Figure 71)

IV.4.3.2. PARP PB-V5

In PARP overexpressing cells under oxidative stress exposure (Figure 72), signals were observed mainly in a diffuse manner within the cytosol, with partial co-localisation with MitoTracker probe.

However, no clear nuclear localization was detected. To summarize all these data, PARP in S2 cells was mainly localized in nuclear compartment, but also present at a lower level in the mitochondrial surroundings. A PARP overexpression led to a higher nuclear targeting, but maintained the signal in "mitochondria".

IV.5. Sub-cellular fractionation and PARP detection

In order to confirm the results obtained by microscopy, another approach was performed using sub-cellular fractionation followed by Western-blot analysis on pure fractions. Two sub-cellular fractions were tested: nuclear and mitochondrial fractions (Figure 73).

Prior to further analyses, samples were qualified on purity basis. All mitochondrial samples did not show any histone protein suggesting the lack of nuclear contaminants.

On the opposite, nuclear samples still displayed pyruvate dehydrogenase (PDH) signals except for sucrose treated fractions. Using a specific PARP polyclonal antibody, two faint populations were observed in nuclear fractions purified (115 and 80 kDa).

As well, crude nuclear fraction presents very low quantity of PARP. In mitochondrial extracts, only low molecular weights proteins were detected at 70 and 80 kDa. These results were observed either with crude or purified fractions.

These results confirmed those obtained by ¨in situ¨ detection on mitochondrial localization but also showed for the first time the presence of different PARP isoforms between nuclear and mitochondrial compartments. (1,[START_REF]→ Global analysis of BER system in Drosophila melanogaster[END_REF]3,[START_REF]IX. Recombinant protein production[END_REF]5,[START_REF]REFERENCES AND ANNEXES Adamietz P. « Poly(ADP-Ribose) Synthase Is the Major Endogenous Nonhistone Acceptor for poly(ADP-Ribose) in Alkylated Rat Hepatoma Cells[END_REF]7,8) analyzed by mass spectrometry.

After mass spectrometry analyses, no PARP detection was observed from derived peptides for all the 8 samples, leading to the question of the exact nature of these bands. The samples analyzed were obtained without anti-proteases in extraction buffer and also any protein purification protocol was adapted to PARP protein.

IV.7. Potential caspase cleavage sites in PARP

In normal cells, PARP behavior is modulated through various post-translational modifications. Among them, poly-(ADP-ribosylation) and inactivation by caspase cleavage are well known [START_REF] Boulares | Role of poly(ADP-Ribose) Polymerase (PARP) Cleavage in Apoptosis. Caspase 3-Resistant PARP Mutant Increases Rates of Apoptosis in Transfected Cells[END_REF]. In this chapter, we investigated Drosophila PARP potential to be cleaved by caspases.

IV.7.1. Human Parp-1

Human Parp1 can be cleaved by caspase 3 and_7 in position 214 and by caspase 1 in position 800 (from N terminus) (Figure 76). Both cleavages show identical profile of 24KDa and 89 KDa. deoxyribose bound in the resulting abasic site is then processed by apuric/apyrimidic (AP) endonuclease such as APE1 enzyme in mammals (Tsuchimoto D et al., 2001), which is active not only in nuclei, but also in mitochondria. A heterologous protein RRP1 has been characterized in the fruitfly in the nucleus [START_REF] Sander | Drosophila Rrp1 Protein: An Apurinic Endonuclease with Homologous Recombination Activities[END_REF], but its mitochondrial location was still unknown.

EthA and Tg alterations were also strongly recognised with both compartments extracts.

They arise from ROS attack on adenine and thymine residues, which are both highly abundant in mtDNA particularly in the AT-rich sequence [START_REF] Anderson | Sequence and Organization of the Human Mitochondrial Genome[END_REF]. EthA results from lipid peroxidation [START_REF] Speina | Chemical Rearrangement and Repair Pathways of 1,N6-Ethenoadenine[END_REF]. These etheno adducts are recognized by MPG (methyl-purine glycosylase) [START_REF] Asaeda | Substrate Specificity of Human Methylpurine DNA N-Glycosylase[END_REF]. Neil 1, 2 and 3 glycosylases could also metabolize modified adenine and guanine. They have been identified in nuclei and also detected in mitochondria [START_REF] Hu | Repair of Formamidopyrimidines in DNA Involves Different Glycosylases: Role of the OGG1, NTH1, and NEIL1 Enzymes[END_REF]van Loon B et Samson LD., 2013). Tg is the main thymine oxidative product of ROS exposure [START_REF] Dianov | Single Nucleotide Patch Base Excision Repair Is the Major Pathway for Removal of Thymine Glycol from DNA in Human Cell Extracts[END_REF].

We found in our present study that the thymine glycol removal was quantitatively strong in purified mitochondria and higher than that observed in purified nuclei. Some reports have shown that the bifunctional glycosylase NTH1 may be responsible for Tg recognition and excision in mice [START_REF] Karahalil | Compromised Incision of Oxidized Pyrimidines in Liver Mitochondria of Mice Deficient in NTH1 and OGG1 Glycosylases[END_REF].

Uracil (paired with A), often encountered in DNA may result from incorporation of dUMP during replication, or be the consequence of cytosine oxidative deamination by ROS, resulting in U:A pairs or U:G mismatch. This DNA damage is controlled by different UNG glycosylases. In humans, two UNG transcripts produced by differential splicing give rise to a nuclear and a mitochondrial [START_REF] Nilsen | Nuclear and Mitochondrial Uracil-DNA Glycosylases Are Generated by Alternative Splicing and Transcription from Different Positions in the UNG Gene[END_REF]. This glycosylase may be the only one able to process uracil residue in mitochondria. In fruitflies, mitochondrial location of this protein was still unknown.

In our experimental conditions, on one side no A removal paired with 8oxoG was detected with crude mitochondrial fractions or pure mitoplast fractions. Also, this activity was quite high in nuclear fractions (27% excision rate in young fruitfly nuclear fractions). In mammals, MYH enzyme catalyzed this activity and has been detected in both nuclear and mitochondrial extracts [START_REF] Karahalil | Compromised Incision of Oxidized Pyrimidines in Liver Mitochondria of Mice Deficient in NTH1 and OGG1 Glycosylases[END_REF]. To our knowledge, this activity has never been described in fruitfly mitochondria.

On the other side, 8oxoG removal was not observed in our study in mitochondrial compartments, despite the fact that many studies have pointed out high 8oxoG excision activity in mitochondria (ie from mouse liver [START_REF] Sander | Drosophila Rrp1 Protein: An Apurinic Endonuclease with Homologous Recombination Activities[END_REF]. The lack of this activity in mitochondria in our study could be the result of a low recognition in our conditions of the modified oligonucleotide attached on the microarray or a low activity in Drosophila mitochondria.

In a previous study, we described the process of aging in Drosophila melanogaster 8 weeks after emerging [START_REF] Dubessay | Aging Impact on Biochemical Activities and Gene Expression of Drosophila Melanogaster Mitochondria[END_REF]. mtDNA mutations frequently increased during aging (de Souza-Pinto [START_REF] De Souza-Pinto | Repair of 8-Oxodeoxyguanosine Lesions in Mitochondrial Dna Depends on the Oxoguanine Dna Glycosylase (OGG1) Gene and 8-Oxoguanine Accumulates in the Mitochondrial Dna of OGG1-Defective Mice[END_REF]. This could be correlated with an increase in ROS production [START_REF] Lee | Mitochondria and Aging[END_REF] but also, repair mechanisms involved in DNA maintenance might be concerned.

Our results effectively showed changes in repair activities in both nuclear and mitochondrial fractions during aging. AP site cleavage decreased in mitochondrial fractions.

By contrast, we observed an increase in U (paired with G) excision.

In nuclear fractions from old fruitflies, excision activities of A, 8oxoG, and Hx declined significantly. The same results were observed for U (paired with G) and 8oxo G-C, but were not significant. Further, no nuclear glycosylase activity was increased during aging in fruitfly.

In mouse liver mitochondrial fractions, a high 8oxoG excision activity increase was observed during aging, while a decrease was noted with nuclear extracts (de Souza-Pinto NC et al.,

2001).

Other measured activities such as U-G glycosylase and endonuclease did not change in either compartment. Most of the observed activities were a consequence of the presence of specific mitochondrial isoforms from various BER glycosylases. In silico analyses were performed to investigate the ability of candidates proteins to be involved in such activities. These proteins were described in several organisms mitochondria but it was less known for drosophila model.

II. .Mitochondrial addressing predictions

In silico analysis of mitochondrial addressing prediction of certain proteins from Drosophila melanogaster involved in BER pathway would help to understand the addressing footprints in protein that maybe play a role in mtDNA repair in extending Drosophila model until human. According to Table 13 mitochondrial addressing studies were made using PSORT II and MitoProtII predictors. Certain proteins from Drosophila melanogaster such as OGG1, RRP1, XRCC1, PARP-B, PARP-C, PARG-B were analyzed.

The 100% of nuclear addressing was reported to Rrp1-PB considered as negative control of mitochondrial addressing. The RpL32-A (named Rp49) was the mitochondrial positive control with 100% of mitochondrial prediction (data not shown).

IDH reported 90% of mitochondrial prediction as was expected for one mitochondrial

III. .Base excision repair genes expression in Drosophila

The RT-q-PCR analysis results demonstrated that the parp mRNA expression profile was susceptible to regulation in the three key parts of BER pathway such as DNA base excision, AP site removal and DNA ligation.

The Figure 79 shows the possible regulation of parp mRNA through the role of OGG1, XRCC1 and RRP1 proteins, the two first having a positive effect and the last one a negative effect. We are not clear if regulatory mechanism of parp expression was carried out directly by OGG1, RRP1 and XRRC1 proteins or through intermediate molecules. Interesting the parg expression profile (mRNA) increase 40% in mutant context rrp1 -, xrcc1 -, and ogg1 -/xrcc1 -; however in ogg1 -context parg expression profile was similar to WT. This results guides that the lack of OGG1 protein did not interfere with parg mRNA expression, compared to RRP1 and XRCC1 proteins which led to a change for this expression. Role for glycosylases as regulators of other genes expression has already been described with knock-out models. In ogg1-mutant mice, a general metabolic perturbation was observed leading to changes in genes expression such as down-regulation of carnitine palmitoyl transferase-1, and the integral transcriptional co-activator Pgc-1 [START_REF] Sampath | 8-Oxoguanine DNA Glycosylase (OGG1) Deficiency Increases Susceptibility to Obesity and Metabolic Dysfunction[END_REF]. XRCC1 deficiency was classicaly described as a severe BER deficiency. In fruitfly, this deficiency does not interfere with viability compared to the same deficiency in mouse, but in our study all tested genes expressions in fruitfly were reduced except for parg.

IV. .PARP isoforms relative expression

IV.1 It was illustrated in Drosophila rrp1 -(Flybase code 10213) mRNA expression of parp and parg expressions about 40%. The two parp isoforms were quantified using primers adapted to common region among genes. In order to understand parp isoforms regulation in rrp1 knockout, parp mRNA variants were quantified using specific primers. The parp-B expression is over-expressing in 50% while parp-C expression was similar WT; also, parg expression was about 70%. Thus, the RRP1 protein can be implicated in parp-B and parg mRNA depletion (these results were discussed above). Nevertheless, western blot anti-with addition of H 2 O 2 (40mM) incubation 30 min, evidenced that the transcript of parp-B increased over-expression until 30% and ER similar to WT. This results guides that copper and H 2 O 2 are possible synergetic actions. In S2 DNA recovery conditions (after Cu 2+ / H 2 O 2 stress incubation followed by 30 min at 24 °C in normal conditions), we showed depletion in both isoformes about 40% compared to basal S2 cells. As well, over-expression of parp is also associated in apoptotic pathway activation [START_REF] Chaitanya | PARP-1 Cleavage Fragments: Signatures of Cell-Death Proteases in Neurodegeneration[END_REF]. We show in silico casp1 digestion profile of Parp-B-V5 the peptides about 16 KDa, 55 KDa, 82 KDa, we illustrated by western blot anti-V5 the protein of 82 KDa in J2 cells.

It

V.2 cells. tion profile of Parp-B-V5 Drosophila melanogaster

Human PARP-1 is a nuclear protein identified as an enzyme that performs central roles in the repair of damaged DNA, PARP-1 participates in initiating base excision repair (BER) [START_REF] Dantzer | Base Excision Repair Is Impaired in Mammalian Cells Lacking Poly(ADP-Ribose) Polymerase-1[END_REF]. Recently, in Human, mitochondrial localization has been shown in HeLa cells [START_REF] Rossi | Mitochondrial Localization of PARP-1 Requires Interaction with Mitofilin and Is Involved in the Maintenance of Mitochondrial DNA Integrity[END_REF].

We localized by microscopy in S2 cells Parp in nucleus and mitochondria. The oxidative stress increases highly the parp-B presence in nucleus and mitochondria.

The western blot anti-PARP results using polyclonal (PARP S/L (dN-16)) identified proteins about 70KDa and 82KDa in mitochondrial samples and about 113KDa in culot

CONCLUSIONS

The project performed during this PhD thesis was able to give some interesting informations regarding mitochondrial DNA repair in Drosophila.

According to the initial first question, we showed that the PARP-B isoform is present both in nuclear fraction. We also showed a possible new isoform about 70 kDa in mitochondria, that still needs to be confirmed.

We focused on PARP proteins under cellular stresses studying both regulation and subcellular localization. The oxidative stress lets to modulate the level of each parp isoforms, but also the ratio of these two isoforms. Some data suggested that PARP was for a part a mitochondrial protein. This aspect needs to be confirmed prior going further. Finally we hypothesized that PARP-B over-expression may lead to caspase 1 activation with consequently PARP cleavage.

Regarding PARP regulation in BER mutants, we showed that PARP-B mRNA was modulated in ogg1-/-, rrp1-/-, xrcc1-/-and ogg1-/xrcc1-mutants. Except in rrp1-/-mutant, PARP-B mRNA decreased in every tested mutant. Molecular analyses performed in mutant flies contexts gave some examples of co-regulations, pointing out the functional interactions existing between various glycosylases or adaptator proteins (XRCC1) in the BER process.

Such an approach needs to be further analysed, in order to investigate and understand the exact role each protein could have on other BER partners.

The last question regarding BER activities during aging was answered using a comprehensive approach of Base Excision Repair processes in fly mitochondria. This technology could be performed on fly model and was efficient to determine which BER activity were present, but also the level and intensity of this activity. In our study, few activities were high in Drosophila mitochondria and directed against THF-A, Eth-A, Tg-A and U-G modifications. These activities were mostly different from those observed in nucleus. Interestingly, aging effects were quantified on mitochondrial BER activities, raising the question of the role of mitochondrial BER mechanisms in aging processes.

  ............................................................................................................. a) Analysis of key BER enzymes activities during aging.

  ... MATERIAL AND METHODS ........................................................................................................................................

  2. cDNA production .......................................................................................................................................................... IV.1.3. RT-q-PCR ......................................................................................................................................................................... IV.2. PCR prior to cloning ....................................................................................................................................................... IV.3. PARP-B-target construction ........................................................................................................................................ IV.4. DNA electrophoresis ...................................................................................................................................................... IV.5. Bacteria transformation ............................................................................................................................................... IV.5.1. Competent cells preparation .................................................................................................................................. IV.5.2. Transformation ............................................................................................................................................................. IV.5.3. Cloning control .............................................................................................................................................................. V.→ In silico analyses .

  .. CONCLUSIONS ............................................................................................................................................................... PERSPECTIVES .............................................................................................................................................................. REFERENCES AND ANNEXES .................................................................................................................................

Figure 1 :(

 1 Figure 1: Endosymbiosis theory: The first eukaryote may have originated from an ancestral prokaryote that had undergone membrane proliferation, compartmentalization of cellular function (into a nucleus, lysosomes, and an endoplasmic reticulum), and the establishment of endosymbiotic relationships with an aerobic prokaryote, and, in some cases, a photosynthetic prokaryote, to form mitochondria and chloroplasts, respectively. (modified from http://mrkingbiochemistry.files.wordpress.com/2013/02/theory-ofendosymbiosis.gif?w=300&h=166 )

Figure 2 :

 2 Figure 2: Electron microscopy of mitochondrial structure (modified from Herrmann JM. et Riemer J., 2010).

Figure 4 :

 4 Figure 4:The pre-protein import machinery of mitochondria. Abbreviations: MIA, mitochondrial intermembrane space import and assembly; SAM, sorting and assembly machinery; TIM, translocase of inner membrane; TOM, translocase of outer membrane[START_REF] Bohnert | A Dynamic Machinery for Import of Mitochondrial Precursor Proteins[END_REF].

Figure 6 :

 6 Figure 6: Schematic representation of ATP synthesis. Three H + pumping sites create variation in the inner

Figure 7 :

 7 Figure 7: Schematic representation of the human mtDNA (Wanrooij S et Falkenberg M., 2010).

Figure 8 :

 8 Figure 8: Schematic representations mtDNA replication modes. (A) Strand-displacement replication mode. (B) RITOLS (left pathway) and strand coupled (right pathway) mode of mtDNA replication (Antoshechkin I, et al., 1997).

Figure 9 :

 9 Figure 9: The bootlace model of mtDNA replication. Preformed (L-strand) transcripts, including complementary tRNA and mRNA hybridize to the template lagging strand of mammalian mtDNA as leading strand DNA synthesis proceeds. RNA recruitment is an ongoing process

Figure 10 :

 10 Figure 10: Proteins at the human mtDNA replication for (Wanrooij S et Falkenberg M., 2010).

  DNA replication (OH), it generates the RNA primers used to initiate leading-strand mtDNA synthesis (Wanrooij S. et al., 2008 ; Chang DD et Clayton DA., 1985 ; Chang DD, Hauswirth WW, et Clayton DA., 1985). About 100 nucleotides are primed in LSP region leading strand DNA synthesis (Figure 11).

Figure 11 :

 11 Figure 11: Schematic representation of the D-loop regulatory region. The three conserved sequence blocks (CSBI, CSBII and CSBIII) are located just downstream of light-strand promoter (LSP). Transition from RNA primer to the newly synthesized DNA has been mapped to sequence within or near CSB II (Kang D et al., 1997). The conserved termination-associated sequence ( Pham XH. et al., 2006) (TAS) elements are located at the 3' of the nascent D-loop strand and are proposed to be a major regulation point of mtDNA replication.Abbreviations: HSP, heavy-strand promoter; LSP, light-strand promoter; OH, origin of H-strand; F, tRNA phenylalanine; P, tRNA prolin(Wanrooij S. et al., 2008).

Figure 12 : 2 .

 122 Figure 12: Schematic model of DNA strands synthesized: (A) the leading strand (H-strand) DNA replication machinery approaches the OL sequence (yellow). (B) the upon passage, single-stranded OL is exposed and adopts a stem-loop structure. MtSSB is unable to bind to the stem-loop and POLRMT can initiate primers from the poly-dT stretch in the single-stranded region of OL (C). After about 25 nucleotides (dotted red), POLRMT is replaced by POLγ, and lagging-strand DNA (L-strand) synthesis[START_REF] Fusté | Mitochondrial RNA Polymerase Is Needed for Activation of the Origin of Light-Strand DNA Replication[END_REF].

Figure 13 :

 13 Figure 13: CoQ10 partially preserves mitochondrial structure of ONH astrocytes against oxidative stressinduced mitochondrial fission. (Noh YH, et al., 2013).

Figure 14 :Figure 15 :

 1415 Figure 14: Mitochondrial DNA localizes to punctuate structures that are distributed throughout the mitochondrial compartment. (A) or after a 20 hour pulse with BrdU (B). Insets 1 and 2 depict enlargements of the boxed areas. (A) Monoclonal antibodies against DNA label punctuate structures that are distributed throughout mitochondria labeled with antibodies against the inner membrane protein COX2. Only a few mitochondria appear devoid of DNA-labeling (arrowheads). (B) Antibodies against the thymidine analogue BrdU label the nucleus as well as punctuate mitochondrial structures labeled with DNA-antibodies. Some of the mitochondrial DNA-positive structures are devoid of BrdU (arrowheads) and very few BrdU-positive structures escape detection with DNA-antibodies (arrows). CoQ10, coenzyme Q10; H 2 O 2 , hydrogen peroxide. Scale bars, m

Figure 16 :

 16 Figure 16: Models for mitochondrial fusion in yeast Saccharomyces cerevisiae a) Mfn1 single protein interaction and b) Fzo1p-Mgm1p-Ugo1p complex protein interaction (OkamotoK et al., 2005).

Figure 17 :

 17 Figure 17:The MMM complex may mediate at least three processes: actin-mitochondria attachment (left), formation of tubular mitochondria (middle), and anchoring of mtDNA nucleoids (right)[START_REF] Okamoto | Mitochondrial Morphology and Dynamics in Yeast and Multicellular Eukaryotes[END_REF]

  Figure 20 represents a typical maternal mode of inheritance of diseases caused by mtDNA mutations (Figure 21).

Figure 20 :Figure 21 :

 2021 Figure 20: Typical pedigree of three filial generations. The black color was used in order to illustrate the illness phenotype. The maternal inheritance was shown in the third filial generation where all illness phenotype was the maternal origin. Nevertheless, the males (II: 1, 2 and 3) did not transmit the illness phenotype to generation III. http://t3.gstatic.com/images?q=tbn:ANd9GcSGVALk3oCWEGsz3rEj0bsWt40YOiWjnonukMHFi_3MonfWDeuh Agc18lJIvg

Figure 22 :

 22 Figure 22: Schematic representation of exogenous and endogenous sources of mitochondrial and nuclear DNA damages involving different cell responses.

  pathway that is initiated by members of the DNA glycosylase family (Barnes DE et Lindahl T., 2004). The DNA glycosylases are responsible for initial recognition of the lesion. They flip the damaged base out of the double helix, and cleave the N-glycosidic bond of the damaged base, leaving an apurinic/apyrimidinic (AP) site. (Figure 23) There are two categories of glycosylases: monofunctional glycosylases have only glycosylase activity, whereas bifunctional glycosylases also possess AP lyase activity. Therefore, bifunctional glycosylases can convert a base lesion into a single-strand break without the need for an AP endonuclease. In human cells, oxidative pyrimidine lesions are generally removed by hNTH1, hNEIL1, or hNEIL2, whereas oxidative purine lesions are removed by hOGG1. At least 11 distinct mammalian DNA glycosylases are known. One example of DNA bifunctional glycosylases is OGG1 that recognizes 8-oxoG paired with cytosine in DNA. Similar fonction was identified in Escherichia coli as MutM (Fpg) (van der Kemp PA. et al., 1996).

  Figure 23 illustrates short-patch and long-patch BER pathways in the nucleus and mitochondria.

Figure 23 :

 23 Figure 23: Short-patch and long-patch BER pathways in the nucleus and mitochondria of mammalian cells. The major factors involved are highlighted. Factors common to both compartments are displayed with an orange background, factors specific for the nucleus are on a blue background and factors specific for mitochondria are on a yellow background. *DNA2 is present in the nucleus but its putative role in nuclear BER is not clear so far. APEX1, AP endonuclease; POL , , , , DNA polymerase , , , ; RFC, replication factor C; PCNA, proliferating cell nuclear antigen; DNA2, endonuclease/helicase; FEN1, flap endonuclease 1; LIG1, LIG3, DNA ligase 1, 3; XRCC1, X-ray crosscomplementing protein-1(Boesch P. et al., 2011).

Figure 25 :

 25 Figure 25: MUTYH-initiated BER of A:8-oxo-G lesions[START_REF] Markkanen | MUTYH DNA Glycosylase: The Rationale for Removing Undamaged Bases from the DNA[END_REF].

Figure 26 ,

 26 Boesch and collaborators summarized the dialogue between nucleus and mitochondria in mtDNA repair under mitochondrial oxidative stress in human and plants cells. They schematized how probably post-translational modifications in DNA repair proteins can be involved in mitochondrial damage signals. One of the more important function of mitochondria is ATP production involved ROS generation and damage of mtDNA repair mechanisms are unclear until nowadays. Due to ROS level in mitochondria BER mechanism remains like one of the more often studied mechanism (Lopez-Contreras AJ, et Fernandez-Capetillo O., 2012). They demonstrated Figure 27 that mitochondrial damage signaling may compete with nuclear damage signaling for the recruitment of monofunctional and bifunctional glycosylases such as UNG1, MUTYH, OGG1, NTHL1, NEIL1 and NEIL2 where most of the BER proteins are also membrane-bound. The Short-patch BER, by example, is mediated by OGG1 for 8-oxoG repair in coordination with APEX1, the DNA polymerase POL and the DNA ligase (LIG3). Besides, CSB recruits the BER proteins to the membrane; CSA and CSB interact with the single-stranded DNA-binding protein SSB and the glycosylase; the tumor suppressor p53 stimulates the glycosylase and POL ; PARP-1 modulates BER. Long patch BER pathway is carried out in addition the endonuclease/helicase DNA2 and the endonuclease FEN1. The transcription factor and packaging protein TFAM binds with high affinity to damaged DNA; TFAM binding is modulated by p53 and the thioredoxin TRX2 and p53 itself can hydrolyse oxidized nucleotides at DNA 3′-ends and the reaction is enhanced by SSB (Lopez-Contreras AJ, et Fernandez-Capetillo O., 2012).

Figure 26 :

 26 Figure 26: Organization of oxidative DNA damage repair processes and factors in mammalian mitochondria, Main repair enzymes are in green; further factors are in yellow or purple; core DNA-interacting proteins are in blue. Modified from[START_REF] Boesch | DNA Repair in Organelles: Pathways, Organization, Regulation, Relevance in Disease and Aging[END_REF]

Figure 27 :

 27 Figure 27: Mitochondrial BER in mammalian cells. Three different sub-pathways of SP-BER converge at the gap filling step. SP-BER (left) and LP-BER (right) converge at the nick-sealing step. In addition to FEN1, DNA2 is required for efficient flap processing during LP-BER in mitochondria (Lopez-Contreras AJ, et Fernandez-Capetillo O., 2012).

Figure 28 :

 28 Figure 28: Schematic representation of DSBs repair mechanisms. A) NHEJ.and B) HR. Both mechanism are in response to ROS DNA damage. B) HR (Lopez-Contreras AJ, et Fernandez-Capetillo O., 2012).

Figure 29 :

 29 Figure 29: Interactions between NHEJ proteins. Physical interactions between NHEJ components are summarized (Rooney S, et al., 2004).

  The exposed 3' ssDNA tails are coated by single-strand DNA binding proteins (SSBs) to prevent the formation of secondary structures. The recombination mediator, (not show in figure) Rad52 then displaces SSB and recruits the Rad51/RecA-type recombinase to form presynaptic helical nucleoprotein filaments. These filaments then initiate homology search and catalyze ATP-dependent strand invasion within duplex DNA templates. The invading strand provides a free 3' end for priming DNA replication that allows the restoration of genetic information missing from the dsDNA breaks. The Rad52 protein has a second function in this pathway, which is to capture the second end through its single-strand annealing activity. After DNA synthesis and ligation, double Holliday junctions are formed.The Holliday junctions can be resolved by different molecular strategies with or without DNA strand crossover. Without the capturing of the second end by Rad52, the invading strand may be dissociated from the D-loop and reannealed to the ssDNA on the second end, a process known as synthesis-dependent strand annealing[START_REF] Chen | Association between Single-Nucleotide Polymorphisms of OGG1 Gene and Pancreatic Cancer Risk in Chinese Han Population[END_REF].Recently studies reveal a recombination protein of likely bacteriophage origin in mitochondria and support the notion that recombination is indispensable for mtDNA integrity[START_REF] Mbantenkhu | Mgm101 Is a Rad52-Related Protein Required for Mitochondrial DNA Recombination[END_REF].II. 4. DNA repair in mitochondriaFrom 1974 Clayton and collaborators show that mitochondria are unable to repair pyrimidine dimers and some types of alkylation damage(Miyaki, et al., 1977). Several years took for discovery of mitochondrial BER(Pettepher, et al., 1991). Recently Gredilla 2010 and Boesch and collaborators in 2011 proved that DNA repair mechanisms are present in nucleus but are also in mitochondria illustrating different pathways which need further confirmation with a hatched background. Just the Nucleotide Excision Repair (NER) pathway was not detected in mitochondria. Nevertheless Homologous recombination (HR) and Nonhomologous end-joining (NHEJ) were associated with Double Strand Break Repair (DSBR) of mtDNA. The Figure 30 shows the DNA repair pathways highly conserved among nucleus and mitochondria (Wilson S, et al., 2012). The Figure 31 shows a possible PARP action in strand break with blocking groups at one or both exposed ends is unable to be repaired by simple short patch-BER.

Figure 30 :

 30 Figure 30: Nuclear and mitochondrial DNA repair shares common proteins. However not all nuclear DNA repair pathways are present in the mitochondria. BER proteins, isoforms of nuclear relatives (Wilson S, et al., 2012).

Figure 32 :

 32 Figure 32: Molecular modeling of the Mgm101 core domain (from A115 to V237) based on the Rad52 structure (1H2I). (A) The positions of N150, F153, and F235, which are highly conserved in Rad52-related proteins, are highlighted. Also indicated are C216 and C217, which form a putative redox sensor for regulating Mgm101 function. (B) Surface representation model of a 14-mer ring formed by Mgm101115-237 (Chen., 2013).

Figure 33 :

 33 Figure33:A model for activation of Mgm101 in mtDNA repair. The Mgm101 rings are proposed to be the storage form of the protein in mt-nucleoids. In response to oxidative stress or other signals, the C216/C217 cysteine pair is modified. This may induce structural remodeling in the rings, which stimulates ssDNA binding and the repair of dsDNA breaks(Chen., 2013).

Figure 34 :

 34 Figure 34: A model of the evolutionary history of recA/RAD51 gene family (Lin Z.et al., 2006).

(

  [START_REF] Ahnesorg | XLF Interacts with the XRCC4-DNA Ligase IV Complex to Promote DNA Nonhomologous End-Joining[END_REF][START_REF] Buck | Cernunnos, a Novel Nonhomologous End-Joining Factor, Is Mutated in Human Immunodeficiency with Microcephaly[END_REF][START_REF] Andres | Crystal Structure of Human XLF: A Twist in Nonhomologous DNA End-Joining[END_REF].As C-NHEJ is essential for V(D)J recombination, defects in previous characterized NHEJ factors completely block lymphocyte development at the progenitor stage and also cause T -and B -severe combined immunodeficiency (T -B -SCID) in both human and mouse models[START_REF] Rooney | The Role of the Non-Homologous End-Joining Pathway in Lymphocyte Development[END_REF][START_REF] Lieber | The Mechanism of Double-Strand DNA Break Repair by the Nonhomologous DNA End-Joining Pathway[END_REF][START_REF] Li | Lymphocyte-Specific Compensation for XLF/cernunnos End-Joining Functions in V(D)J Recombination[END_REF]. XLF mutations cause a less severe lymphocytopenia than SCID in human patients. In the case of XLF-deficient mice, the lymphocytes numbers are reduced with not stage-specify blockade. In agreement, chromosomal V(D)J recombination occur normally in XLF-deficient lymphocyte but not in XLF-deficient murine embryonic stem cells. This observation suggested that more than one repair pathway(s) might exist in developing lymphocytes to compensate XLF deficiency during V(D)J recombination[START_REF] Lieber | The Mechanism of Double-Strand DNA Break Repair by the Nonhomologous DNA End-Joining Pathway[END_REF].Recently, it has been reported that chromosomal V(D)J recombination in XLF-deficient lymphocytes requires ATM and its kinase activity(Zha S. et al., 2011) ATM kinase is a master regulator of the DNA damage response. Although ATM promotes efficient DNA repair during CJ formation(Bredemeyer A L et al., 2006), ATM is not required for V(D)J recombination and lymphocyte development[START_REF] Barlow | Atm-Deficient Mice: A Paradigm of Ataxia Telangiectasia[END_REF]. nucleus and ADP ribosyl-acceptor hydrolase (ARH3) and in mitochondria (Pankotai E, et Lacza Z., 2009). In nucleus the proteins targeted such as H1, H2B and TRF2 are involved in numerous biological processes, including DNA repair, chromatin structure and transcription. Poly(ADPribosyl)ation of acceptor proteins has functional consequences such as DNA-break signaling, chromatin relaxation and recruitment of DNA repair proteins (Yelamos J et al., 2011). Recently, in order to clarify the Poly(ADP-ribose) metabolism, using siRNA in PARG knock-down it was shown that the poly(ADP-ribosyl)ation of proteins in the cells was lost, revealing that PARP1 expression and activity is involved with PARG expression in knockdown cells. (Uchiumi F, et al., 2013) Thus, poly(ADP-ribose) in a cell is regulated under the control of PARP1/PARG gene expression balance. Structural and enzymological evidences of PARP family lead to propose a new consensus nomenclature for all ADP-ribosyltransferases (ARTs) based on the catalyzed reaction and on structural features. ARTs members are classified in two big subfamilies such as: the DNA-dependent PARPs, (PARP1, PARP2 and PARP3) and the tankyrases (Hottiger MO et al., 2010).

  ), PARP-1 accounts for more than 90% of the poly(ADP-ribosyl)ating capacity of the cells The domains of each member are indicated: According to protein sequence from NH2 to COOH terminal in PARP1 The ART domain is the catalytic core required for basal ART activity. The WGR domain named after a conserved central motif (W-G-R) is also found in a variety of polyA The PARP regulatory domain (PRD) might be involved in regulation of the PARPbranching activity. polymerases and in proteins of unknown function. The sterile alpha motif (SAM), a widespread domain found in signaling and nuclear proteins, can mediate homo-or heterodimerization. The ankyrin repeat domains (ARD) mediate protein-protein interactions in diverse families of proteins. The vault protein inter-alpha-trypsin (VIT) and von Willebrand type A (vWA) domains are conserved domains found in all inter-alpha-trypsin inhibitor (ITI) family members. The WWE domain is named after three conserved residues (W-W-E), and is predicted to mediate specific protein-protein interactions in ubiquitin-and ADP-ribose conjugation systems. The Macro or A1pp domains are structurally related to the catalytic domain of enzymes that process ADP-ribose-1′-phosphate, a reaction product derived from ADP-ribose 1′-2′ cyclic phosphate generated by Terminal Phosphotransferase (TpT). The Macro domain can serve as ADPr or O-acetyl-ADP-ribose binding module The BRCT domain (BRCA1 carboxy-terminal domain) (Amé JC, et al., 2004).

Figure 36 :

 36 Figure 36: Schematic comparison of the ARTD (PARP) family. ZF: zinc finger domains. SAP: SAF/Acinus/PIAS-DNA-binding domain, MVP-ID: Major-vault particle interaction domain, NLS: nuclear localization signal. CLS: centriole-localization signal. HPS: Histidine-proline-serine region. RRM is an RNAbinding/recognition motif. UIM: ubiquitin interaction motif. MVP-ID: M-vault particle interaction domain. TPH: Ti-PARP homologous domain. GRD: glycine-rich domain. Within each ART domain, the region that is homologous to the PARP signature (residues 859-908 of PARP1) as well as the equivalent of the PARP1 catalytic E988 is shaded. The WWE domain is a protein-protein interaction motif. TM: transmembrane domain (HottigerMO et al., 2010).

Figure 38 :Figure 39 :

 3839 Figure 38: Schematic representation 3D of PARP-1 zinc fingers. F1 and F2 were structurally independent.(modified from[START_REF] Eustermann | The DNA-Binding Domain of Human PARP-1 Interacts with DNA Single-Strand Breaks as a Monomer through Its Second Zinc Finger[END_REF]

Figure 40 :

 40 Figure 40: (a) Human PARP-1 clevage site. (a) During apoptosis, PARP-1 is cleaved by caspase-3 (common with caspase7) into a 24-kDa and an 89-kDa fragment. (Modified from Eustermann S. et al., 2011).

Figure 41 :

 41 Figure 41: Relationship between caspase-3 activation and PARP cleavage during apoptosis (Liang PY. et al., 2013).

Figure 42 :

 42 Figure 42: Schematic representation of chromatin superstructure relaxation due to PARP action: relationship between SSB and PARP-1 activation. The position of covalent histone-tail poly(ADP-ribosyl)ation is indicated below the sequences of rat histones[START_REF] Schreiber | Poly(ADP-Ribose): Novel Functions for an Old Molecule[END_REF].

  Figure42shows poly(ADP-ribose) synthesis at the DNA-damage site triggers both the immediate recruitment of the SSB repair (SSBR) scaffold protein XRCC1, but also the relaxation of chromatin superstructure as mediated by poly(ADP-ribosyl)ation of histone H1 and H2. This modification facilitates repair enzymes access to the DNA lesion and interaction with XRCC1.III.2.3. PARP-1 role in few DNA repair pathwaysPARP-1 has a role in SSBs repair. PARP-1 knock down with siRNA or PARP-1 activity inhibition with small molecules reduce SSB repair and the absence of PARP1 increase the quantity of DSBs.The SSB accumulated is transformed in DBSs as well, HR system can be modulated due to the new level of DBSs, thus PARP-1 can be involved in different mechanisms such as: BER, SSB, NHEJ and HR mechanisms(Godon C, et al., 2008).III.3. The structure-function of PARP-2 ambiguityPARP-2 was discovered as a result of the presence of residual DNA-dependent PARP activity in embryonic fibroblasts derived from PARP-1-deficient mice[START_REF] Shieh | Poly(ADP-Ribose) Polymerase Null Mouse Cells Synthesize ADP-Ribose Polymers[END_REF].

Figure 43 :III. 2 . 2 .

 4322 Figure 43: The domain structure of PARP-2, showing the PARP signature sequence (in dark blue within the F catalytic domain). NLS nuclear localization signal; NoLS nucleolar localization signa (Mégnin-Chanet F, et al., 2010).

  TIPARP: containing the TPH domain (Cys-Cys-Cys-His) and Trp-Trp-Glu domain, (Figure 36) which can exhibit PAR-binding activity (Katoh M, et Katoh M., 2003). The macroPARPs: containing macrodomain folds, which are ADP-ribose-binding modules that can facilitate the localization of these ARTs to sites of poly and mono(ADP-ribosyl)ation (Gibson BA et Kraus WL., 2012).

Figure 44 :Figure 45 :

 4445 Figure 44: Model of PARP inhibitors action in single strand nick. PARP inhibitors and truncated PALB2 pathway leads to error prone repair of DNA.(Metzger MJ, et al., 2013).

Figure 46 :

 46 Figure 46: Schematic representation of PARP isoformes in Drosophila. ParpPB (113 kDa) is encoded by the full transcript and ParpPC (92 kDa) by the truncated transcript. Two main actors of the Poly(ADP-ribose) metabolism were shown in nucleus from Drosophila melanogaster cells. Boamah and collaborators showed the presence of PARP-B and PARG in nucleus. PARP-B is essential for fly development. Deletion or disruption of PARP-B protein functions disintegrates nucleolus structure (Figure 47). Thus, PARP-B controls nucleolar structural integrity (Boamah EK, et al., 2012).DNA repair pathways in Drosophila melanogaster can be associate with larval diet.

  Figure 47: A-B: PARP-B protein localizes to nucleoli in all Drosophila tissues, including polytene chromosomes of larval salivary glands (A) and diploid nuclei of larval brain (B). The dissected larval salivary glands and brains expressing PARP1-DsRed (red) were stained with the DNA binding dye Draq5 (green). Positions of nucleoli are indicated with arrows. SGlarval salivary gland; BRlarval brain. C-D. PARP-B deletion displaces nucleoli protein as detected by nucleoli-specific antibody AJ1 (red). AJ1 detects nucleoli in every cell of Drosophila mid-intestine in wild-type second-instar larvae (C), but only in a few cells in ParpCH1 mutants (D). DNA is detected with OliGreen dye (green). E-H. Deletion or disruption of PARP-B proteinfunctions disintegrates nucleolus structure. Salivary glands from wild-type (E), ParpRNAi expressing (F), hypomorphic Parp mutant, ParpC03256 (G), and overexpressing antagonist of PARP-B, PARG (H) 3rd instar larvae were stained for the nucleolar specific protein Fibrillarin (red)[START_REF] Boamah | Poly(ADP-Ribose) Polymerase 1 (PARP-1) Regulates Ribosomal Biogenesis in Drosophila Nucleoli[END_REF].

Figure 48 :

 48 Figure 48: Representation of double mutant production strategy. The pBac DNA insertion ogg1[f08013] and xrcc1[f03685] were represented in red color. Eyes colors were indicated with thin arrows in all fruitflies.

Figure 49 :

 49 Figure 49: Schematic representation of glycosylase action detection on microarray. Step 1 represents incubation of oligonucleotides modified with biological extract; step 2 represents the glycosylase and endonuclease actions enzyme and the step 3 washing . Microarray analyses show in green color the glycosylase action dot.

Figure 50 :

 50 Figure 50: Schematic representation of the Real Time quantity PCR amplification conditions.

Figure 51 :

 51 Figure 51: Schematic representation of PCR amplification conditions of Parp-B, GFP-targ and (Hist) 6 -V5-targ using Pfu enzyme during 30 cycles. Denaturation at 90°C, annealing at 60°C and DNA synthesis at 72°C were represented in purple, green and blue colors respectively.

Figure 52 :Figure 54 :

 5254 Figure 52: J2 plasmid: PARP-B-V5 cloned in pMT-puro vector

1 .

 1 Competent cells preparationCompetent cells were prepared according to protocol used in our laboratory. A single fresh colony E. coli DH5was inoculated into 3 mL of LB broth and cultured 6 hours at 37°C.Then the LB broth volume was increased to 200 mL and growth was carried out overnight at 37 C and 200 cycles/min in a rotary shaker. Next, 500 L of the culture was transferred into 50 mL of LB broth and incubated under the same conditions until culture OD 600 reaches 0.3-0.4. Growth was stopped by transfer into a 50-mL polypropylene tube stored at 0 C during 10 min. The tube was centrifuged at 1,700 g for 15 min at 4 C. Pellet was resuspended in 10 mL of filter-sterilized ice-cold 0.1 M CaCl 2 and stored for 5 min on ice. Then suspension was centrifugated at 1,700 g for 15 minutes at 4 C. After discarding the supernatant the tube was inverted for 1 min to allow the last traces of media to drain away. Finally cell pellet was resuspended in 2 mL of filter-sterilized ice-cold 0.1 M CaCl 2 and store for 2-3 hours on ice.The total volume was divided into 200 L of competent E. coli DH5  suspension in sterile microcentrifuge tubes and used immediately. Alternatively cells were stored at -80 C.

(

  ExPASy) http://web.expasy.org/peptide_cutter/ In natural substrates, he prediction and characterization of proteolytic cleavage sites can lead to understand these structural relationships. (Garay-Malpartida HM, et al., 2005) V.4. Physico-chemical properties Molecular Weight (MW) and isoelectric point (pI) were determined using the program: Compute pI/MW (ExPASy) http://web.expasy.org/compute_pi/ VI. .PARP sub-cellular localization VI.1. S2 cells culture According to the manufacturer's protocol (modified) S2 cells were incubated in complete Schneider medium (Invitogen) containing Fetal Calf Serum (10%) antibiotics (Penicillin and Streptomycin 1%) at 24˚C until density reaches 6 to 20 x 10 6 cells/mL.

Day 2 :

 2 Transfection: Solution A (1 µg plasmid in 100 µl of incomplete SDM) was slowly added to 8% Cellfectin in incomplete SDM and gently vortexed. The mix was incubated 20 min at room temperature. It was added to S2 cell culture where SDM complete medium was previously replaced by incomplete one. Incubation was performed during 5 hours at 24 °C. centrifuged at 4°C, (12000 g, 15 min) and finally diluted in sterile water. DNA quantification was in Nanodrop 1000 spectrophotometer. The aliquots were stored at -80°C.

  mass spectrometry was performed on the local Metabolomics platform facility (INRA,

  proteins (Figure56, lanes CN) such as PDH and Rieske. Another purification step using a sucrose cushion led to the almost complete disappearance of mitochondrial contaminants in purified nuclear fraction (Figure56, lanes PN). These results prove that BER activities measured in the purified nuclear fractions were exclusively of nuclear origin.

Figure 56 :

 56 Figure 56: Western blot analyses of various sub-cellular fractions. Mitochondrial (A) and nuclear (B) protein fractions were analyzed: 15 mg of crude mitochondrial fraction (CM), Percoll gradient purified mitochondria (PM) and purified mitoplast fraction (Mpl) from young (1 week) and old (8 weeks) fruitflies were separated by SDS PAGE, transferred on membrane and analyzed by Western blot. Antibodies directed against complex III Rieske subunit (mitochondrial component) or against lamin and histone H3 (nuclear components) were used.

Figure 57 :

 57 Figure 57: Enzymatic activity measurements in various sub-cellular fractions. Cytochrome oxidase (A) and citrate synthase (B) activities were measured in various subcellular compartments: purified nuclear fraction (PN), crude mitochondrial fraction (CM) and purified mitoplast fraction (Mpl). Fractions from young (black) and old (gray) fruitflies were analyzed. Error bars represent the SD of independent experiments (n = 12, Student's test: ** p < 0.01).

Figure 58 :

 58 Figure 58: Cleavage rate measurements using lesion-containing oligonucleotide microarrays. The cleavage rate represents the percentage of loss fluorescence at each lesion spot after incubation with purified nuclear fractions (A), crude mitochondrial fraction (B) and purified mitoplasts (C). The different lesion substrates are indicated: ethenoadenine paired with T (Eth-A), hypoxanthine paired with T (Hx-T), uracil paired with G (U-G), tetrahydrofuran paired with A (THF-A), 8oxoguanine paired with C (8oxoG-C), A paired with 8oxoguanine (A-8oxoG), thymine glycol paired with A (Tg-A), uracil paired with A (U-A). Fractions from young (white) and old (black) fruitflies were analyzed. (n = 3-7, Student's test: * p < 0.05). Error bars represent the SD of independent experiments.

(

  XRCC1-PA), two isoforms of poly-(ADP-ribose) polymerase (Parp-PB and PC), poly-(ADPribose) glycohydrolase (Parg-PB), and on the other hand a known mitochondrial protein : isocitrate dehydrogenase (idh-PC).

Figure 58 :

 58 Figure 58: Relative mRNA expression of 5 days old male Drosophila melanogaster .from FlyBase Data and in this study RT-q-PCR results (n=3).

Figure 59 :

 59 Figure 59: Relative mRNA expression of ogg1, rrp1, xrcc1, parp and parg in ogg1 -mutants flies compared to WT. The results are the mean ± deviation (Student's test: *** p < 0.001). n = 3

Figure 60 :III. 4 .

 604 Figure 60: Relative mRNA expression of ogg1, rrp1, xrcc1, parp and parg in rrp1 -mutants flies compared to WT. The results are the mean ± deviation. Student's test: ***p<0.001, **p<0.01 ,n = 3

Figure 61 :

 61 Figure 61: Relative mRNA expression of ogg1, rrp1, xrcc1, parp and parg in Xrcc1 -mutants flies compared to WT The results are the mean ± deviation. Student's test: ***p<0.001, n= 3.

Figure 62 :

 62 Figure 62: Relative mRNA expression of ogg1, rrp1, xrcc1, parp and parg in ogg1 -/xrcc1 -mutants flies compared to WT. The results are the mean ± deviation Student's test: ***p<0.001 (n = 3).

Figure 63 :

 63 Figure 63: Relative mRNA expressions of PARP isoforms and PARG in wild type (WT) or Rrp1 -mutant (Rrp1 -) Drosophila using RT-q-PCR experiments. All values are expressed as percentages using PARG expression level in WT context as a reference. The results are the mean ± deviation Student's test: ***p<0.001, **p<0.01, n = 3.

Figure 64 :

 64 Figure 64: Relative mRNA expressions of PARP isoforms in S2 control and S2 under cooper stress using RT-q-PCR experiments. All values are expressed as percentages using PARP B expression level in S2 context as a reference The results are the mean ± deviation. Student's test: *p<0.05, **p<0.01, n = 3.

Figure 65 :

 65 Figure 65: Relative mRNA expressions of PARP isoforms in S2 control and S2 under H 2 O 2 stress. Using RT-q-PCR experiments. The results are the mean ± deviation. Student's test: *p<0.05, **p<0.01 (n = 3).

Figure 66 :

 66 Figure 66: Relative mRNA expressions of PARP isoforms in S2 control and S2 under copper/H 2 O 2 stress. Using RT-q-PCR experiments. The results are the mean ± deviation. Student's test: *p<0.05, **p<0.01 (n = 3).

Figure 67 :

 67 Figure 67: Relative mRNA expressions of PARP isoforms in S2 control and S2 after copper/H 2 O 2 stress S2 DNA repairing). The results are the mean ± deviation. Student's test, **p<0.01 (n = 3)..

  ) fusion protein to characterize ¨in situ¨ the subcellular localization of the PARP protein. Both inserts have been tested through enzymatic digest (annexes 6), then fully sequenced (annexes 7; annexes 8). They appear conform to the expected sequences. These plasmids were transfected into S2 cells. The stable cell lines obtained after puromicin selection were named J2 and C4 according to the name of transfected plasmids. Genomic integration and mRNA expression of parp-B-V5 and parp-B-GFP were validated by PCR amplification from genomic DNA and from total RNA extraction. PB in basal conditions The first set of experiments was performed on S2 cells in basal conditions. In Figure 68, using IDH-GFP construct as a positive control, a punctuated signal was observed in the cytosol and co-localized with Mitotracker staining, suggesting a mitochondrial localization.

Figure 68 :

 68 Figure 68: Mitochondria were identified in red color by MitoTracker detection, nucleus in blue color with Hoechst reactive and parp-B was identified in green color using antibody anti-parp-alexa488. S2 untransfected cells were used as a negative control (Merge MitoTracker + Hoechst). The scale of picture dimension was represented with yellow line of 2.5 m (n = 3).

Figure 69 :

 69 Figure 69: Mitochondria were identified in red color by MitoTracker detection, nucleus in blue color with Hoechst and parp-B-V5 was identified in green color using antibody alexa488 anti-V5 monoclonal antibody S2 untransfected cells were used as a negative control (Merge MitoTracker + Hoechst). Isocitrate dehydrogenase was used as a positive control for mitochondrial addressing (IDH-GFP). The yellow bar indicates 2.5 m, n = 3.

Figure 70 :IV

 70 Figure 70: Mitochondria were identified in red color by MitoTracker detection, nucleus in blue color with Hoechst reactive and Parp-B-GFP was identified in green color thanks to GFP signal). S2 untransfected cells were used as a negative control (Merge MitoTracker + Hoechst). Isocitrate dehydrogenase was used as a positive control for mitochondrial addressing (IDH-GFP). The yellow bar indicates 3.5 m. n = 3

Figure 71 :

 71 Figure 71: Mitochondria were identified in red color by MitoTracker detection, nucleus in blue color with Hoechst reactive and Parp-B-GFP was identified in green color thanks to GFP signal). S2 untransfected cells were used as a negative control (Merge MitoTracker + Hoechst). Isocitrate dehydrogenase was used as a positive control for mitochondrial addressing (IDH-GFP). The yellow bar indicates 2.5 m. (n = 3)

Figure 72 :

 72 Figure 72: Mitochondria were identified in red color by MitoTracker detection, nucleus in blue color with Hoechst reactive and Parp-B-GFP was identified in green color thanks to GFP signal). S2 untransfected cells were used as a negative control (Merge MitoTracker + Hoechst). Isocitrate dehydrogenase was used as a positive control for mitochondrial addressing (IDH-GFP). The yellow bar indicates 2.5 m. n = 3

Figure 73 :

 73 Figure 73: Western blot analysis of PARP isoforms in subcellular fractions from Drosophila. (a) crude mitochondria, (b) crude nucleus, (c) purified mitochondria, (d) purified nucleus. The red arrows show the main proteins identified in mitochondrial samples and green arrow the protein visualized in nuclear sample.

Figure 74 :

 74 Figure 74: Western blot analysis on S2 cells extracts using either anti-PARP (left) or anti-V5 (right) antibodies. (a) J2 cells (S2 cells transfected with parp-B-V5) after 0.7mM cooper incubated 24 hours, (b) J2 cells after 24h incubation with 0.7mM copper followed by 40mM H 2 O 2 during 30 min, (c) C4 cells [S2 cells transfected with parp-B-GFP] after 0.7mM cooper incubated 24 hours, (d) S2 cells in basal conditions. The red band corresponds to the 70KDa band(Prestained Protein Ladder; thermo scientific)

Figure 75 :

 75 Figure 75: Protein coloration in gel using coomassie Brilliant Blue (A) Mitochondria fraction from Drosophila, (B) Nuclear fraction from Drosophila, (C) J2 cells extract after 24 hours incubation with 0.7mM copper . Red arrows show the bands(1,[START_REF]→ Global analysis of BER system in Drosophila melanogaster[END_REF]3,[START_REF]IX. Recombinant protein production[END_REF]5,[START_REF]REFERENCES AND ANNEXES Adamietz P. « Poly(ADP-Ribose) Synthase Is the Major Endogenous Nonhistone Acceptor for poly(ADP-Ribose) in Alkylated Rat Hepatoma Cells[END_REF]7,8) analyzed by mass spectrometry.

Figure 76 :

 76 Figure 76: Schematic hypothetical hydrolysis of human parp1 by caspases. Red color represents caspase 3 and 7 action and green color caspase 1 action.

Figure 78 :

 78 Figure 78: Western Blot on various protein fractions from bacteria overexpressing PARP protein (extract) and from purified fractions (fractions 1 and 2). "Wash" fraction represents the washing buffer after passage through the columnn; n=1

  protein. OGG1 showed by PSORTII, a mitochondrial and nuclear addressing prediction very similar and by MitoProtII, a 44% mitochondrial addressing. The XRCC1, PARP-B, PARP-C, PARG-B mitochondrial addressing prediction was poor about 1-20% by MitoProtII analysis. The human PARP1 show 8% of mitochondrial addressing. Nevertheless, Rossi and collaborators in 2009 showed that PARP1 was present in human mitochondria, but also that was related to the mitofilin receptor controlling its entry. In Drosophila, mitofilin receptor (Flybase code CG6455) was discovered (2005) before studies of human mitofilin receptor and Parp1 mitochondrial internalization. In silico studies guide to possible presence of main BER actors such as OGG1 and PARP-B in Drosophila mitochondria: OGG1 with potential MTS and PARP-B with potential receptor in IMM.

Figure 79 :

 79 Figure 79: Schematic representation of mRNA parp regulation in BER system. The induction of mRNA parp expression was represented in purple and the inhibition in red. Full arrows described a positive action and dotted lines, an indirect action.

  was demonstrated in human cell with severely damaged DNA have amplified PARP-1 activity resulting in high NAD + consumption depleting ATP pools and this activity inevitably leads to passive necrotic cell death (due to prolonged ATP depletion) (Eguchi Y, Shimizu S, et Tsujimoto Y., 1997; Lemaire C et al., 1998). In cells expressing caspase-resistant PARP-1 would be more clear the necrosis induction and an increased of apoptosis associate with depletion of NAD + and consequently in ATP level (Herceg Z et Wang ZQ., 1999). Interesting recent reports show that caspases are not only involved in apoptosis pathways. They are implicated in forms of cell proliferation in a Drosophila model. The caspase Dcp-1 in Drosophila represents caspase-3 in human (Fan Y et Bergmann A., 2008 ; Kondo S. et al., 2006).

  .. II.→.Sub-cellular fractionation ...................................................................................................................................... II.1. Sub-cellular fractions purification (nucleus and mitochondria) ................................................................. II.2. Mitochondrial fractions validation ............................................................................................................................ II.2.1. Western blot analysis .................................................................................................................................................. II.2.2. Enzyme assays ................................................................................................................................................................

III.→Comprehensive BER repair analysis through microarray technology ............................................. IV. →Molecular Analysis ...................................................................................................................................................

IV.1. Expression analyses ....................................................................................................................................................... IV.1.1. mRNA extraction .......................................................................................................................................................... IV.1.

. et al., 2010). Codon Universal code Human mitochondrial code UGA STOP Trp AGA Arg STOP AGG Arg STOP AUA IIe MetTable 1 :

 1 Comparison of mitochondrial genetic code to universal code(Osawa S

	The universal arginine codons AGG
	and AGA became in STOP, nevertheless UAG (universal STOP) became in Trp. Further,
	AUA has been reassigned to Met rather than serving as an Ile codon (Smits P

et al., 1992) I.2. Mitochondria Distribution

  COX2 and/or BrdU under normal conditions. The merge DNA/COX2 illustrated the co-localization mtDNA and IMM. Besides, the merge DNA/BrdU shows that mitochondria fission and mtDNA replication are not depending mechanisms.

	Mitochondria exist in two interconverting forms; as organelle isolated or/and as extended filaments, networks
	or clusters connected with intermitochondrial junctions (Skulachev VP., 2001). Mitochondria distribution
	is very variable in human cell. The Figure 13 shows primary skin fibroblasts labeled with
	antibodies against DNA,

  shows mitochondrial diseases due to mitochondrial (mt) tDNA and nuclear

	(n) DNA (nDNA) damage
	Genetic and biochemical classification of the mitochondrial diseases
	Genome Gene	[mtDNA] Biochemistry	Clinical phenotype
	mtDNA	single Δ x prot. synth.	KSS; ocular myopathy; PS
	tRNA Leu(UUR)		↓ prot. synth	MELAS
	tRNA Lys		↓ prot. synth	MERRF
	other tRNAs		↓ prot synth.	multiple phenotypes
	ATPase6		↓ATP synth.	NARP/MILS
	ND1, ND4, ND6		↓complex I	LHON
	ND1, ND4		↓ complex I	myopathya
	Cyt b		↓ complex III	myopathya
	COX III		↓ complex IV myopathya
	nDNA
	NDUF		↓ complex I	LS
	SDHA		↓complex II	LS
	BCS1L		↓ complex III	GRACILE
	SURF1		↓ complex IV LS
	SCO1		↓ complex IV hepatoencephalomyopathy
	SCO2		↓ complex IV cardioencephalomyopathy
	COX10		↓ complex IV nephroencephalomyopathy
	COX 15		↓complex IV	cardioencephalomyopathy
	ATP 12		↓ complex V	fatal infantile multisystemic
	TP	multiple Δ	↓ _	MNGIE
	ANT1	multiple Δ	↓ prot. synth.	adPEO-plus b
	Twinkle	multiple Δ	↓ prot. synth	adPEO-plus b
	POLG	multiple Δ	↓ prot. synth.	ad/arPEO-plus b
	dGK	depletion ↓ prot. synth.	hepatocerebral syndrome
	TK2	depletion ↓prot. synth	myopathy; SMA
	TAZ		↓ cardiolipin	Barth syndrome
	OPA1		↓ mit. Motility ad optic atrophy

Table 2 :

 2 Representation

of mitochondrial diseases due to mtDNA and nuclear DNA (nDNA) damage. Abbreviations and symbols: Δ, deletion; , decrease, KSS, Kearns-Sayre syndrome; PS, Pearson syndrome; MELAS, Mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes; LS, Leigh syndrome; MILS, maternally inherited LS; GRACILE, growth retardation, aminoaciduria, cholestasis, lactacidosis, early death; adPEO, autosomal dominant progressive external ophthalmoplegia arPEO, autosomal recessive PEO; SMA, spinal muscular atrophy. [mtDNA] indicates changes of mtDNA secondary to nDNA mutations (defects of intergenomic signaling). a Mutations in cyt b and COX genes can also cause multisystemic diseases. b Plus refers to proximal weakness, neuropathy, psychiatric disorders, parkinsonism (DiMauro S., 2004).

Table 3 :

 3 List

of certain enzymes DNA glycosylases present in nucleus and mitochondria from human cells. Chromosomal location, cellular localization and major substrates are represented

[START_REF] Barnes | Repair and Genetic Consequences of Endogenous DNA Base Damage in Mammalian Cells[END_REF]

.

Table 4 :

 4 Proteins known to affect DNA recombination in yeast, mammalian, and plant mitochondria

	Organism and protein Molecular function	Mutant phenotype on recombination
	Yeast
	Abf2	high-mobility-group	Increases Holliday junctions,
		protein primarily for mtDNA packaging reduces repeat-mediated recombination
	Exo5	5'-3' exonuclease	Unknown
	Hmi1	3'-5' DNA helicase	mtDNA fragmentation
	Mgm101	Single-strand annealing protein	Decreases repeat-mediated recombination
	Mhr1	Homologous pairing in vitro	Decrease gene conversion
	MRX complex	5'-3' exonuclease	Decrease repeat-mediated recombination
	Msh1	Mismatch repair-like protein	Increases repeat-mediated recombination
	Nuc1	Endo/exonuclease	Decreases mtDNA recombination
	Pif1	5'-3' DNA helicase	Reduce recombination in p+ x p-crosses
	Rim1	Single-strand annealing	Suppresses pif1 mutation

Table 5 :

 5 Different D. melanogaster strains used in this study. Number beside the mutant name indicates the FlyBase code.

Table 7 :

 7 Primers

	used for Parp-B, GFP and (Hist)6-V5 PCR amplification. Parp-B and GFP primers contained
	restriction site (small letters). (Hist)6-V5 Tag was amplified without restriction site.

Table 8 :

 8

Summary of clones for PARP-B construct: clon-19 J-2 represents GFP cloned in pGEM7z(+) vector in SmaI/BamHI positions; clon-1 represents Parp-B cloned in clon-19 in KpnI/SmaI positions; clon-7 represent clon-1 inserted with targ (Hist)6-V5 PCR product in SmaI position; J2 plasmid represents Parp-B-(Hist)6-V5 cloned in pMT puro vector in KpnI/EV positions; C-4 plasmid represent Parp-B-GFP cloned in pMT puro vector in KpnI/EV position and the plasmid A-8 represents Parp-B-(Hist)6-V5 cloned in pBAD24 vector in HindIII (treatment with klenow)/KpnI positions.

Table 9 :

 9 List of plasmids used in this study.

Table 11 :

 11 Incubation conditions for oxidative stress induction in S2 cells using different H 2 O 2 concentrations.

Table 12 :

 12 Gels composition.

Table 13 :

 13 Table 13 shows PsortII and MitoProtII addressing prediction of certain protein such as: ogg1, rrp1, xrcc1, parp-B, parp-C, parg-B, Idh-C and RpL32-A from Drosophila melanogaster and parp-1 from human. Red color represents very high nuclear addressing prediction and green color very high mitochondrial addressing prediction. ND: non determined. Mitochondrial addressing prediction of some proteins involved in BER mechanism from Drosophila and human species.

	Name	Addressing prediction
		Nuclear	Mitochondrial
	FlayBase code	PsortII	MitoProtII
	Ogg1-PA	35	22	44
	Rrp1-PA	92	ND	19
	Rrp1-PB	100	ND	27
	XRCC1-	78	9	19
	Parp-PB	78	ND	5
	Parp-PC	83	ND	2
	Parg-PB	61	4	1
	Idh-PC	ND	61	90
	Human	67	4	8
	Parp1

In the mitochondria long-patch BER dependant multi nucleotide excision appears to be driven by EXOG as part of an APE1 complex however the presence of FEN-1 and DNA2 in the mitochondria suggests a secondary long patch-BER pathway, potentially to excise lesions resistant to EXOG activity. After the lesion has been resolved nucleotides are inserted by Additionally, it was demonstrated that blockade of IgG in bladder cancer cell T24 resulted in up regulation of cleaved caspase-3 and cleaved poly(ADP-ribose) polymerase (PARP) [START_REF] Liang | Overexpression of Immunoglobulin G Prompts Cell Proliferation and Inhibits Cell Apoptosis in Human Urothelial Carcinoma[END_REF]. The Poly(ADP-ribosyl)ation is considered a reversible posttranslational protein modification and is involved in the regulation of a number of biological functions such as: maintenance of genomic stability, transcriptional regulation, energy metabolism, centromere function, telomere dynamics, mitotic spindle formation during cell division, concluding, poly(ADP-ribosyl)ation is involved in aging and cell death (Aguilar-Quesada R. et al., 2007 ; [START_REF] Lee | Apoptosis Induction of Human Prostate Carcinoma Cells by Cordycepin through Reactive Oxygen Species-mediated Mitochondrial Death Pathway[END_REF].

III.1. Poly(ADP-ribose) metabolism

The metabolism of Poly(ADP-ribose) is a dynamic process mediated by ARTs and poly(ADP-ribose) glycohydrolase (PARG) enzymes [START_REF] Bonicalzi | Regulation of poly(ADP-Ribose) Metabolism by poly(ADP-Ribose) Glycohydrolase: Where and When?[END_REF]. Polymers with chain lengths about 200 ADP-ribose units are rapidly degraded by PARG poly(ADPribose) and / or ADP ribosyl-acceptor hydrolase (ARH3) [START_REF] Isabelle | Investigation of PARP-1, PARP-2, and PARG Interactomes by Affinity-Purification Mass Spectrometry[END_REF]. The Figure 35 shows the Poly(ADP-ribosyl)ation regulation reversed by the activities of poly(ADP-ribose)polymerase PARP and poly(ADP-ribose) glycohydrolase (PARG) in

III.2. The structure-function of human PARP-1

The member PARP-1 has been studied most extensively; it is a monomeric enzyme, of 116-kDa highly conserved [START_REF] Amé | The PARP Superfamily[END_REF], (Figure 37). This molecule is involved in the polyADP-ribosylation carried out by poly(ADP-ribose)polymerase (PARP) enzymes, of which the first discovered and most important is PARP-1, a very highly abundant nuclear protein found in higher eukaryotes [START_REF] Amé | The PARP Superfamily[END_REF]. The main targets for addition of PAR by PARP-1 are histones and PARP-1 itself (Adamietz P., 1987 ;[START_REF] Poirier | Poly(ADP-Ribosyl)ation of Polynucleosomes Causes Relaxation of Chromatin Structure[END_REF]. although there are many other targets including HMG proteins [START_REF] Tanuma | ADP-Ribosylation of Chromosomal Proteins and Mouse Mammary Tumor Virus Gene Expression. Glucocorticoids Rapidly Decrease Endogenous ADP-Ribosylation of Nonhistone High Mobility Group 14 and 17 Proteins[END_REF], topoisomerase I (Krupitza G et Cerutti P., 1989) and p53 [START_REF] Kanai | Inhibition of Crm1-p53 Interaction and Nuclear Export of p53 by poly(ADP-Ribosyl)ation[END_REF]. According with Figure 37 the DNA binding domain contains three zinc finger motifs, the two first enclosing the motif A showed in Figures 38 and 39 by F1 and F2 domains [START_REF] Eustermann | The DNA-Binding Domain of Human PARP-1 Interacts with DNA Single-Strand Breaks as a Monomer through Its Second Zinc Finger[END_REF] and the third isolating zinc finger motifs is localized among the motifs B and C. The two first zinc finger domains are responsible for DNA interaction and genome screening, while the third zinc finger domain is involved in PARP-1 dimerisation [START_REF] Langelier | A Third Zinc-Binding Domain of Human poly(ADP-Ribose) Polymerase-1 Coordinates DNA-Dependent Enzyme Activation[END_REF].

III.2.1. PARP-1 structure

Motif B contains a bipartite nuclear localization signal (NLS) responsible for the nuclear addressing of PARP-1, and also, motif B enclose a caspase3/7 cleavage site [START_REF] Eustermann | The DNA-Binding Domain of Human PARP-1 Interacts with DNA Single-Strand Breaks as a Monomer through Its Second Zinc Finger[END_REF], Figures 40 and41).

All amplifications were made according to the manufacturer's protocol using kit MESA GREEN qPCR MasterMix Plus; SYBR PCR, (Eurogentec). The specific oligonucleotides are described in Table 6. The Rp49 gene was used as a reference and all cDNA amplifications were made in Mastercycler-Pros PCR machine (BioRad) at Tm 60°C and 45 cycles (Figure 50).

IV.2. PCR prior to cloning

All amplifications were made at 60°C Tm during 30 cycles using Pfu polymerase (Promega). Amplifications were performed with specific primers (Table 7) and protocol presented in Figure 51. Then medium was changed to complete SDM. For stable transfected cells lines, a selection was performed using puromycin antibiotic (5 µg/mL) during 6 weeks.

Day 3: Expression of protein of interest: Cells were incubated in presence of 0.7 mM Cu 2 SO 4 during 24 hours at 24°C to induce the expression of the specific protein.

VI.3. Validation of stable cells lines

All PCR conditions were shown on Table 10. GoTaqPol (0.5 U) polymerase (Promega)

was used with the following conditions: Tm 60°C, 72°C for 3 min, 30 cycles. ). According to Karin Andersson protocol's, (Amersham Pharmacia Biotech, Uppsala, Sweden) a single fresh colony A8 was inoculated into LB ampicillin (100ng/ml) and cultured overnight at 37 C with 200 cycles/minutes in a rotary shaker. Next, 500 L of the culture was transferred into 50 ml of LB broth and it was incubated at the same conditions. When culture OD 600 reached 0.5-0.6, expression induction was performed using L-Arabinose (with various concentrations ranging from 0.002% to 0.02%) during 3 hours. Bacteria were centrifuged at 1,700 g for 15 min at 4 C and the cell pellets was recovered.

Sample

The pellets were resuspended in 10 ml of appropriate buffer (0.16 M phosphate buffer 4 M NaCl, 25 mM imidazole pH 7.4). Bacterial lysis was performed at ice-cold using ultrasound ten times 0.5 minutes pulses. Centrifugation at 1,700 g for 15 minutes at 4 C. Recombinant protein was purified on a 0.2 m filter (Millipore).

IX.2. Protein purification

The poly-histidine tagged recombinant protein was purified manually using a column HisTrap Chelating, 1mL (amersham pharmacia biotech). Firstly the column was equilibrated with 3 volume of binding buffer (0.16 M phosphate buffer, 4 M NaCl, 25 mM Imidazole pH 7.4),

The E.coli extracts (10mL) was charged into the column and was washed with 10 ml of binding buffer and subsequently a second wash with 10 mL of biding buffer containing 100mM Imidazole.

The protein was recovered with 1mL of eluting buffer (0.16 M phosphate buffer, 4 M NaCl 250 mM Imidazole pH 7.4). The first mL collected corresponds to wash buffer that remained in column. Two fractions about 500 L were collected and stored at -80C.

V. .Production of recombinant PARP protein

The production of PARP protein was planned to have a tool to:

 Investigate the molecular weight of a standardized PARP protein  Generate polyclonal antibodies against the whole PARP protein

The pBAD24 expression vector was chosen to produce PARP-B in E. coli. The construct used is illustrated (annexes 3) and was fully sequenced (annexes 9). It appeared conform to the expected PARP sequence After recombinant E. coli generation and PARP protein induction through L-arabinose incubation, extract was purified on His-Trap column and subsequent fractions were analyzed by western blot using an anti-His antibody.The Figure 78 represents parp-B-His 6 expression in E. coli. The western Blot (WB) anti-Histidine was made with bacterial extracts, fraction 1 and fraction 2 following purification on affinity column. In every sample, a single protein with a molecular weight of 85 kDa was detected.

DISCUSION I. .BER activities in mitochondria

Research on mtDNA repair mechanisms goes back many years, and numerous studies have characterized mitochondrial BER activities, especially glycosylases [START_REF] Dianov | Base Excision Repair in Nuclear and Mitochondrial DNA[END_REF][START_REF] Maynard | Mitochondrial Base Excision Repair Assays[END_REF][START_REF] Boesch | DNA Repair in Organelles: Pathways, Organization, Regulation, Relevance in Disease and Aging[END_REF]. The most frequent method used to identify and characterize these activities was based on the excision of a modified oligonucleotide carrying a 32 P label, followed by electrophoresis and autoradiography (Gredilla R. et Stevnsner T.., 2012). Such an approach appeared less suitable for comparison of BER activities between various samples.

The use of a microarray technology, which has already been tested and validated [START_REF] Pons | Age-Associated Modifications of Base Excision Repair Activities in Human Skin Fibroblast Extracts[END_REF] on nuclear BER activities could constitute a powerful analytical tool for another subcellular compartment. However, due to the specificity and sensitivity of this technique, a mitochondrial purification procedure has been developed to remove any nuclear residual contaminant. This protocol has been validated through the detection of specific protein components from each compartment. It has also been chosen due to its ability to preserve inner mitochondrial activities.

We demonstrated that crude mitochondrial fractions displayed strong excision abilities for EthA, Tg, U (paired with G) and THF (abasic sites). However, other base modifications resulting from ROS oxidation were slightly or not recognized by all the mitochondrial fractions. Interestingly, repair activities observed with purified mitoplast fractions were both qualitatively and quantitatively equivalent to those measured with crude mitochondrial fractions, but also revealed more discrete activity (U paired with A).

In purified nuclear fractions, repair activities were partly different from those observed with mitochondria. THF and U (paired with G) were still strongly recognised as already described for human nuclear extracts [START_REF] Prasad | High-Resolution Two-Dimensional Electrophoresis of Nuclear Proteins[END_REF]. Compared with mitochondria samples, Hx and A (paired with 8oxoG) excision activities were high, but EthA and U (paired with A) excision activities were less noteworthy for the mitochondrial counterparts.

High abasic site excision activities in nuclear or mitochondrial fractions were not surprising. This is in fact the middle step of the base excision repair process following specific glycosylase action detecting and removing modified bases. The new phosphate- The mRNA expression ratio (ER) of ParpB/ParpC was disrupted under certain condition.

After copper incubation ER=1.4 compared with S2 control (ER about 1) with very high significance.

Copper is an essential but potentially toxic trace element in Drosophila [START_REF] Balamurugan | Copper Homeostasis in Drosophila by Complex Interplay of Import, Storage and Behavioral Avoidance[END_REF].

II)

In order to identify the BRCT domain in nuclear and mitochondrial samples.

Firstly, we suggest the production of recombinant PARP-B with aim to obtain polyclonal antibodies which will be used in protein immunoprecipitation in nuclear and mitochondrial fractions.

In long term: