Iron sulfur (FeS) clusters are ubiquitous metallocofactors required by a large number of proteins involved in myriad cellular processes. Nuclear and cytosolic FeS proteins depend on the cytosolic iron sulfur cluster assembly (CIA) pathway for cluster acquisition. The CIA pathway begins with a scaffolding complex, comprising Nbp35 and Cfd1 in Saccharomyces cerevisiae. Nbp35 and Cfd1 each harbor a deviant Walker A domain for nucleotide hydrolysis that is essential for their FeS cluster scaffolding activity. Since there is little information about the CIA scaffold’s nucleotide hydrolysis activity, it has been challenging to discern the role nucleotide is playing in FeS cluster biogenesis. This thesis investigates the nucleotide driven steps of FeS cluster assembly and transfer, and the individual roles of the scaffold subunits Nbp35 and Cfd1. First addressed was answering the question of why two different scaffold subunits are needed for CIA function, and identifying the scaffold’s quaternary structure. Size exclusion chromatography revealed that the CIA scaffold exists as homodimers and heterodimers. Only Nbp352 and Nbp35-Cfd1 exhibited detectable ATPase activity. Though Cfd12 did not have detectable ATPase activity, it bound nucleotide with an affinity comparable to Nbp352 and Nbp35-Cfd1. Site directed mutagenesis and nucleotide binding studies revealed that the Cfd1 subunit is the high affinity binding site for ATP in Nbp35-Cfd1, and that the Nbp35 subunit binds nucleotide at saturating concentrations. Cfd1 therefore controls nucleotide binding in Nbp35-Cfd1. Additionally, it was found that the Cfd1 subunit is hydrolysis competent when complexed with Nbp35, identifying Nbp35 as an activator of Nbp35-Cfd1’s ATPase activity. Next, ATP’s role in FeS cluster biogenesis by CIA was identified. Mutation of the ATPase domain of Nbp35 impaired the ability of the scaffold to assemble and transfer FeS clusters in vivo. Four phenotypes were identified by observing how each mutation affected the scaffold’s nucleotide binding and hydrolysis. In vitro experiments established that cluster occupancy of the bridging cluster site of Nbp35-Cfd1 decreased the scaffold’s affinity for nucleotide. These results support a model of FeS cluster biogenesis in which nucleotide binding and FeS cluster binding regulate one other, with the bridging cluster site translating information to the ATPase site and vice versa. Nucleotide binding is also proposed to drive a conformational change that mediates interaction with another CIA component, later identified as Dre2. Dre2 was found to stimulate the rate of ATP hydrolysis by Nbp35-Cfd1 in an FeS cluster dependent manner. It is likely that nucleotide hydrolysis is then needed for the scaffold to assemble and/or transfer the FeS cluster. The results of these experiments have allowed us to describe the critical role of nucleotide in FeS biogenesis by CIA and explain the requirement for two distinct scaffold subunits. Finally, a fluorescent [Fe4S4] cluster sensor based on bacterial FNR (fumarate and nitrate reductase transcription factor) was designed, developed, and tested for practicality. FNR was fused to a SNAP tag protein which was then covalently labeled with a fluorescent molecule. The loss of cluster by the sensor resulted in an increase in fluorescence intensity, due to the cluster’s ability to quench fluorescence. As such, cluster decay rates could be measured as a function of increasing fluorescence intensity. The rates observed via fluorescence followed the same trends as the rates obtained by measuring the decay of clusters via absorbance. Encouragingly, the rates observed for the cluster decay were similar to decay rates determined previously via alternative methods.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

The fidelity of DNA replication prevents mutations that may lead to cancer predisposition or neurodegenerative diseases. One mechanism that enhances DNA replication fidelity is DNA mismatch repair, which corrects mismatches and small insertion/deletion loops that have escaped polymerase proofreading. In all eukaryotes and most prokaryotes, MutL (a key mismatch repair protein) has an intrinsic endonuclease activity that nicks the newly synthesized strand and recruits downstream factors to remove and correct errors. It has been proposed that ATP binding promotes a series of conformational changes that induce structural order within MutL and stimulates its endonuclease activity. The C-terminal domain of MutL, which harbors the endonuclease site, does not bind to DNA. This has prevented the molecular characterization of its endonuclease activity. In this thesis, we first show that MutL in B. subtilis exhibits asymmetric conformations similar to yeast and human MutL homologs. We also devise a novel approach to bypass the binding defect of the C-terminal domain by using fusion proteins. We find that these fusions bind to DNA specifically and, in the presence of the processivity clamp, can nick DNA. One of these fusion proteins in particular stimulates the nicking activity much more efficiently than the C-terminal domain alone. This work lays the foundation for the mechanistic characterization of the MutL endonuclease and provides a method to stabilize transient protein-DNA interactions. / Thesis / Master of Science (MSc)

ATP-citrate lyase (ACLY), upregulated in chronic kidney disease (CKD), catalyzes the synthesis of acetyl-coA from citrate. Acetyl-CoA is a vital precursor for lipid/cholesterol synthesis and histone acetylation that regulates gene expression. In renal cells, ACLY regulates fibrogenic, lipogenic and inflammatory gene expression; its inhibition reduced fibrosis in the unilateral ureteral obstruction (UUO) model. The ACLY metabolic by-product malonyl-coA is also an important inhibitor of fatty acid oxidation (FAO), and defective FAO in proximal tubular epithelial cells (PTEC) is now established as a major contributor to fibrosis. Here we tested the efficacy of a novel ACLY inhibitor on reducing fibrosis and its potential role in improving FAO in UUO. 8-week-old male C57BL/6J mice underwent UUO surgery and were treated orally with an ACLY inhibitor (EVT0185, Espervita Therapeutics) for 10 days. Kidneys were assessed by immunohistochemistry, immunoblotting, and RNAseq. Effects of ACLY inhibition were tested on the HK2 PTEC cell line and primary renal fibroblast responses to TGFβ1 (5ng/ml, 48h), a cytokine known to promote fibrosis and reduce FAO. Lipid accumulation was assessed by Oil Red O staining and LC/MS analysis. ACLY inhibition significantly and dose-dependently decreased fibrosis in the UUO model determined by trichrome, PSR, fibronectin, and α-smooth muscle actin (SMA) expression. ACLY inhibition decreased macrophage (F4/80) infiltration including that of the profibrotic M2 phenotype marked by CD206. RNAseq analysis showed upregulation of FAO-related hallmark pathways and reduction in inflammation pathways with ACLY inhibition. Defective FAO is known to result in PTEC apoptosis and lipid accumulation. ACLY inhibition reduced both apoptosis, as assessed by the presence of cleaved caspase 3, as well as lipid accumulation, with a particular decrease in cholesteryl esters. In HK2 cells and renal fibroblasts, TGFβ1-induced fibrotic protein expression was inhibited by ACLY inhibition, and lipid accumulation was reduced in PTECs. ACLY inhibition reduced renal fibrosis, apoptosis, and lipid accumulation in UUO mice. ACLY inhibition also prevented profibrotic responses to TGFβ1 in PTECs and fibroblasts. Current studies are ongoing to confirm beneficial effects on restoring FAO. / Thesis / Master of Science (MSc) / Chronic kidney disease (CKD) is the leading cause of kidney failure in Canada, affecting 4 million Canadians. There is no cure for CKD and current treatments are only able to slow down disease progression. CKD is caused by scarring in the kidney. The kidney requires a lot of energy to do its job filtering our blood and creating urine, and with CKD the ability to create and use energy is reduced. The protein ATP-citrate lyase (ACLY) that is present in the kidney contributes to CKD. Research has shown that people and mice with CKD have higher levels of this protein than healthy individuals. ACLY creates a molecule called acetyl-coA that is likely to cause our kidneys to produce less energy. This study will test if ACLY is causing the kidneys to produce and use less energy. This will be done by using mice with CKD and blocking the activity of ACLY using a drug to see if this will help the kidney create more energy for itself. The kidneys of the mice will be tested to see if the drug worked in increasing energy levels and if it prevented kidney scarring. A type of cell in the kidney, called tubular cells, makes up most of the kidney and requires a lot of energy to function. We performed experiments with tubular cells and gave them stressors, like those found in CKD, and ACLY-blockers to test if the energy levels are restored and if scarring was reduced. This study is important because there is no cure for CKD and many patients will eventually develop end-stage kidney disease, requiring dialysis or transplant. Research needs to be done to create new medications for those suffering from CKD. Current studies are testing ACLY-blocking drugs to treat heart disease. If our study is successful, this drug is well-positioned to be developed into a new treatment for CKD.

No description available.

Existe una gran cantidad de patologías que afectan al esqueleto, en las que se ve comprometido el balance entre la formación y la resorción ósea. Generalmente, las alternativas terapéuticas apuntan a la modulación de la resorción ósea actuando a nivel del osteoclasto. Sin embargo, estos abordajes terapéuticos no resultan suficientes, generando la necesidad de implementar nuevas estrategias farmacológicas con blanco de acción en la fisiología de la célula formadora de hueso, el osteoblasto. Para ello, un extensivo y detallado conocimiento tanto de la función como de los procesos de proliferación, migración y diferenciación, de la citada célula, son necesarios. El sistema de señalización purinérgico, donde los nucleótidos son liberados por las células, de manera controlada, y actúan como mensajeros extracelulares activando receptores purinérgicos en la membrana plasmática de la célula blanco es conocido desde hace unos 45 años. Desde entonces, se ha reportado un número creciente de células capaces de liberar nucleótidos, de manera controlada, al medio extracelular, lo que sumado al hecho de que, virtualmente, todas las células del cuerpo poseen algún tipo de receptor purinérgico (purinoceptor), hace de este sistema de señalización uno de los más importantes respecto de la regulación de la homeostasis del cuerpo. El papel de los nucleótidos y sus receptores en los procesos de proliferación y diferenciación de los osteoblastos ha sido estudiado desde hace unos 25 años. Sin embargo, aún no se ha alcanzado la dilucidación completa de los mecanismos regulatorios de estos procesos. El objetivo de esta tesis fue determinar los efectos de la activación de receptores purinérgicos, por nucleótidos extracelulares, en los procesos de migración, proliferación y diferenciación celular de cultivos primarios de calvaria de rata neonata. Se investigó también, el efecto de una concentración elevada de calcio extracelular en la proliferación y diferenciación celular y su influencia en la regulación de dichos procesos por la estimulación purinérgica. Además, se evaluó el rol de la enzima GSK3 (por inhibición con Litio), en la regulación de la proliferación y diferenciación celular. Por último, se examinó el efecto de los agonistas mencionados sobre la expresión y localización de β-catenina. La migración celular fue evaluada mediante “ensayo de la herida”. El tratamiento con los nucleótidos mostró estimular la migración de las células calvariales. Para estudiar el proceso de proliferación se recurrió a citometría de flujo, recuento en cámara de Neubauer y tinción con Cristal Violeta. Los resultados obtenidos le atribuyen efectos positivos a los nucleótidos extracelulares. Por otro lado, el tratamiento con Litio provocó disminuciones en la capacidad proliferativa de las células. Mientras que no se observaron cambios en la proliferación por incubación de las células en un medio con elevada concentración de Calcio extracelular. El análisis de la diferenciación de las células a un fenotipo osteoblástico se llevó a cabo mediante mediciones de actividad enzimática de Fosfatasa Alcalina, cuantificación de ARNm de marcadores de maduración/diferenciación osteoblástica por QRT-PCR (reacción en cadena de la polimerasa en tiempo real cuantitativa) y detección de mineralización por tinción con rojo de Alizarina. Al respecto, el tratamiento con ATPγS exhibió un rol pro-osteogénico, al elevar la actividad enzimática de FAL, la expresión génica de marcadores de diferenciación y la capacidad mineralizante. En cambio, el agonista UTP no indujo cambios en la actividad de la enzima y, en la mayoría de los casos, reprimió la transcripción de genes osteogénicos. Por su parte, la exposición a Litio incrementó la actividad de FAL pero reprimió la expresión de genes osteogénicos y disminuyó la mineralización de los cultivos. Una elevada concentración de Calcio extracelular mostró efectos positivos a nivel de diferenciación, al incrementar la actividad FAL y la mineralización de los osteoblastos. Finalmente, el tratamiento de los cultivos con nucleótidos extracelulares, en un medio conteniendo elevada concentración de Calcio, mostró leves efectos aditivos/sinergísticos en la estimulación de la diferenciación a células formadoras de hueso. La expresión de β-catenina resultó aumentada bajo tratamiento con nucleótidos extracelulares, Litio o Calcio. Asimismo, la translocación a núcleo se vio incrementada por dichos tratamientos. De acuerdo a los resultados obtenidos en esta tesis, puede atribuirse un rol regulatorio sobre la fisiología osteoblástica al sistema de señalización purinérgico y GSK3/ β-catenina. / There are a great number of pathologies that affect the skeleton, in which the balance between formation and bone resorption is compromised. Generally, the therapeutic alternatives point to the modulation of the bone resorption acting at the level of the osteoclast. However, these therapeutic approaches are not enough, generating the need to implement new pharmacological strategies targeting the physiology of the bone-forming cell, the osteoblast. To this end, an extensive and detailed knowledge of both the function and processes of proliferation, migration and differentiation of the aforementioned cell are necessary. Purinergic signaling system, wherein nucleotides are released by cells in a controlled manner, and act as extracellular messengers activating purinergic receptors in the plasma membrane of the target cell is known for about 45 years. Since then, it has been reported an increasing number of cells capable of releasing nucleotides, in a controlled manner, to the extracellular medium, which together with the fact that virtually all cells in the body have some type of purinergic receptor (purinoceptor), makes of this signaling system one of the most important regarding the regulation of body homeostasis. The role of nucleotides and their receptors in the proliferation and differentiation processes of osteoblasts has been studied for about 25 years. However, the complete elucidation of the regulatory mechanisms of these processes has not yet been reached. The aim of this work was to determine the effects of the activation of purinergic receptors by extracellular nucleotides in the processes of cell migration, proliferation and differentiation in neonatal rat calvaria primary cultures. We also investigated the effect of a high concentration of extracellular calcium on cell proliferation and differentiation and its influence on the regulation of these processes by purinergic stimulation. In addition, the role of the GSK3 enzyme (by inhibition with lithium) was evaluated in the regulation of cell proliferation and differentiation. Finally, the effect of the mentioned agonists on the expression and localization of β-catenin was examined. Cell migration was assessed by "wound assay". Treatment with nucleotides showed to stimulate the migration of the calvaria cells. The proliferation process was studied by using flow cytometry, Neubauer chamber count and Crystal Violet staining. The results obtained attribute positive effects to the extracellular nucleotides. On the other hand, treatment with Lithium caused decreases in the proliferative capacity of the cells. While no changes in proliferation were observed by incubation of the cells in medium with high concentration of extracellular calcium. The analysis of cell differentiation to an osteoblastic phenotype was carried out by measurements of Alkaline Phosphatase (FAL) enzyme activity, quantification of mRNA of osteoblastic maturation / differentiation markers by QRT-PCR (quantitative real-time polymerase chain reaction) and detection of mineralization by Alizarin red staining. In this regard, treatment with ATPγS exhibited a pro-osteogenic role, by raising the enzymatic activity of FAL, the gene expression of differentiation markers and the mineralizing capacity. In contrast, the UTP agonist did not induce changes in enzyme activity and, in most cases, repressed the transcription of osteogenic genes. On the other hand, exposure to lithium increased FAL activity but repressed the expression of osteogenic genes and decreased the mineralization of the cultures. A high concentration of extracellular calcium showed positive effects at the level of differentiation, increasing the FAL activity and mineralization of osteoblasts. Finally, the treatment of cultures with extracellular nucleotides in medium containing high calcium concentration showed slight additive / synergistic effects in the stimulation of differentiation into bone-forming cells. Expression of β-catenin was increased under treatment with extracellular nucleotides, Lithium or Calcium. Also nucleus translocation was increased by these treatments. According to the results obtained in this work, a regulatory role on osteoblastic physiology can be attributed to the purinergic signaling system and GSK3 / β-catenin.

El conocimiento de los mecanismos moleculares implicados en la modulación de las funciones biológicas de los osteoblastos, células responsables de la formación de hueso, es indispensable como punto de partida para el diseño de nuevas estrategias terapéuticas aplicables en las patologías óseas. La acción del trifosfato de adenosina (ATP) sobre los receptores purinérgicos de la membrana plasmática ha mostrado regular importantes funciones celulares de los osteoblastos. Los resultados expuestos en esta Tesis describen por primera vez, el efecto del ATP sobre la expresión de proteínas inductoras de formación de hueso en osteoblastos de calvaria de rata (OBCs), como así también la modulación de dicho suceso por la infección con los virus de Sarampión (MEV) y Junín (JUNV). Mediante diferentes técnicas, como ensayos de proliferación, determinación de deposiciones de calcio, citometría de flujo y reacción en cadena de la polimerasa en tiempo real, se demostró que la activación por ATP de los receptores P2Y2 estimula la proliferación y diferenciación de los OBCs. Además, se observó que estos receptores modulan positivamente la expresión de proteínas asociadas a la diferenciación osteoblástica como las proteínas morfogenéticas óseas (BMP-2, 4, 5 y 7), sialo-proteína ósea, colágeno y fosfatasa alcalina. Se demostró la participación de la vía de la fosfatidil inositol 3 quinasa / proteína quinasa B (PI3K/AKT) en la estimulación inducida por el ATP sobre la proliferación y diferenciación de los OBCs. También, el empleo de diferentes nucleótidos sugiere que la expresión génica de BMP-1, 3, y 6 estaría regulada por los subtipos P2Y6, P2Y4,1,12,13 y P2Y6,1,12,13 respectivamente. Además, por técnicas de microscopía convencional y de fluorescencia, western blot y ensayos de infectividad, se demostró que MEV o JUNV pueden infectar a los OBCs y modular la expresión de varias proteínas relacionadas con la diferenciación de los osteoblastos. La infección con MEV mostró analogía con datos reportados en la literatura, de biopsias de pacientes con otosclerosis, enfermedad de los huesecillos del oído que causa sordera. Sin embargo, a diferencia de lo observado para el tratamiento con ATP, esta modulación no ocurre a través de la vía PI3K/AKT. La activación de los receptores P2Y1, 12, 13 inhibe la multiplicación o liberación de MEV. En cambio en el caso de los OBCs infectados con JUNV, los tratamientos con nucleótidos no modificaron los títulos virales, sin embargo, generaron péptidos truncados derivados de la nucleoproteína viral característicos de la infección persistente de cultivos celulares por JUNV. Asimismo, se comprobó la capacidad de JUNV de infectar persistentemente a los OBCs. Estos resultados permiten especular acerca de las probables alteraciones inducidas por el virus en la respuesta de los OBCs a estímulos externos y ubica a estos sistemas virus-célula como modelos de estudio de enfermedades óseas de probable etiología viral como la otosclerosis. Los resultados aquí presentados contribuyen a la comprensión de los mecanismos involucrados en la modulación de la proliferación y maduración de osteoblastos por receptores purinérgicos y también como respuesta a la infección viral. / The knowledge of molecular mechanisms involved in the modulation of osteoblasts biological functions, cells responsible for bone formation, is essential as a starting point to design new therapeutic strategies applicable in bone diseases. The adenosine triphosphate (ATP) action on purinergic receptors of plasma membrane has shown to regulate important cellular functions in osteoblasts. The results presented in this thesis describe, for the first time, the ATP effect on the expression of proteins related to bone formation in rat calvarial osteoblasts (OBCs), as well as the modulation of this event when infected with Measles (MEV) and Junin (JUNV) virus. Using different techniques, such as proliferation trials, calcium deposition determination, flow cytometry and realtime quantitative polymerase chain reaction, it was demonstrated that the P2Y2 receptor activation by ATP, stimulates OBCs proliferation as well as differentiation. Moreover, it was observed that these receptors positively modulate those proteins associated with osteoblast differentiation such as bone morphogenetic proteins (BMP-2, 4, 5 and 7), bone sialoprotein, collagen and alkaline phosphatase. It has been demonstrated that the phosphatidyl inositol 3 kinase / protein kinase B (PI3K/AKT) pathway is involved in the proliferation and differentiation of OBCs. The use of different nucleotides also suggests that BMP-1, 3, and 6 gene expression would be regulated by P2Y6, P2Y4, 1, 12, 13 and P2Y6, 1, 12 and 13 subtypes receptors respectively. In addition, by conventional microscopy and fluorescence techniques, western blot and infectivity assays, it was demonstrated that both viruses are able to infect OBCs and modulate several proteins related to these cells differentiation. These results are in accordance with reports dealing with biopsies from patients suffering otosclerosis, a human disease of the ear ossicles which causes hearing impairment. However, by contrast to that observed for the treatment with ATP, viral modulation does not occur via PI3K/AKT. The P2Y1, 12, 13 receptors activation inhibit the MEV release or multiplicity; however in the case of OBCs infected with JUNV, the treatments with different nucleotides did not modify the viral titer but it induced the appearance of truncated peptides derived from the viral nucleoprotein, a salient feature of persistent infection of JUNV in vitro. In addition and accordance to the results shown here, JUNV was able to infect OBCs, persistently. On the other hand, results allow speculation about the viral modulation of OBCs response to external stimuli and the perspectives of viruscell systems as useful models in the study of otosclerosis, a bone disease associated to viral infection. The results presented here contribute to understand the mechanisms involved in the modulation of proliferation and maturation in osteoblasts by purinergic receptors as well as in response to viral infection.

Living organisms rely on many different mechanisms to adapt to changes within their environment. Protein phosphorylation and dephosphorylation events are one such way cells can communicate to generate a response to environmental changes. In the Kennelly laboratory we hope to gain insight on phosphorylation events in the domain Archaea through the study of the acidothermophilic organism Sulfolobus solfataricus. Such findings may provide answers into evolutionary relationships and facilitate an understanding of phosphate transfer via proteins in more elaborate systems where pathway disturbances can lead to disease processes. A λ-phage expression library was generated from S. solfataricus genomic DNA. The immobilized expression products were probed with a purified protein kinase, SsoPK4, and radiolabeled ATP to identify potential native substrates. A protein fragment of the ORF sso0563, the catalytic A-type ATPase subunit A (AtpA), was phosphorylated by SsoPK4. Full length and truncated forms of AtpA were overexpressed in E. coli. Additional subunits of the ATPase were also overexpressed and ATPase activity reconstituted in vitro. Phosphoamino acid analysis and MS identified the phosphorylation sites on AtpA. Several variants of AtpA were derived via site-directed mutagenesis and assayed for ATPase activity. Chemical cross-linking was employed to determine possible ATPase subunit interactions; tryptic digests of AtpA and its mutant variants were performed to examine protein folding. The phosphorylated-mimic variant of AtpA, T98D, resulted in an inactive ATPase complex as determined by ATPase activity assays and native-PAGE indicating potential phosphoregulation by SsoPK4 on enzyme activity. Ultimately, any findings would need verification with in vivo studies. / Ph. D.

Extracellular adenosine-5'-triphosphate (ATP), released from damaged cells or coreleased as a cotransmitter from synaptic vesicles, acts on its plasma membrane receptors termed purinergic. Purinergic P2X receptors are ATP-gated cation channels. To date seven P2X isoforms designated P2X1-7 have been cloned that are organized as trimeric homomers or heteromers. All P2X subunits share a similar structure consisting of a large extracellular loop, two transmembrane domains and intracellular N- and C- termini. An additional structural feature is conserved aminoacids, these include ten conserved cysteine residues in the extracellular loop. All ectodomain cysteines form disulfide bonds which are organized in two areas: three disulfide bridges are localized in the N-termini half and two in the C-termini half at P2X receptor. ATP binding pocket is apparently localized between two neighbouring subunits. The aim of this Diploma Thesis was to examine the relevance of ectodomain cysteine residue and/or disulfide bonds for the expression, function and ATP binding properties of the P2X receptor. All ten, one by one, ectodomain cysteines were substituted by alanines and ATP-induced currents was recorded in HEK293 cells expressing wild-type P2X4 receptor and its mutants. Low responsible or nonfunctional mutants...

Malgré la relocalisation au noyau d'une majorité des gènes du procaryote ancestral à l'origine des mitochondries, une poignée de gènes réside encore dans l'organite après près de deux milliards d'années d'évolution. Les raisons du maintien d'un génome mitochondrial sont mal comprises. Je me suis intéressé à cette problématique via des expériences de relocalisation artificielle de gènes mitochondriaux chez la levure Saccharomyces cerevisiae. Nous avons réussi, pour la première, à exprimer de manière fonctionnelle depuis le noyau le gène ATP9 qui encode une petite protéine hydrophobe essentielle au canal à protons de l'ATP synthase. Majoritairement mitochondrial chez les eucaryotes, comme S.cerevisiae, ce gène est retrouvé dans le génome nucléaire de la majorité des métazoaires, des algues vertes chlorophycées et des champignons filamenteux ascomycètes tel que Podospora anserina. Nos résultats montrent que l'hydrophobicité de la sous-unité Atp9p doit être diminuée pour qu'elle puisse être importée dans la mitochondrie depuis le cytosol. Nous avons également identifié un certain nombre d'autres adaptations pour optimiser l'expression du gène ATP9 relocalisé. Il apparaît donc que si le transfert du gène ATP9 est en principe possible chez la levure, il s'agit d'un processus très complexe. Une telle évolution n'a donc que peu de chances de se produire et d'être maintenue par la sélection naturelle, à moins que le transfert du gène ATP9 au noyau ne confère quelque avantage à l'organisme. Nous avons confirmé cette hypothèse par une étude menée chez P.anserina où nous avons montré que la relocalisation au noyau du gène ATP9, qui s'est produite naturellement au cours de l'évolution, a permis la mise en place de régulations spécifiques permettant d'ajuster les besoins en ATP synthase au cours du cycle de vie de ce champignon. Les résultats de cette étude nous amènent à introduire une nouvelle hypothèse selon laquelle les variations de contenu en gènes des génomes mitochondriaux ne sont pas influencées uniquement par des contraintes au niveau de la structure de leur produits, mais aussi par le mode de vie de l'organisme. / Despite the nuclear relocation of most genes of the ancestral procaryotic genome which gave birth to mitochondria, a small set of genes still remains into the organite after 2 billions years of evolution. The reasons for this maintenance of mitochondrial genome are currently not clear. I studied these questions by experimenting artificial relocations of mitochondrial genes in the yeast Saccharomyces cerevisiae. We managed, for the first, to functionally express the ATP9 gene from the nucleus, which encodes a small hydrophobic essential subunit of the proton chanel of the ATP synthase. Mostly mitochondrial within eukaryotes like S.cerevisiae, this gene can be found in the nuclear genome in most metazoans, chlorophyceans green algae and ascomycota filamentous fungi like Podospora anserina. Our results show that the hydrophobicity of the Atp9p subunit has to be decreased to be imported into the mitochondria from the cytosol. We also identified some adaptations optimizing the expression of the relocated ATP9 gene. It seems that if the ATP9 gene relocation is possible within the yeast, yet it is a complex and difficult process. Such an evolution has only few chances to occur and to be maintained by natural selection, unless it could confer some advantage to the organism. We have confirmed this hypothesis in a study made on P.anserina, in which we showed that the natural ATP9 relocation to the nucleus that appeared during its evolution allowed the setting up of specific regulations modulating the ATP synthase needs during the life-cycle of this fungus. The results presented here lead us to introduce a new hypothesis postulating that the variations of the set of genes contained in the mitochondrial genome are influenced not only by the constraints generated by their products structure, but also by the lifestyle of the organism.