A fibrose hepática é uma patologia que acompanha outras doenças crônicas do fígado como a cirrose e o hepatocarcinoma. As células estreladas hepáticas (HSC, do inglês hepatic stellate cells) compõem uma população celular heterogênea que se caracteriza por transitar entre dois fenótipos. As células com fenótipo quiescente possuem a capacidade de armazenar vitamina A em gotas lipídicas. Os insultos ao fígado desencadeiam uma resposta inflamatória que gera estímulos parácrinos e autócrinos mediados por citocinas e espécies reativas. Neste contexto, as HSC assumem um fenótipo ativado fibrogênico e tornam-se responsáveis pela cicatrização hepática. Danos crônicos ao fígado levam a uma deposição de matriz extracelular exagerada que configura o estado patológico da fibrose. O resveratrol (RSV – 3,4’,5-tri-hidroxi-trans-estilbeno) é uma fitoalexina produzida por algumas espécies de plantas. Inúmeros efeitos benéficos à saúde são atribuídos ao RSV por causa do seu potencial antioxidante, antiinflamatório e pró-apoptótico. Estudos anteriores mostraram que tratamento da GRX, uma linhagem murina de HSC ativadas, com concentrações de RSV próximas as biodisponíveis (0,1 a 1 μM) resultou em parada do ciclo na fase S com consequente inibição de proliferação celular, um efeito associado à citotoxicidade e que pode favorecer a resolução da fibrose hepática. Neste estudo, por técnicas espectrofotométricas, foi demonstrado que tratamento da GRX por 24 horas com concentrações entre 0,1 a 50 μM de RSV promoveu um efeito pró-oxidante que causa uma citotoxicidade dependente da dose, bastante aumentada no grupo tratado com a concentração mais alta. Os efeitos citotóxicos atenuados encontrados nas células tratadas por 120 horas sugerem que a GRX pode se tornar resistente a estes efeitos. O potencial pró-oxidante do RSV foi o ponto de partida para investigar a possibilidade de que esta fitoalexina provocasse uma alteração no metabolismo mitocondrial da GRX. Para isso, os efeitos do RSV (1 a 50 μM) na função mitocondrial, na indução de morte mediada por estas organelas e na autofagia/mitofagia foram investigados por técnicas de espectrofotometria, de imunocitoquímica, de citometria de fluxo, de microscopia confocal e de microscopia eletrônica de transmissão em GRX tratadas por 24 e 120 horas. Foi demonstrado que todas as concentrações de RSV promovem apoptose por meio da ativação de caspases, alteram a dinâmica/função mitocondrial e induzem o aumento de autofagia/mitofagia na GRX. No entanto, o RSV provocou biogênese de mitocôndrias nos grupos tratados com 1 e 10 μM, enquanto que o tratamento com 50 μM causou dano celular evidente na GRX, sem induzir biogênese de mitocôndrias. Desta forma, é possível que a citotoxicidade “dose-dependente” do RSV, que causa a morte celular e dano oxidativo em 24 horas de tratamento, esteja relacionada com o desequilíbrio entre a indução concomitante de apoptose mediada por dano mitocondrial, autofagia/mitofagia e biogênese de mitocôndrias. Por fim, foi investigada a liberação de TNF-α, Interleucina-6 e Interleucina-10 pela GRX tratada por 24 e 120 horas com RSV (0,1 a 50 μM), considerando o papel antiinflamatório do RSV e o papel das HSC ativadas na sinalização autócrina que contribui para a modulação fenotípica destas células. Foi demonstrado que o tratamento da GRX com RSV por 24 e 120 horas induziu a redução da liberação de Interleucina-6; enquanto que a liberação de TNF-α e Interleucina-10 foi aumentada. Estes resultados confirmam um efeito antiinflamatório do RSV que deve contribuir na prevenção da ativação ou da perpetuação do estado ativado das HSC por meio de sinalização autócrina. Ainda que a concentração do RSV seja importante para efetivamente induzir a morte das HSC ativadas, o tratamento com esta fitoalexina pode ser promissor para a resolução da fibrose hepática por diminuir a população de células ativadas e, possivelmente, prevenir a perpetuação do estado fenotípico ativado. Estudos avaliando indicadores de quiescência em células tratadas são ainda necessários para desvendar completamente os efeitos do RSV quanto às possibilidades de inibição da perpetuação ou reversão fenotípica das HSC ativadas. / Liver fibrosis is a disease that accompanies other hepatic chronic diseases such as cirrhosis and hepatocellular carcinoma. Hepatic stellate cells (HSC) are a heterogeneous cell population characterized by transiting between two phenotypes. Cells with a quiescent phenotype are able to store vitamin A into lipid droplets. Damage to the liver trigger an inflammatory response that generates paracrine and autocrine stimulation mediated by cytokines and reactive species. In this context, HSC assume an activated and fibrogenic phenotype responsive for hepatic wound-healing. Chronic insults to the liver lead to an excessive deposition of extracellular matrix that configures the pathological state of fibrosis. Resveratrol (RSV – 3,4’,5-tri-hidroxi-trans-stilbeno) is a phytoalexin produced by some species of plants. Several beneficial effects are attributed to this molecule due to its antioxidant, antiproliferative and pro-apoptotic potential. Previous studies showed that treatment with bioavailable concentrations of RSV (0.1 to 1 μM) promoted an arrest cycle at the S phase in GRX, a murine activated HSC model, leading to cell proliferation inhibition, a cytotoxic effect that contributes to the liver fibrosis resolution. In this study, it was shown by spectrophotometric techniques that GRX treatment for 24 hours at concentrations between 0.1 to 50 μM of RSV promoted a fairly clear pro-oxidant effect that causes a dose-dependent cytotoxicity that was higher in the group treated with 50 μM. The attenuated cytotoxicity found after 120 hours of GRX treatment suggest that these cells became resistant to this effect. The pro-oxidant potential of RSV was the starting point for investigating the possibility that this phytoalexin would cause a change in the GRX mitochondrial metabolism. Thus, the effects of RSV (1 to 50 μM) on altering the mitochondrial function, on inducing mitochondrial-mediated cell death, and autophagy/mitofagia were investigated in GRX treated for 24 and 120 hours by spectrophotometric techniques, immunocytochemistry, flow cytometry, confocal microscopy, and transmission electron microscopy. All the RSV concentrations promote cell apoptosis through caspases activation, alter the mitochondrial dynamics and function, and induce an increase of autophagy/mitofagia. Curiously, only 1 and 10 μM of RSV induced mitochondrial biogenesis in GRX, while the highest concentration caused an evident cell damage without inducing mitochondrial biogenesis. Thus, it is possible that the "dose-dependent" cytotoxicity of RSV, which causes cell death and oxidative damage in 24 hours of treatment, is related to an imbalance between the concomitant induction of mitochondrial-mediated apoptosis, autophagy/mitofagia, and mitochondrial biogenesis. Finally, it was investigated the release of TNF-α, Interleukin-6 and Interleukin-10 by GRX treated for 24 and 120 hours with RSV (0.1 to 50 μM), considering the anti-inflammatory role of RSV and the autocrine signalling role of HSC that contributes to the perpetuation of its activated phenotype. It was demonstrated that GRX treatment with RSV for 24 and 120 hours reduced the release of Interleukin-6 in the culture medium; whereas the release of TNF-α and Interleukin-10 was increased. These results confirm the anti-inflammatory properties of RSV and may contribute to the prevention of HSC activation through autocrine signalling. Although RSV concentration is important to effectively induce activated HSC death, cells treatment with this phytoalexin may be promising for liver fibrosis resolution through decreasing the population of activated cells or through preventing the perpetuation of activated state of HSC. Future studies evaluating the quiescence indicators of GRX under RSV treatment are still needed to fully unravel the effects of this phytoalexin on inhibiting the perpetuation of activated HSC or reversing its activated phenotype.

The present thesis focuses on the elucidation of human mitochondrial inner membrane presequence-translocation machinery with implications on cancer cell proliferation. Mitochondria are the endosymbiotic organelles in an eukaryotic cell performing a vast repertoire of functions and require approximately 1500 proteins. However, the mitochondria genome contains only 13 protein-coding genes primarily transcribing the complexes of the electron transport chain. Therefore, it is evident that most of the mitochondrial proteome is encoded by the nucleus and synthesized on cytosolic ribosomes. Chapter 1: Mechanism of mitochondrial inner membrane protein translocation and its oncogenic connection. Mitochondria consist of different routes of directing proteins to their intramitochondrial destinations. The presequence pathway, mediated by the inner membrane TIM23 complex, is responsible for the import of matrix and a number of single transmembrane helixes containing inner membrane proteins. This pathway accounts for approximately 60% of the total proteome imported into the organelle and hence, is the major focus of discussion in the present study. The components of the TIM23 complex can be subdivided into two groups, the protein conducting channel and the import motor. The initial translocation across the TIM23 channel utilizes the electrochemical membrane potential that exists across the inner membrane whereas the final step of the translocation process is driven by energy from ATP hydrolysis. MtHsp70 forms the central component of the import motor, and its function is regulated by the J-proteins. Pam18 stimulates the ATPase activity of mtHsp70. Pam16, on the other hand, forms a subcomplex with Pam18 and exerts an inhibitory effect its ATPase stimulatory activity, in turn regulating the activity of the import motor. The stoichiometric coupling with the substrate binding-release cycle of mtHsp70 drives the import process. Although the organization of presequence translocation machinery and its functional annotations have been described in detail in yeast system, little information is available on its organization in human. It is difficult to contemplate the existence of similar machinery in human mitochondria with complex and diversified functions. Human mitochondria apart from regulating the metabolic pathways are involved in progression of cancer, neurodegenerative disorders, responses to xenobiotic stress and induction of apoptosis. Numerous reports have shown that mutations and overexpression of human orthologs of translocase components are associated with various cancer subtypes. Such disease condition also involves targeting of specific cell signaling molecules that reprogram organellar functions and alter the cellular phenotype. Based on this evidence we defined our study into four broad objectives – 1) identify the components of human presequence translocase as Chapter two and three, 2) characterize the subunit organization of human presequence translocation machinery in Chapter four, 3) determine the functional connection between the translocase components and the cancer phenotype in Chapter four and five and 4) understand how the functions of J-proteins have evolved across the species as Chapter six. Chapter 2: Unraveling the role of Magmas in human mitochondrial protein transport. Pam16 plays a critical role in regulation of import process by governing the activity of the import motor. Proteins orthologous to Pam16 had been reported earlier to be overexpressed in various metabolically active tissues and cancer subtypes. We found that in humans a protein named as Mitochondria Associated Granulocyte Macrophage colony Stimulating factor signaling molecule (Magmas) showed significant sequence similarity with yeast Pam16 at its C-terminal region. Magmas was initially discovered as a protein that was overexpressed in neoplastic prostrate and when the cells were exposed to GM-CSF. Our experiments suggested that Magmas localized in human and yeast mitochondria and it was associated with the inner mitochondrial membrane. Magmas could complement the growth of yeast cells that were deleted for the essential gene PAM16 and could import precursor proteins into the mitochondria. Like Pam16, Magmas was able to form a stable heterodimeric subcomplex with yeast Pam18 and human Pam18 ortholog DnaJC19 (JC19). We found that J-domain forms the minimal region required for heterodimer formation between Magmas and Pam18/JC19. Mutations in Magmas J-like domain resulted in temperature sensitive growth phenotypes in yeast cells and associated import defect in translocating precursor proteins into the organelle due to inability to form a stable subcomplex with Pam18 and JC19, resulting in loss of import function. Loss of subcomplex formation leads to dissociation of Pam18 from the translocation machinery highlighting the importance of Magmas in tethering Pam18/JC19 to the presequence translocase. Magmas, showing characteristic of a J-like protein, was unable to stimulate the ATPase activity of mtHsp70. However, it exerted an inhibitory effect on the ATP stimulatory effect of the J-protein Pam18/JC19, indicating that Magmas has a regulatory effect on the overall activity of import motor. In contrast Magmas mutants those are incapable of forming a stable heterodimer with Pam18 were unable to regulate the activity of Pam18 resulting in import defects. In summary, our results highlight that Magmas is an ortholog of yeast Pam16 performing similar functions at the import channel. Chapter 3: Existence of two J-protein subcomplexes at the translocation channel with distinct physiological functions. JC19 has been regarded as the human ortholog of Pam18 whose loss of function was associated with dilated cardiomyopathy and ataxia syndrome. However, immunoprecipitation analysis using anti-Magmas antibody revealed the presence of a second J-protein identified as DnaJC15 (JC15) that shared a highly similar J-domain with JC19. JC15 was initially identified as a protein whose loss in expression resulted in development of a chemoresistant phenotype in ovarian carcinoma cells exposed to chemotherapeutic treatment. We found that JC15 localizes in mitochondria where it was associated with the inner membrane. Similar to Pam18 and JC19, JC15 heterodimerized with Magmas/Pam16 through its J-domain and associated with the presequence translocase of the inner membrane. A loss of function mutation at the J-domain of JC15 destabilizes its interaction with Magmas resulting in protein translocation defects and temperature-sensitive growth phenotype in yeast cells. The JC15 mutant showed inability to get associated with the translocation channel and had dysregulated stimulation of mtHsp70 activity leading to decreased mitochondria biogenesis and loss of mitochondrial membrane potential. In summary, our results showed that JC15 is the second human ortholog of Pam18 with similar functions. In contrast to yeast, in human mitochondria JC15 and JC19 were found to form two separate and distinct J-protein subcomplexes with Magmas at the mitochondrial import motor. The essentiality of the J-proteins for normal human mitochondria function was addressed through siRNA mediated downregulation of Magmas, JC19 and JC15. We found that Magmas and JC19 are essential for normal mitochondrial function and cell viability whereas JC15 is dispensable and might have a supportive role. Interestingly, both JC19 and JC15 interacted with Magmas with equal affinity and stimulated mtHsp70’s ATPase activity by equivalent levels. This shows that both JC19 and JC15 share similar properties in terms of their functions at the import channel, and the differences might be in a much broader perspective in terms of their association with the translocation channel. Chapter 4: Architecture of human mitochondrial inner membrane presequence -translocation machinery. In yeast, there exists a single J-protein subcomplex formed by Pam16 and Pam18, which is recruited to the sole translocase. However, humans present a completely different scenario where there exists a two distinct subcomplexes formed by Magmas with either of the J-proteins. So the question arises how the individual subcomplexes is recruited to the translocation machinery; whether they are associated to one or differentially recruited to two different translocases. We identified the existence of three distinct translocases in the human system constituted by the two J-proteins along with the Tim17 paralogs. JC15 along with Tim17a forms the translocase A of size similar to that of the yeast system, and it forms the ancestral translocase in the humans. Tim17b isoforms, on the other hand, associates with JC19 to form mammalian specific translocases B1 and B2. The association of the J-proteins at the translocation channel was found to be mediated by Magmas as a subcomplex. Downregulation of Magmas resulted in dissociation of both the J-proteins, and its overexpression resulted in redistribution of J-proteins at the translocases. We found that translocase B imported precursor proteins at a comparatively higher rate as compared to translocase A. Disruption of translocase B had deleterious effects on cell viability, respiratory chain complex's activities, Fe-S cluster biogenesis, mitochondria morphology, regulation of free radical levels and maintenance of mitochondrial genome. In contrast, depletion of translocase A did not significantly alter the survivability of cells, mitochondrial activity and maintenance of organellar morphology. This shows that translocase B is essential and performs the constitutive import function in the mammalian system whereas translocase A is dispensable and might have a supportive role in maintenance of mitochondrial function. However, translocase A play a specific role in human mitochondria in context to cancer cells. We observed that the elevated level of Tim17a found in cancer cells is responsible for maintenance of higher mitochondrial DNA copy number and higher proliferative potential of cancer cells. Additionally, translocase A also plays a specific role in translocation of cell signaling proteins that lack a mitochondrial targeting sequence into the mitochondria, highlighting the possible role of this translocase in neoplastic transformation. Chapter 5: Mechanistic insights into the role of JC15 as a part of translocase A in chemoresistant phenotype. JC15 had been initially identified to be associated with development of chemoresistance in cancer cells. However, the molecular mechanism followed by the protein has not been elucidated yet. Our studies have shown that overexpression of JC15 leads to increased sensitivity of cells to chemotherapeutic drug cisplatin and are coupled with complete loss of membrane potential, mitochondrial swelling and cytochrome c release. However, this chemosensitive phenotype was partially ameliorated upon preexposing the cell to cyclosporine A which is an inhibitor of cyclophilin D, a critical component of mitochondrial membrane transition pore (MPTP) complex. A similar reversal of phenotype was observed upon depleting cyclophilin D even under JC15 overexpressing background. This highlighted a possible functional connection between these two proteins. In order to check this hypothesis other way around, we overexpressed cyclophilin D in the cells which resulted in constitutive opening of the MPTP complex, enhanced mitochondrial swelling and reduced cell viability. In contrast, the gain of function anomalies of cyclophilin D overexpression was significantly reversed upon JC15 depletion. We observed through co-immunoprecipitation analysis that JC15 activates cyclophilin D by releasing it from the inhibitory effects of TRAP1 and couples it to the MPTP complex. Additionally, we have also shown that the J-domain of JC15 is critical for its interaction with cyclophilin D and loss of function mutation at the J-domain of JC15 disrupts its interaction with cyclophilin D. As a result the JC15 mutant is not able to mount a chemosensitive response to cisplatin drug. Chapter 6: Identification of regions determining the divergence of J-proteins functions at the mitochondrial import motor. The above studies show ample evidence to suggest that the two human J-proteins have undergone significant divergence in their function in human mitochondria in spite of having a highly similar J-domain. Therefore, we asked the question that how the human J-proteins have evolved and diversified from the primitive yeast protein Pam18 and what are the regional determinants in the protein sequence that dictate the function of the J-domain. We utilized a purely genetic approach to address the problem. We observed that JC19 was unable to rescue the growth of yeast cells deleted for the essential gene Pam18 and JC15 expression resulted in cold sensitive phenotype. We used JC15 as the model protein for our assays and applied three methodologies. First, generation and isolation of a series of mutations in JC15 that could rescue the cold sensitive phenotype, and the growth of the cells were similar to the wild type. Second, to identify the regulatory residues by isolation of second site suppressors that could be the suppressor the mutant phenotypes isolated earlier. Third, we utilized a purely evolutionary approach by swapping the individual domains between the three J-proteins- Pam18, JC19 and JC15. Our genetic data support the idea that the partial loss of function of human J-protein in the yeast system is due to altered subcomplex dynamics with Pam16. The altered dynamics of the subcomplex is mainly regulated by the residues in the arm, linker and helical regions of the J-domain, especially the helix II regions. Our analysis has also uncovered a critical role of the targeting (T) region of J-proteins which along with inter-membrane space (IMS) domain share significant sequence diversity among J-proteins in yeast and humans. The T-region in conjunction with the IMS domain plays a crucial role in regulating the J-domain’s function across the kingdoms and within the species. Although, our genetic data needs to be supplemented with biochemical evidence, this study provides significant insights into the diversity of J-protein function across the species and mode of their regulation through regions flanking the J-domain.

Mitochondrion is an endosymbiotic organelle synthesizing ~1% of its proteome, while remaining ~99% of the proteins are encoded by the nuclear genome and translated on the cytosolic ribosome. Therefore active mitochondrial biogenesis requires efficient protein transport destined for the different sub-compartments. Mitochondrion contains specialized translocation machineries in the outer and in the inner membrane known as TOM40 and TIM23-complex respectively. Import of a majority of mitochondrial proteome is mediated by inner membrane presequence translocase (TIM23 complex). However, the structural organization of Tim23-complex and mechanisms of mitochondrial inner membrane protein translocation is still elusive. Therefore, the present thesis addresses above elusive questions. Chapter 2 highlights the functional significance of different segments of Tim23 in regulating the conformational dynamics of the presequence-translocase- Tim23 is the central channel forming subunit of the presequence-translocase which recruits additional components for the assembly of the core complex. However the functional significance of different segments of Tim23 was not understood due to the lack of suitable conditional mutants. Our study has reported many conditional mutants from different segments of Tim23 which are precisely defective in the organization of the core complex and in the recruitment of the import motor component which enhances our understanding of protein translocation across mitochondrial inner membrane. Chapter 3 highlights the functional cooperativity among mtHsp70 paralogs and orthologs using Saccharomyces cerevisiae as a model organism- mtHsp70s are implicated in a broad spectrum of functions inside the mitochondria. In case of lower eukaryotes gene duplication event has given rise to multiple copies of Hsp70s thereby presenting an opportunity of division of function among these paralogs. The mitochondria of yeast Saccharomyces cerevisiae contains three Hsp70s, including Ssc1, Ssq1 and Ssc3 (Ecm10). The Ssc1 is essential for protein translocation and de novo protein folding functions while Ssq1 is needed for the Fe/S cluster biogenesis inside the mitochondria. Although it has been proposed earlier that, Ssc1 and Ssc3 possesses overlapping functions in protein translocation as a part of import motor in the Tim23-complex. However the physiological relevance and experimental evidences in favor above hypothesis was not established clearly. Our study has reported Ssc3 as an ‘atypical chaperone’ which cannot perform the generalized chaperone functions due to the conformational plasticity associated with both the domains of Ssc3 resulting into weaker client protein affinity, altered interaction with cochaperones and dysfunctional allosteric interface. Additionally, we have also highlighted the role of Nucleotide-binding domain in determining the functional specificity among Hsp70 paralogs and orthologs.

Chez les eucaryotes, l’information génétique est transcrite en ARN messager qui subit plusieurs étapes de maturation et évènements d’assemblage avant d’être exporté hors du noyau. Ces modifications du transcrit sont effectuées par de nombreux facteurs protéiques recrutés au transcrit naissant, formant ainsi une particule ribonucléoprotéique (mRNP). La biogenèse du mRNP est étroitement liée avec la transcription et le contrôle qualité afin d’assurer l’efficacité et l’exactitude de la production de mRNPs matures. Des études récentes suggèrent que les membres du complexe THO-Sub2 pourraient être des facteurs cruciaux dans le couplage de la transcription, de la biogénèse du mRNP et de l’export. Dans notre groupe, nous avons mis en oeuvre un essai novateur pour étudier la biogénèse du mRNP et le contrôle qualité, basé sur l’expression du facteur Rho bactérien dans Saccharomyces cerevisiae. Rho interfère avec l’assemblage adéquat du mRNP et génère des transcrits aberrants qui sont dégradés par la machinerie de dégradation nucléaire. Dans cette étude, nous avons utilisé le système expérimental Rho pour mieux comprendre Rrp6 et l’implication de l’exosome dans la dégradation des transcrits liée au contrôle qualité, ainsi que pour mieux caractériser le rôle et la fonction du complexe THO-Sub2 dans le processus de biogénèse du mRNP. Les résultats obtenus révèlent une différence intéressante dans le comportement des membres du complexe THO sous l’action de Rho et dévoilent leur dépendance à la liaison à l’ARN, ce qui n’aurait pas pu être observé avec d’autres techniques expérimentales. Cela confirme le potentiel attendu du système expérimental basé sur Rho dans l’étude des facteurs protéiques impliqués dans la biogénèse et le contrôle qualité du mRNP. / In eukaryotes, the genetic information is transcribed into messenger RNA which undergoes various processing and assembly events prior to its export from the nucleus. These transcript modifications are performed by numerous protein factors recruited to the nascent transcript, thus making a messenger ribonucleoprotein particle (mRNP). mRNP biogenesis is tightly interconnected with both transcription and quality control to ensure efficiency and accuracy in production of mature mRNPs. Recent findings suggest that members of THO-Sub2 complex might be crucial factors in coupling transcription, mRNP biogenesis and export. In our group, we have implemented an innovative assay to study mRNP biogenesis and quality control, based on the expression of the bacterial factor Rho in Saccharomyces cerevisiae. Rho interferes with proper mRNP assembly and generates aberrant transcripts degraded by the nuclear degradation machinery. In this study, we use Rho experimental system to expand our findings on Rrp6 and exosome involvement in quality control degradation of transcripts, as well as to better characterize the role and function of THO-Sub2 complex in the process of mRNP biogenesis. Obtained results reveal an interesting difference in behavior of THO complex members upon Rho action and disclose their dependence on binding to the RNA, which could not be observed by other experimental techniques. This substantiates the expected potential of Rho-based experimental system in the study of protein factors involved in mRNP biogenesis and quality control.

Eukaryotic ribosome synthesis is a complex process that consumes a lot of energy and involves several hundreds of trans-acting factors that transiently associate with nascent ribosomes. Biogenesis of ribosomal subunits (the small 40S and the large 60S) starts with transcription of a long precursor ribosomal RNA (pre-rRNA) by RNA polymerase I (Pol I) in the nucleolus. This is a key step that globally controls yeast ribosome synthesis. The pre-rRNA, ‘the 35S transcript’, encodes the mature sequence (18S, 5.8S, and 25S) rRNA constituents of both the 40S and 60S subunits. The 35S transcript is subsequently modified, cleaved (processed) and assembled with numerous structural ribosomal proteins and ribosome synthesis factors (trans-acting factors) to form various ribosomal particles (pre-ribosomes, precursors to the 40S and 60S subunits) along ribosome assembly pathway. <p>In the budding yeast Saccharomyces cerevisiae, it has been reported recently that the processing of the 35S nascent transcript and the assembly of pre-ribosomes occur concomitantly with Pol I transcription in the nucleolus. In this process, the growing Pol I transcript gradually assembles with pre-40S structural ribosomal proteins and ribosomal synthesis factors to form the so-called ‘SSU-processome’ or ‘90S pre-ribosome’, the earliest precursor of the 40S subunit. The SSU-processome/90S pre-ribosome localizes to the nucleolus and consists of the 35S pre-rRNA, the U3 small nucleolar (sno) RNA, about a dozen of 40S ribosomal proteins and more than forty ribosome synthesis factors. The U3 snoRNA and pre-40S ribosome synthesis factors are all implicated in the processing of the 35S precursor (at sites A0, A1 and A2) and therefore in the synthesis of the 18S rRNA component of the 40S subunit. Significantly, the association of the U3 snoRNA with the growing 35S transcript is important for pre-40S assembly, whereas its dissociation from the processed transcript (following cleavage at sites A0-A2) is crucial for the overall structural remodeling of the 18S rRNA and for the formation of pre-40S ribosomes from the earliest precursor 90S particles. <p>This thesis mostly addresses the identification and functional characterization of Esf2 and Bfr2, two novel 40S synthesis factors, components of the SSU-processome/90S pre-ribosome in yeast. Both proteins localize to the nucleolus and their genetic depletions lead to failure in the production of 40S subunits. In the absence of either factor, the 35S pre-rRNA is not processed at sites A0-A2 and the 18S rRNA is not synthesized. Also, pre-ribosome assembly is affected and pre-40S ribosomes fail to mature properly. Strikingly, in the absence of either factor, the U3 snoRNA remains associated with unprocessed 35S transcript within pre-ribosomes indicating that Esf2 and Bfr2 are required to dissociate U3 from pre-ribosomes. This process also involves RNP (ribonucleoprotein particle) unwinding activities of the putative RNA helicase Dbp8. <p>La biogenèse du ribosome eucaryote est un processus complexe qui consomme beaucoup d’énergie et implique plusieurs centaines de facteurs trans qui s’associent de manière transitoire avec les pré-ribosomes en cours de formation. La biogenèse des sous-unités ribosomiques (la petite sous-unité 40S et la grande sous-unité 60S) débute dans le nucléole par la synthèse d’un long précurseur d’ARN ribosomique (le pré-ARNr, dit 35S chez la levure Saccharomyces cerevisiae) par l’ARN Polymérase I (Pol I). Ceci constitue une étape clé dans le contrôle global de la synthèse du ribosome chez la levure. Le pré-ARNr 35S renferme les séquences des ARNr matures 18S (ARNr de la sous-unité 40S) et 5.8S et 25S (deux des trois ARNr de la sous-unité 60S). Le pré-ARNr 35S subit un long processus de maturation et d’assemblage au cours duquel il est modifié, clivé (on parle du « processing » du pré-ARNr) et s’assemble avec des protéines ribosomiques (« RP », composants structuraux des sous-unités ribosomiques matures) et de nombreux facteurs de synthèse (facteurs trans) pour former différentes particules pré-ribosomiques (précurseurs des sous-unités 40S et 60S).<p><p>Chez la levure S. cerevisiae, il a récemment été montré que le processing du pré-ARNr 35S et l’assemblage des pré-ribosomes se produisent de manière concomminante avec la transcription Pol I dans le nucléole. Ainsi, le transcrit Pol I en cours de synthèse s’assemble progressivement avec des facteurs de synthèse ainsi que des RP pour former le « SSU processome » ou « pré-ribosome 90S », tout premier précurseur de la petite sous-unité 40S. Le SSU processome/pré-ribosome 90S est localisé dans le nucléole et est consisté du pré-ARNr 35S naissant, du petit ARN nucléolaire (snoRNA) U3, d’une douzaine de RP de la petite sous-unité 40S et de plus de 40 facteurs de synthèse. Le snoRNA U3 et ces facteurs de synthèse sont tous impliqués dans les clivages du pré-ARNr 35S aux sites A0, A1 et A2, et donc dans la biogenèse de l’ARNr 18S. L’association du snoRNA U3 avec le pré-ARNr 35S naissant est importante pour l’assemblage du SSU processome/pré-ribosome 90S. Par ailleurs, sa dissociation après les clivages aux sites A0-A2 permet un remodelage structural général de l’ARNr 18S et la formation du « pré-ribosome 40S » à partir de la particule précoce 90S.<p><p>Au cours de cette thèse, nous avons identifié et caractérisé fonctionnelement chez la levure deux nouveaux facteurs de synthèse de la petite sous-unité 40S et composants du SSU processome/pré-ribosome 90S: Esf2 et Bfr2. Ces deux protéines sont localisées dans le nucléole. Leur déplétion entraîne une incapacité à produire la sous-unité ribosomique 40S. En l’absence d’Esf2 ou Bfr2, le pré-ARNr 35S n’est plus clivé aux sites A0-A2 et l’ARNr 18S mature n’est plus produit. L’assemblage des pré-ribosomes est aussi affecté, notamment la formation du pré-ribosome 40S. De manière importante, en l’absence de l’un ou l’autre de ces facteurs, le snoRNA U3 reste associé au pré-ARNr 35S non clivé au sein des pré-ribosomes, indiquant qu’Esf2 et Bfr2 sont requises pour la dissociation d’U3 des pré-ribosomes. Ce processus implique aussi Dbp8, une hélicase à ARN présumée.<p> / Doctorat en sciences, Spécialisation biologie moléculaire / info:eu-repo/semantics/nonPublished

Bien que les statines forment la classe d'hypolipidémiants la plus utilisée, une toxicité musculaire a été reportée, pouvant ainsi provoquer l’apparition d’une myopathie. Dans la première partie, nous avons montré chez l’Homme et l’animal que les statines inhibent directement la chaine respiratoire mitochondriale, et induisent la production de radicaux libres dérivés de l’oxygène (RLO), qui active les voies apoptotiques dans les muscles glycolytiques, alors que les muscles oxydatifs ne sont pas atteints. Nous avons ensuite montré in vitro que le stress réducteur peut engendrer une oxydation mitochondriale, pouvant conduire à une activation de la voie de biogenèse mitochondriale. De plus l’augmentation du contenu mitochondrial induite a permis de protéger les cellules contre l’apoptose induite par les statines. Enfin, nous avons montré in vivo que l’induction des voies de biogenèse mitochondriale est nécessaire à la tolérance des statines dans les muscles oxydatifs. En conclusion, le phénotype mitochondrial, tant au niveau quantitatif que qualitatif, semble être un facteur clé dans l’apparition de la myopathie aux statines. / Although statins are the most prescribed class of lipid-lowering agents, adverse muscular toxicity has been reported, which can lead to the appearance of a myopathy. In the first part, we showed in Humans and animals that statins inhibit directly the mitochondrial respiratory chain, and induce the production of reactive oxygen species (ROS), that trigger apoptotic pathways in glycolytic skeletal muscles, whereas oxidative muscles are not impaired. We then showed in vitro that reductive stress can provoke mitochondrial oxidation, that could lead to an activation of mitochondrial biogenesis pathways. Moreover, the consequent increase in mitochondrial content enabled to protect cells against statin-induced apoptosis. Finally, we showed in vivo that the induction of mitochondrial biogenesis is necessary for statin tolerance in oxidative skeletal muscles. In conclusion, mitochondrial phenotype, both quantitatively and qualitatively, seems to be a key factor in the appearance of statin myopathy.

Abstract Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) and AMP-activated protein kinase (AMPK) are major factors regulating energy homeostasis. In this study, we aimed to investigate how energy flux affects several hepatic functions mediated by these factors. We define a novel role of PGC-1α and AMPK as modulators of the immune system in the liver. We show that PGC-1α is involved in the regulation of a cluster of genes related to the immune system, most importantly Interleukin 1 receptor antagonist (IL1Rn). Since PGC-1α is responsive to energetic stress associated with fasting or physical exercise, the same stimuli promote IL1Rn in hepatocytes. We identify AMPK as an independent inducer of IL1Rn and hypothesise that it could account for the anti-inflammatory effect of the antidiabetic drug metformin. We also demonstrate that metformin reduces expression of Sirtuin 3 (SIRT3) in hepatocytes and promotes acetylation of mitochondrial protein. We suggest that this mechanism, in spite of increased mitochondrial biogenesis, contributes to reduced ATP synthesis in metformin-treated samples. In addition, we demonstrate that Pregnane X receptor (PXR) is induced in the liver during fasting and by PGC-1α in hepatocytes. Furthermore, we describe a negative regulatory mechanism involving SIRT1, activated by pyruvate and interfering with PXR signaling. We show that SIRT1 attenuates PGC-1α-mediated co-activation of PXR and its target genes, i.e. Cyp3a11, with possible implications for drug and xenobiotic metabolism. In conclusion, we demonstrate how energetic stress affects various hepatic functions mediated by PGC-1α and AMPK. Moreover, we describe SIRT1 and metformin as factors capable of modulating this response. / Tiivistelmä Peroksisomiproliferaattori-aktivoituvan reseptori gamman koaktivaattori 1α (PGC-1α) ja AMP:n aktivoima proteiinikinaasi (AMPK) ovat keskeisiä energiametabolian säätelijöitä. Tässä tutkimuksessa oli tavoitteena selvittää kuinka energiataso vaikuttaa useisiin, näiden tekijöiden säätelemiin maksan toimintoihin. Osoitamme että PGC-1α ja AMPK tekijöillä on ennestään tuntematon merkitys immuunijärjestelmän säätelyssä maksassa. Näytämme myös, että PGC-1α säätelee joukkoa geenejä, joiden tehtävä liittyy immuunijärjestelmään, tärkeimpänä Interleukiini 1 reseptori antagonistia (IL1Rn). Paastoon ja fyysiseen aktiivisuuteen liittyvä energiastressi aktivoi PGC-1α:aa ja näiden samojen stimuluksien havaittiin lisäävän myös IL1Rn tasoa hepatosyyteissä. Havaitsimme AMPK:n olevan itsenäinen IL1Rn indusori ja hypoteesimme mukaan tämä voi välittää diabeteslääkkeenä käytettävän metformiinin anti-inflammatorisia vaikutuksia. Osoitamme myös, että metformiini alentaa Sirtuiini (SIRT) 3:n ekspressiota maksasoluissa ja lisää mitokondriaalisten proteiinien asetylaatiota. Uskomme tämän mekanismin, huolimatta lisääntyneestä mitokondrioiden biogeneesistä, myötävaikuttavan vähentyneeseen ATP synteesiin metformiinikäsitellyissä näytteissä. Lisäksi osoitamme, että paasto ja PGC-1α indusoivat Pregnaani X reseptorin (PXR) ilmentymistä maksasoluissa. Kuvaamme myös PXR signalointiin vaikuttavan ja pyruvaatin aktivoiman, SIRT1:n välitteisen, negatiivisen säätelymekanismin. SIRT1 estää PGC-1α välitteistä PXR koaktivaatiota ja kohdegeenien, kuten Cyp3a11, aktivaatiota, millä voidaan olettaa olevan merkitystä lääkeaineiden ja vierasaineiden metaboliaan. Yhteenvetona osoitamme, että energiastressi PGC-1α:n ja AMPK:n välittämänä vaikuttaa useisiin maksan toimintoihin. Lisäksi näytämme, että SIRT1 ja metformiini voivat moduloida näitä vaikutuksia.

CSIR / Translation initiation is a rate limiting step during protein biosynthesis. Initiation occurs by formation of an initiation complex comprising 30S subunit of ribosome, mRNA, initiator tRNA, and initiation factors. The initiator tRNA has a specialized function of binding to ribosomal P site whereas all the other tRNAs are selected in the ribosomal A site. The presence of a highly conserved 3 consecutive G-C base pairs in the anticodon stem of the initiator tRNA has been shown to be responsible for its P-site targeting. The exact molecular mechanism involved in the P-site targeting of the initiator tRNA is still unclear and focus of our study. Using genetic methods, we obtained mutant E. coli strains where initiator tRNA mutants lacking the characteristic 3-GC base pairs can also initiate translation. One such mutant strain, A30, was selected for this study. Using standard molecular genetic tools, the mutation was mapped and identified to be a mutation in a transcription remodeling factor, RapA (A511V). RapA is a transcription recycling factor and it displaces S1 when it performs its transcription recycling activity. We found this mutation to cause an increase in the S1-depleted ribosomes leading to decreased fidelity of translation initiation as the mutant RapA inefficiently displaces S1 from RNA polymerase complex. The mutation in the RapA was also found to cause changes in the transcriptome which leads to downregulation of major genes important for methionine and purine metabolism. Using mass spectrometric analysis, we identified deficiencies of methionine and adenine in the strain carrying mutant RapA. Our lab had previously reported that methionine and S-adenosyl methionine deficiency cause deficiency of methylations in ribosome which in turn decreases the fidelity of protein synthesis initiation. We used strains deleted for two newly identified methyltransferases, namely RlmH and RlmI, for our study and these strains also showed decreased fidelity of initiation. RlmH and RlmI methylate 1915 and 1962 positions of 23S rRNA respectively. We found that deletion of these methyltransferases also caused defects in ribosome biogenesis and compromised activity of ribosome recycling factor. We constructed phylogenetic trees of the initiator tRNA from 158 species which distinctly assembled into three domains of life. We also constructed trees using the minihelix or the whole sequence of species specific tRNAs, and iterated our analysis on 50 eubacterial species. We identified tRNAPro, tRNAGlu, or tRNAThr (but surprisingly not elongator tRNAMet) as probable ancestors of tRNAi. We then determined the factors imposing selection of methionine as the initiating amino acid. Overall frequency of occurrence of methionine, whose metabolic cost of synthesis is the highest among all amino acids, remains almost unchanged across the three domains of life. Our results indicate that methionine selection, as the initiating amino acid was possibly a consequence of the evolution of one-carbon metabolism, which plays an important role in regulating translation initiation. In conclusion, the current study reveals the importance of methylations in ribosome biogenesis and fidelity of translation initiation. It also strongly suggests a co-evolution of the metabolism and translation apparatus giving adaptive advantage to the cells where presence of methionine in the environment can be a signal to initiate translation with methionine initiator tRNA.

Atrial natriuretic factor (ANF) is a polypeptide hormone produced in heart atria, stored in atrial secretory granules and released into the circulation in response to various stimuli. Proper sorting of ANF at the level of the trans-Golgi network (TGN) is required for the storage of ANF in these specific granules, and this sorting of hormones has been found to be associated with G-proteins. Specifically, the Go protein alpha-subunit (Gαo) was established to participate in the stretch-secretion coupling of ANF, but may also be involved in the transporting of ANF from the TGN into atrial granules for storage and maturation. Based on knowledge of Gαo involvement in hormone production in other endocrine tissues, protein-protein interactions of Gαo and proANF and their immunochemical co-localization in granules, the direct involvement of these two proteins in atrial granule biogenesis is probable. In this study, mice were created using the Cre/lox recombination system with a conditional Gαo knockout in cardiocytes to study and characterize ANF production, secretion and granule formation. Deletion of this gene was successful following standard breeding protocols. Characterization and validation of cellular and molecular content of the knockout mice through mRNA levels, protein expression, peptide content, electron microscopy, and electrocardiography determined that a significant phenotypic difference was observed in the abundance of atrial granules. However, Gαo knockout mice did not significantly alter the production and secretion of ANF and only partially prevented granule biogenesis, likely due to incomplete Gαo knockout. These studies demonstrate an involvement of Gαo in specific atrial granule formation.

A fibrose hepática é uma patologia que acompanha outras doenças crônicas do fígado como a cirrose e o hepatocarcinoma. As células estreladas hepáticas (HSC, do inglês hepatic stellate cells) compõem uma população celular heterogênea que se caracteriza por transitar entre dois fenótipos. As células com fenótipo quiescente possuem a capacidade de armazenar vitamina A em gotas lipídicas. Os insultos ao fígado desencadeiam uma resposta inflamatória que gera estímulos parácrinos e autócrinos mediados por citocinas e espécies reativas. Neste contexto, as HSC assumem um fenótipo ativado fibrogênico e tornam-se responsáveis pela cicatrização hepática. Danos crônicos ao fígado levam a uma deposição de matriz extracelular exagerada que configura o estado patológico da fibrose. O resveratrol (RSV – 3,4’,5-tri-hidroxi-trans-estilbeno) é uma fitoalexina produzida por algumas espécies de plantas. Inúmeros efeitos benéficos à saúde são atribuídos ao RSV por causa do seu potencial antioxidante, antiinflamatório e pró-apoptótico. Estudos anteriores mostraram que tratamento da GRX, uma linhagem murina de HSC ativadas, com concentrações de RSV próximas as biodisponíveis (0,1 a 1 μM) resultou em parada do ciclo na fase S com consequente inibição de proliferação celular, um efeito associado à citotoxicidade e que pode favorecer a resolução da fibrose hepática. Neste estudo, por técnicas espectrofotométricas, foi demonstrado que tratamento da GRX por 24 horas com concentrações entre 0,1 a 50 μM de RSV promoveu um efeito pró-oxidante que causa uma citotoxicidade dependente da dose, bastante aumentada no grupo tratado com a concentração mais alta. Os efeitos citotóxicos atenuados encontrados nas células tratadas por 120 horas sugerem que a GRX pode se tornar resistente a estes efeitos. O potencial pró-oxidante do RSV foi o ponto de partida para investigar a possibilidade de que esta fitoalexina provocasse uma alteração no metabolismo mitocondrial da GRX. Para isso, os efeitos do RSV (1 a 50 μM) na função mitocondrial, na indução de morte mediada por estas organelas e na autofagia/mitofagia foram investigados por técnicas de espectrofotometria, de imunocitoquímica, de citometria de fluxo, de microscopia confocal e de microscopia eletrônica de transmissão em GRX tratadas por 24 e 120 horas. Foi demonstrado que todas as concentrações de RSV promovem apoptose por meio da ativação de caspases, alteram a dinâmica/função mitocondrial e induzem o aumento de autofagia/mitofagia na GRX. No entanto, o RSV provocou biogênese de mitocôndrias nos grupos tratados com 1 e 10 μM, enquanto que o tratamento com 50 μM causou dano celular evidente na GRX, sem induzir biogênese de mitocôndrias. Desta forma, é possível que a citotoxicidade “dose-dependente” do RSV, que causa a morte celular e dano oxidativo em 24 horas de tratamento, esteja relacionada com o desequilíbrio entre a indução concomitante de apoptose mediada por dano mitocondrial, autofagia/mitofagia e biogênese de mitocôndrias. Por fim, foi investigada a liberação de TNF-α, Interleucina-6 e Interleucina-10 pela GRX tratada por 24 e 120 horas com RSV (0,1 a 50 μM), considerando o papel antiinflamatório do RSV e o papel das HSC ativadas na sinalização autócrina que contribui para a modulação fenotípica destas células. Foi demonstrado que o tratamento da GRX com RSV por 24 e 120 horas induziu a redução da liberação de Interleucina-6; enquanto que a liberação de TNF-α e Interleucina-10 foi aumentada. Estes resultados confirmam um efeito antiinflamatório do RSV que deve contribuir na prevenção da ativação ou da perpetuação do estado ativado das HSC por meio de sinalização autócrina. Ainda que a concentração do RSV seja importante para efetivamente induzir a morte das HSC ativadas, o tratamento com esta fitoalexina pode ser promissor para a resolução da fibrose hepática por diminuir a população de células ativadas e, possivelmente, prevenir a perpetuação do estado fenotípico ativado. Estudos avaliando indicadores de quiescência em células tratadas são ainda necessários para desvendar completamente os efeitos do RSV quanto às possibilidades de inibição da perpetuação ou reversão fenotípica das HSC ativadas. / Liver fibrosis is a disease that accompanies other hepatic chronic diseases such as cirrhosis and hepatocellular carcinoma. Hepatic stellate cells (HSC) are a heterogeneous cell population characterized by transiting between two phenotypes. Cells with a quiescent phenotype are able to store vitamin A into lipid droplets. Damage to the liver trigger an inflammatory response that generates paracrine and autocrine stimulation mediated by cytokines and reactive species. In this context, HSC assume an activated and fibrogenic phenotype responsive for hepatic wound-healing. Chronic insults to the liver lead to an excessive deposition of extracellular matrix that configures the pathological state of fibrosis. Resveratrol (RSV – 3,4’,5-tri-hidroxi-trans-stilbeno) is a phytoalexin produced by some species of plants. Several beneficial effects are attributed to this molecule due to its antioxidant, antiproliferative and pro-apoptotic potential. Previous studies showed that treatment with bioavailable concentrations of RSV (0.1 to 1 μM) promoted an arrest cycle at the S phase in GRX, a murine activated HSC model, leading to cell proliferation inhibition, a cytotoxic effect that contributes to the liver fibrosis resolution. In this study, it was shown by spectrophotometric techniques that GRX treatment for 24 hours at concentrations between 0.1 to 50 μM of RSV promoted a fairly clear pro-oxidant effect that causes a dose-dependent cytotoxicity that was higher in the group treated with 50 μM. The attenuated cytotoxicity found after 120 hours of GRX treatment suggest that these cells became resistant to this effect. The pro-oxidant potential of RSV was the starting point for investigating the possibility that this phytoalexin would cause a change in the GRX mitochondrial metabolism. Thus, the effects of RSV (1 to 50 μM) on altering the mitochondrial function, on inducing mitochondrial-mediated cell death, and autophagy/mitofagia were investigated in GRX treated for 24 and 120 hours by spectrophotometric techniques, immunocytochemistry, flow cytometry, confocal microscopy, and transmission electron microscopy. All the RSV concentrations promote cell apoptosis through caspases activation, alter the mitochondrial dynamics and function, and induce an increase of autophagy/mitofagia. Curiously, only 1 and 10 μM of RSV induced mitochondrial biogenesis in GRX, while the highest concentration caused an evident cell damage without inducing mitochondrial biogenesis. Thus, it is possible that the "dose-dependent" cytotoxicity of RSV, which causes cell death and oxidative damage in 24 hours of treatment, is related to an imbalance between the concomitant induction of mitochondrial-mediated apoptosis, autophagy/mitofagia, and mitochondrial biogenesis. Finally, it was investigated the release of TNF-α, Interleukin-6 and Interleukin-10 by GRX treated for 24 and 120 hours with RSV (0.1 to 50 μM), considering the anti-inflammatory role of RSV and the autocrine signalling role of HSC that contributes to the perpetuation of its activated phenotype. It was demonstrated that GRX treatment with RSV for 24 and 120 hours reduced the release of Interleukin-6 in the culture medium; whereas the release of TNF-α and Interleukin-10 was increased. These results confirm the anti-inflammatory properties of RSV and may contribute to the prevention of HSC activation through autocrine signalling. Although RSV concentration is important to effectively induce activated HSC death, cells treatment with this phytoalexin may be promising for liver fibrosis resolution through decreasing the population of activated cells or through preventing the perpetuation of activated state of HSC. Future studies evaluating the quiescence indicators of GRX under RSV treatment are still needed to fully unravel the effects of this phytoalexin on inhibiting the perpetuation of activated HSC or reversing its activated phenotype.