A restrição calórica (RC) estende a expectativa de vida de muitos organismos por mecanismos ainda em estudo. Entre os vários efeitos fisiológicos da RC encontra-se o aumento na biogênese mitocondrial, dependente de óxido nítrico (NO•), sintetizado pela enzima óxido nítrico sintase endotelial (eNOS). Um dos indutores fisiológicos mais potentes da eNOS é a insulina, cujos níveis plasmáticos são consideravelmente reduzidos nos organismos em RC. O objetivo deste trabalho foi investigar os mecanismos associados ao aumento da sinalização por NO• durante a RC in vivo e in vitro, e as conseqüências celulares do aumento de massa mitocondrial no que diz respeito à longevidade e capacidade respiratória celulares. Submetemos camundongos Swiss fêmeas à RC de 40% e observamos um considerável aumento tecido-específico na fosforilação basal de Akt e eNOS em músculo esquelético, tecido adiposo visceral e cérebro, os quais também apresentaram maior massa mitocondrial. A associação entre a sinalização por insulina, NO• e biogênese mitocondrial foi adicionalmente confirmada em um grupo de camundongos tratados com o desacoplador mitocondrial dinitrofenol (DNP), que também reduz a insulinemia e aumenta a longevidade em camundongos. Para o estudo mecanístico deste fenômeno, usamos soros de ratos Sprague-Dawley submetidos à RC de 40% ou alimentados ad libitum (AL) em cultura celular de células vasculares da musculatura lisa (VSMC), reproduzindo um protocolo descrito para RC in vitro. O uso do soro RC aumentou a fosforilação do receptor de insulina e Akt, a expressão de eNOS e nNOS (forma neural da NOS) e a fosforilação de eNOS, o que se refletiu em maior liberação de nitrito (NO2) no meio de cultura. Inibindo-se a Akt, todos os efeitos promovidos pela RC na sinalização por NO• foram revertidos. Ao se imunoprecipitar do soro a adiponectina, citocina conhecida por aumentar a sensibilidade à insulina, aumentada durante a RC, os efeitos do soro RC na via de sinalização de insulina foram abolidos e, conseqüentemente, os efeitos na sinalização por •NO foram prevenidos. Neurônios de células granulosas de cerebelo, que não expressam eNOS, apenas nNOS, foram cultivados com os soros AL ou RC, e também apresentaram considerável aumento na sinalização por •NO. Estas alterações induziram a biogênese mitocondrial e capacidade respiratória, e foram associadas à maior longevidade celular. Os mesmos efeitos mitocondriais foram observados em células secretoras de insulina, INS1, entretanto a secreção de insulina em resposta à glicose tornou-se inibida, por um mecanismo desconhecido, porém associado a reduzidos níveis intracelulares de espécies oxidantes, moléculas-chave para a secreção de insulina; e à alteração da morfologia mitocondrial, provavelmente devido à maior expressão de mitofusina-2 (Mfn-2). Ao se nocautear a Mfn-2, houve um aumento na geração de EROs e as células em RC passaram a secretar insulina a níveis comparáveis aos das células controle. Concluímos que durante a RC a maior sensibilidade à insulina aumenta a atividade de eNOS, via Akt, associada à maior biogênese mitocondrial. A adiponectina é uma molécula-central nestes eventos. A expressão de nNOS também é afetada, por mecanismos desconhecidos. O aumento de biogênese mitocondrial eleva a capacidade respiratória celular e impacta positivamente a longevidade in vitro. A alteração da morfologia mitocondrial associa-se a alterações na produção de oxidantes intracelulares e mudanças na secreção de insulina. / Calorie restriction (RC) is known to extend the lifespan in many organisms, and its mechanisms of action are still under investigation. Enhanced mitochondrial biogenesis driven by nitric oxide (•NO), synthesized by the endothelial nitric oxide synthase (eNOS), is proposed to be a CR central effect. Insulin is one of the most potent physiological activators of eNOS. However, plasmatic insulin levels are dramatically reduced in organisms under CR. The goal of this work was uncover the mechanisms associated with enhanced •NO signaling during CR, in vivo and in vitro, as well as the cellular consequences of increased mitochondrial mass, regarding lifespan and reserve respiratory capability. Female Swiss mice were submitted to 40% of CR. A tissue-specific (skeletal muscle, abdominal adipose tissue and brain) increment in basal Akt and eNOS phosphorylation, which was related to enhanced mitochondrial biogenesis, was observed. Indeed, this association was also verified in tissues from mice treated with low doses of a mitochondrial uncoupler, dinitrophenol (DNP). To unveil the mechanism behind the insulin signaling effects on •NO levels, serum from Sprague-Dawley rats submmited to 40% of CR was used to culture in VSMC cells, an in vitro CR protocol. CR sera enhanced insulin receptor (IR) and Akt phosphorylation, as well as nitrite (NO2-) accumulation in the culture media, the expression of eNOS and nNOS (neural NOS isoform) and eNOS phosphorylation. The effects of CR sera were reversed by Akt inhibition. The immunoprecipitation of serum adiponectin, a cytokine known to improve peripheral insulin sensitivity, also reversed the CR serum effects on insulin and •NO signaling. Cerebellar neurons, which do not express eNOS, just nNOS, were also cultured with CR or AL serum and also presented striking increments in •NO signaling, associated with mitochondrial biogenesis, increased reserve respiratory capability and lifespan extension. The mitochondrial effects promoted by CR were also observed in insulin secreting cells (INS1). However, under the CR condition, insulin secretion stimulated by glucose was impaired. The likely explanations are reduced mitochondrial reactive oxygen species (ROS) generation, or the alteration in mitochondrial morphology, associated, in our model, with enhanced mitofusin-2 expression (Mfn-2). In cells which the Mfn-2 was knocked down, insulin secretion in CR and AL groups was responsive to glucose at the same level, and the intracellular oxidants levels were much higher. Overall, CR improves •NO signaling due to enhanced insulin sensitivity, through Akt, and results in mitochondrial biogenesis. Adiponectin is a key molecule in this phenomenon. Increments in mitochondrial mass enhance the cellular reserve respiratory capability and lifespan. Mitochondrial morphology alterations are associated with possible decreases in ROS generation and impaired insulin release, maintained the low levels of plasmatic insulin.

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.

Das Überleben und die Vermehrung der parasitär lebenden Apicomplexa setzen eine effiziente Synthese von Phospholipiden während ihres gesamten Lebenszyklus voraus. In dieser Arbeit nutzten wir zunächst Eimeria falciformis um den Prozess der Lipid-Biogenese in Sporozoiten zu untersuchen. Durch Lipidomics-Analysen wurde das Auftreten von zwei exklusiven Lipiden, Phosphatidylthreonin (PtdThr) und Inositolphosphorylceramid. Der Parasit exprimiert fast das gesamte Lipid-Biogenese- Netzwerk aus eukaryotischen und prokaryotischen Enzymen. Toxoplasma gondii diente als genmanipulierbarer Ersatz für die Untersuchung der Eimeria-Enzyme, mit dem wir ein stark räumlich segmentiertes Netzwerk der Lipidsynthese im Apicoplast, ER, Golgi und Mitochondrium zeigen konnten. Ebenso legte die Komplementierung einer T. gondii-Mutante mit einer PtdThr-Synthase von E. falciformis eine konvergente Funktion von PtdThr für den lytischen Zyklus von Kokzidien-Parasiten nahe. Außerdem setzten wir T. gondii als etablierten Modelorganismus ein, um die De- novo-Synthese und die metabolische Rolle eines bedeutenden Lipidvorläufers, CDP- Diacylglycerin (CDP-DAG), zu untersuchen. Wir konnten zwei phylogenetisch divergente CDP-DAG-Synthase (CDS) Enzyme in T. gondii nachweisen. Das eukaryotisch-typische TgCDS1 und das prokaryotisch-typische TgCDS2 lokalisieren im ER bzw. im Apicoplast. Der konditionierte Knockdown von TgCDS1 bremst das Parasitenwachstum stark ab, was den fast vollständigen Verlust der Virulenz im Mausmodell hervorruft. Das restliche marginale Wachstum der TgCDS1 Mutante wird durch zusätzliche Deletion der TgCDS2 verhindert. Lipidomics-Analysen zeigten eine signifikante und spezifische Abnahme der Phosphatidylinositol (PtdIns)- und Phosphatidylglycerol (PtdGro)-Level bei Verlust der TgCDS1- bzw. TgCDS2-Gene. Zusammengenommen zeigt unsere Arbeit ein Phospholipid-Biogenese-Modell mit erstaunlicher Kooperation verschiedener Organellen und einem extensiven Lipidtransport im Parasiten. / The survival and proliferation of apicomplexan parasites oblige efficient synthesis of phospholipids throughout their life cycles. Here, we first deployed Eimeria falciformis to investigate the process of lipid biogenesis in sporozoites. Lipidomics analyses demonstrate the occurrence of two exclusive lipids phosphatidylthreonine (PtdThr) and inositol phosphorylceramide along with other prototypical lipids. The parasite expresses nearly the entire lipid biogenesis network, which is an evolutionary mosaic of eukaryotic- and prokaryotic-type enzymes. Using Toxoplasma gondii as a gene- tractable surrogate to examine the Eimeria enzymes, we show a highly compartmentalized network of lipid synthesis distributed primarily in the apicoplast, ER, Golgi and mitochondrion. Likewise, trans-species complementation of a T. gondii mutant with a PtdThr synthase from E. falciformis suggests a convergent function of PtdThr in promoting the lytic cycle in coccidian parasites. We also employed the well-established model parasite T. gondii to explore de novo synthesis and metabolic roles of one major lipid precursor CDP-diacylglycerol (CDP- DAG). We report the occurrence of two phylogenetically divergent CDP-DAG synthase (CDS) enzymes in T. gondii. Eukaryotic-type TgCDS1 and prokaryotic-type TgCDS2 reside in the ER and apicoplast, respectively. Conditional knockdown of TgCDS1 severely attenuates parasite growth, which translates into a nearly complete loss of virulence in a mouse model. Residual growth of the TgCDS1 mutant is abolished by subsequent deletion of TgCDS2. Lipidomics analyses reveal significant and specific decline in phosphatidylinositol (PtdIns) and phosphatidylglycerol (PtdGro) upon loss of TgCDS1 and TgCDS2, respectively. Taken together, our work establishes a phospholipid biogenesis model involving significant inter-organelle cooperation and lipid trafficking in apicomplexan parasites.

La biogenèse des ribosomes eucaryotes est un processus complexe qui implique la production et l'assemblage de 4 ARNr et 80 protéines. La production des deux sous-unités du ribosome, 40S et 60S, débute dans le nucléole par la synthèse d'un long précurseur commun contenant les séquences des ARNr matures et se termine dans le cytoplasme où ont lieu les dernières étapes d'assemblage des protéines ribosomiques et de clivage des ARNr. La production de ribosomes nécessite la participation de plus de 200 co-facteurs, qui catalysent les clivages et modifications des ARNr, coordonnent leur repliement et leur association aux protéines ribosomiques, et assurent des étapes de contrôle-qualité. Ces protéines sont associées aux particules en cours de maturation et absentes des sous-unités matures. Cette voie de synthèse, globalement conservée chez les eucaryotes, a été principalement étudiée chez la levure. Cependant, des études récentes ont montré des différences importantes de ce processus entre levure et mammifères. Un des verrous importants pour comprendre la fonction des co-facteurs, est l'absence de données sur la structure des précurseurs des sous-unités ribosomiques. J'ai donc entrepris une étude structurale de l'assemblage cytoplasmique de la petite sous-unité ribosomique chez l'homme par cryo-microscopie électronique à transmission. Le but de ma thèse était de déterminer la structure 3D des précurseurs de la petite sous-unité ribosomique purifiés à différentes étape de leur maturation. Ce travail a été conduit en collaboration avec l'équipe du Pr. Ulrike Kutay (ETH Zurich) pour la purification des particules pré-40S à partir de cellules humaines. La première structure 3D de particule pré-40S intermédiaire purifiée en étiquetant le co-facteur LTV1 a été déterminée à 19Å de résolution. Dans un deuxième temps, la structure 3D de la particule pré-40S tardive purifiée à via RIO1(KD) a aussi été déterminée à 15Å de résolution. Ces données nous ont permis de proposer un modèle de localisation des co-facteurs sur les précurseurs de la petite sous-unité ribosomique et de montrer une nouvelle différence dans la formation de la petite sous-unité chez l'Homme comparé à la levure, du fait de la présence de la protéine RACK1 sur les particules pré-40S humaines. La comparaison des structures des précurseurs de la petite sous-unité obtenues a permis de mettre en lumière l'existence de remodelages structuraux de la particule pré-40S au cours de sa maturation. Ce travail met en lumière les premières structures 3D de particules pré-40S humaines et pose les fondements méthodologiques d'explorations futures de la dynamique structurale des particules pré-ribosomiques. / Ribosome biogenesis is a complex process that requires the production and the correct assembly of the 4 rRNAs with 80 ribosomal proteins. In Human, the production of the two subunits, 40S and 60S, is initiated by the transcription of a pre-ribosomal rRNA precursor to the mature 18S, 5.8S, and 28S rRNAs by the RNA polymerase I, which is chemically modified and trimmed by endo- and exoribonuclease, in order to form the mature rRNAs. The nascent pre rRNA associated with ribosomal proteins, small ribonucleoprotein particles (snoRNP) and so called co-factors leading to the assembly of an initial 90S particle. This particle is then split into pre-40S and pre-60S pre-ribosomal particles that fallow independent maturation to form the mature subunit into the cytoplasm. Production of eukaryotic ribosomes implies the transient intervention of more than 200 associated proteins and ribonucleoprotein particles, that are absent from the mature subunits. Synthesis of ribosome, globally conserved in eukaryotes, has been principally studied in yeast. However, recent studies reveal that this process is more complex in human compared in yeast. An important bottleneck in this domain is the lack of structural data concerning the formation of intermediate ribosomal subunits to understand the function of assembly factors. Determination of the structural remodeling of pre-ribosomal particles is crucial to understand the molecular mechanism of this complex process. So I have undertaken a structural study on the assembly of the small ribosomal subunit using cryo-electron microscopy and image analysis. The goal of my thesis is to determine the 3D structures of human pre-40S particles at different maturation stages to see the structural remodeling that occurs during the biogenesis of the small ribosomal subunit. We are collaborating with the group of Pr Ulrike Kutay at ETH Zurich, who purify human pre-40S particles. The 3D structures of human pre-40S particles purified at an intermediate and late maturation stages, has been determined with a resolution of 19 and 15Å respectively. Supplementary densities, compared to the mature subunit, indicate the presence of assembly factors and show the unexpected presence of the RACK1 protein in the precursor of the human small ribosomal subunit in the cytoplasm. The comparison of the 3D structures of human pre-40S particle allows showing the structural remodeling that occur during the maturation of the small ribosomal subunit. This work provides the first 3D structure of human pre-40S particles and laid the methodological foundations for future exploration of the structural dynamics of pre-ribosomal particles.

The peroxisome is a subcellular organelle that carries out a diverse range of metabolic functions, including the b-oxidation of very long chain fatty acids, the breakdown of peroxide and the a-oxidation of fatty acids. Disruption of peroxisome metabolic functions leads to severe disease in humans. These diseases can be broadly grouped into two categories: those in which a single enzyme is defective, and those known as the peroxisome biogenesis disorders (PBDs), which result from a generalised failure to import peroxisomal matrix proteins (and consequently result in disruption of multiple metabolic pathways). The PBDs result from mutations in PEX genes, which encode protein products called peroxins, required for the normal biogenesis of the peroxisome. PEX1 encodes an AAA ATPase that is essential for peroxisome biogenesis, and mutations in PEX1 are the most common cause of PBDs worldwide. This study focused on the identification of mutations in PEX1 in an Australasian cohort of PBD patients, and the impact of these mutations on PEX1 function. As a result of the studies presented in this thesis, twelve mutations in PEX1 were identified in the Australasian cohort of patients. The identified mutations can be broadly grouped into three categories: missense mutations, mutations directly introducing a premature termination codon (PTC) and mutations that interrupt the reading frame of PEX1. The missense mutations that were identified were R798G, G843D, I989T and R998Q; all of these mutations affect amino acid residues located in the AAA domains of the PEX1 protein. Two mutations that directly introduce PTCs into the PEX1 transcript (R790X and R998X), and four frameshift mutations (A302fs, I370fs, I700fs and S797fs) were identified. There was also one mutation found in an intronic region (IVS22-19A>G) that is presumed to affect splicing of the PEX1 mRNA. Three of these mutations, G843D, I700fs and G973fs, were found at high frequency in this patient cohort. At the commencement of these studies, it was hypothesised that missense mutations would result in attenuation of PEX1 function, but mutations that introduced PTCs, either directly or indirectly, would have a deleterious effect on PEX1 function. Mutations introducing PTCs are thought to cause mRNA to be degraded by the nonsense-mediated decay of mRNA (NMD) pathway, and thus result in a decrease in PEX1 protein levels. The studies on the cellular impact of the identified PEX1 mutations were consistent with these hypotheses. Missense mutations were found to reduce peroxisomal protein import and PEX1 protein levels, but a residual level of function remained. PTC-generating mutations were found to have a major impact on PEX1 function, with PEX1 mRNA and protein levels being drastically reduced, and peroxisomal protein import capability abolished. Patients with two missense mutations showed the least impact on PEX1 function, patients with two PTC-generating mutations had a severe defect in PEX1 function, and patients carrying a combination of a missense mutation and a PTC-generating mutation showed levels of PEX1 function that were intermediate between these extremes. Thus, a correlation between PEX1 genotype and phenotype was defined for the Australasian cohort of patients investigated in these studies. For a number of patients, mutations in the coding sequence of one PEX1 allele could not be identified. Analysis of the 5' UTR of this gene was therefore pursued for potential novel mutations. The initial analyses demonstrated that the 5' end of PEX1 extended further than previously reported. Two co-segregating polymorphisms were also identified, termed –137 T>C and –53C>G. The -137T>C polymorphism resided in an upstream, in-frame ATG (termed ATG1), and the possibility that the additional sequence represented PEX1 coding sequence was examined. While both ATGs were found to be functional by virtue of in vitro and in vivo expression investigations, Western blot analysis of the PEX1 protein in patient and control cell extracts indicated that physiological translation of PEX1 was from the second ATG only. Using a luciferase reporter approach, the additional sequence was found to exhibit promoter activity. When examined alone the -137T>C polymorphism exerted a detrimental effect on PEX1 promoter activity, reducing activity to half that of wild-type levels, and the -53C>G polymorphism increased PEX1 promoter activity by 25%. When co-expressed (mimicking the physiological condition) these polymorphisms compensated for each other to bring PEX1 promoter activity to near wild-type levels. The PEX1 mutations identified in this study have been utilised by collaborators at the National Referral Laboratory for Lysosomal, Peroxisomal and Related Genetic Disorders (based at the Women's and Children's Hospital, Adelaide), in prenatal diagnosis of the PBDs. In addition, the identification of three common mutations in Australasian PBD patients has led to the implementation of screening for these mutations in newly referred patients, often enabling a precise diagnosis of a PBD to be made. Finally, the strong correlation between genotype and phenotype for the patient cohort investigated as part of these studies has generated a basis for the assessment of newly identified mutations in PEX1.

La biogenèse du ribosome est un processus complexe et dynamique qui nécessite de nombreuses étapes de maturation et de modification des ARNr ainsi que l’assemblage et le transport des RNPs précurseurs. Un ribosome mature contient une centaine de pièces, ARN et protéines confondus, mais son assemblage requiert l’intervention de plus de 400 facteurs de synthèse. De part le coût énergétique important de ce processus, plusieurs voies de régulation interviennent pour contrôler la biogenèse des ribosomes en fonction des conditions nutritives. L’une des voies les plus connue est la voie TOR (Target of rapamycin). Cette voie de régulation agît principalement au niveau de la transcription des différents intervenants de la biogenèse : les ARNr, les protéines ribosomiques mais aussi les facteurs de synthèse. Ces facteurs, ayant une action transitoire dans la maturation des ribosomes, sont, par économie, recyclés pour la synthèse de nouveaux ribosomes. Nous nous sommes donc intéressés au devenir de ces facteurs, plus particulièrement de ceux intervenants dans la biogenèse de la petite sous unité, lorsque les conditions environnementales sont inadaptées à la croissance cellulaire. Ainsi, nous avons pu montré, pour quatre facteurs particuliers : Dim2, Rrp12, Hrr25 et Fap7, que leur localisation est dépendante de la synthèse ribosomique. Ainsi, lors de carence en sources nutritives, l’inhibition de la synthèse et de l’activité ribosomique entraîne un confinement de ces facteurs ribosomiques dans le nucléole ou dans des corps cytoplasmiques. En outre, la localisation particulière des facteurs ribosomiques Hrr25 et Fap7 dans les P-bodies en phase de croissance saturée laisse penser que ces corps cytoplasmiques sont le lieu de dégradation des pré-ribosomes lorsque les carences nutritives perdurent.

In mammalian cells, homeostasis and fate during development relies on the proper transport of membrane-bound cargoes to their designated cellular locations. The hetero-tetrameric adaptor protein complexes (APs) are required for sorting and concentration of cargo at donor membranes, a crucial step during targeted transport. AP2, which functions at the plasma membrane during clathrin-mediated endocytosis, is well characterized. In contrast, AP1 a clathrin adaptor mediating the delivery of lysosomal hydrolases via mannose 6-phosphate receptors (MPRs) and AP3 an adaptor ensuring the proper targeting of lysosomal membrane protein are difficult to study by classic cell biology tools. To gain new insights on these APs, our lab has previously designed an in vitro system. Reconstituted liposomes were modified with small peptides mimicking the cytosolic domains of bona fide cargoes for AP1 and AP3 respectively and thereby enabling the selective recruitment of these APs and the identification of the interacting protein network. In the study at hand we utilize above-described liposomes to generate supported lipid bilayers and Giant Unilamellar Vesicles (GUVs), large-scale membrane systems suited for analysis by fluorescence microscopy. By using cytosol containing fluorescently-tagged subunits, we visualized clathrin coats on artificial membranes under near physiological conditions for the first time. Moreover, we demonstrated clathrin-independent recruitment of AP3 coats on respective GUVs. Presence of active ARF1 was sufficient for the selective assembly of AP1-dependent clathrin coats and AP3 coats on GUVs. By using dye-conjugated ARF1, we show that ARF1 colocalized with AP3 coats on GUVs and that increased association of ARF1 with GUVs coincided with AP1-dependent clathrin coats. Our previous study identified members of the septin family together with AP3 coats on liposomes. Here we show on GUVs, that active ARF1 stimulated the assembly of septin7 filaments, which may constrain the size and mobility of AP3 coats on the surface. Subsequent cell biology studies in HeLa cells linked septins to actin fibers on which they may control mobility of AP3-coated endosomes and thus their maturation. An actin nucleation complex, based on CYFIP1 was identified together with AP1 on liposomes before. Here we show on GUVs, that CYFIP1 is recruited on the surface surrounding clathrin coats. Upon supply of ATP, sustained actin polymerization generated a thick shell of actin on the GUV surface. The force generated by actin assembly lead to formation of long tubular protrusions, which projected from the GUV surface and were decorated with clathrin coats. Thereby the GUV model illustrated a possible mechanism for tubular carriers formation. The importance of CYFIP1-reliant actin polymerization for the generation of MPR-positive tubules at the trans-Golgi network (TGN) of HeLa cells was subsequently demonstrated in our lab. The notion that tubulation of artificial membranes could be triggered by actin polymerization allowed us to perform a comparative mass spectrometry screen. By comparing the abundance of proteins on liposomes under conditions promoting or inhibiting actin polymerization, candidates possibly involved in stabilization, elongation or fission of membrane tubules could be identified. Among the proteins enriched under conditions promoting tubulation, we identified type I phosphatidylinositol-4-phosphate 5-kinases. Their presence suggested an involvement of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) in tubule formation. By cell biology studies in HeLa we show, that down regulation of these enzymes altered the dynamics of fluorescently-tagged MPRs, illustrating the importance of locally confined PI(4,5)P2 synthesis during formation of coated carriers at the TGN. Bin–Amphiphysin–Rvs (BAR) domains are known to sense membrane curvature and induce membrane tubulation. Among various BAR domain proteins, Arfaptin2 was enriched under conditions allowing tubulation of liposomes. By microscopy studies on HeLa cells we show, that Arfaptin2 as well as its close paralog Arfaptin1 were present on AP1-coated MPR tubules emerging from the TGN. We further show, that tubule fission occurred at regions were Arfaptin1 is concentrated and that simultaneous down regulation of both Arfaptins lead to increased number and length of MPR tubules. Since fission of coated transport intermediates at the TGN is poorly understood, our findings contribute a valuable component towards a model describing the entire biogenesis of coated post-Golgi carriers. In conclusion, combining artificial membrane systems and cell biology studies allowed us to propose new models for formation as wall as for fission of AP1-coated transport intermediates at the TGN. Further we gained new insights on AP3 coats and the possible involvement of septin filaments in AP3-dependent endosomal maturation.

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Peroxisome biogenesis is complex, involving a diverse array of intracellular pathways and mechanisms that mediate their biogenesis and cellular functions. Relevant to our understanding of peroxisome biogenesis is the utilization of peroxisomal membranes for viral genome replication as observed in plant cells infected by several members of the Tombusviridae family of positive-strand RNA viruses. Tomato Bushy Stunt Virus (TBSV), for instance, usurps an array of host factors that facilitate the transformation of peroxisomes into peroxisomal multivesicular bodies (pMVB) the sites of viral RNA replication. In this study, pMVB topology and biogenesis was investigated using transmission electron and epifluorescence microscopy of tobacco and wildtype or mutant budding yeast that were transformed with TBSV replicase proteins and a defective interfering viral RNA. Overall, the results suggest that host-virus interactions specifically associated with Endosomal Sorting Complex Required for Transport (ESCRT) and lipid metabolism are involved in TBSV replication and pMVB biogenesis.