Les protéasomes jouent un rôle majeur dans la protéolyse intracellulaire non-lysosomale et interviennent de ce fait dans la régulation de toutes les voies biologiques intracellulaires ainsi que dans la production des peptides antigéniques présentés au système immunitaire. La biosynthèse du protéasome 20S (le cœur protéolytique) est un processus complexe qui met en œuvre plusieurs protéines chaperonnes qui facilitent l'assemblage des sous-unités entre elles pour former d’abord des « hémi-protéasomes ». Ces hémi-protéasomes s'unissent ensuite deux à deux pour créer le protéasome 20S. La Protéine de Maturation du Protéasome (POMP) est une chaperonne « clé » de l’assemblage du protéasome: son hyperexpression induit une augmentation des activités protéolytiques du protéasome et une augmentation d’expression de plusieurs de ses sous-unités. Cette surexpression augmente également la longévité de la levure Saccharomyces cerevisiae, et augmente ses capacités de résistance au stress oxydant. L’activité protéolytique et l’expression des sous-unités des protéasomes sont augmentées dans l’épiderme psoriasique. Nos précédents résultats étaient en faveur d’une régulation post-transcriptionnelle de cette expression. Nous avons émis l’hypothèse que POMP était impliquée dans la biosynthèse accrue du protéasome dans la peau psoriasique. L'objectif de cette thèse a donc été d’étudier l’assemblage du protéasome et l’expression de POMP dans la peau psoriasique et au cours de la différenciation kératinocytaire. L'expression des protéasomes et de POMP a été évaluée au niveau protéique et au niveau des ARN messagers par Immuno-Histochimie, Western-blots et RT-PCR. L’assemblage du protéasome a été étudié par électrophorèse en conditions non dénaturantes, à partir de lysats de peaux psoriasiques ou de kératinocytes. L’expression de POMP a été modulée avec des ARN interférents dans des lignées HaCaT. Nous avons observé que les protéasomes 26S et 20S (et leur activité protéolytique) étaient augmentés dans la peau psoriasique lésée, de même que les régulateurs PA28β, PA28γ, PA200 et la sous-unité RPT4 du 19S. Ces résultats suggèrent que toutes les formes majeures des protéasomes sont augmentées dans la peau psoriasique. POMP est surexprimée dans l’épiderme psoriasique et associée à des précurseurs du protéasome. Au cours de la différenciation des cellules HaCaT, l’assemblage des protéasomes 20S et 26S et l’expression de POMP augmentent aux temps précoces (3 premiers jours) alors qu’aux temps plus tardifs les protéasomes 26S se désassemblent. Ce profil de désassemblage (marqué par la diminution du protéasome 26S, l’augmentation du protéasome 20S et l'accumulation du complexe régulateur 19S) est très similaire à celui observé au cours du stress oxydatif. L’inhibition forte de l'expression de POMP a un effet anti-proliferatif et pro-apoptotique via l’inhibition de l’assemblage du protéasome dans les lignées kératinocytaires. Une inhibition plus modérée diminue l’expression des marqueurs de différenciation kératine 10 et involucrine au cours de la différenciation calcium-induite des HaCaT. L’ensemble de ces résultats suggère que l’augmentation de l’assemblage, de l’activité et de la quantité des protéasomes dans l’épiderme psoriasique est liée (au moins en partie) à la surexpression de POMP et que la dérégulation de POMP pourrait altérer la prolifération et la différenciation kératinocytaires. Nos travaux sont donc en faveur de l’implication de POMP et de l’assemblage du protéasome dans la pathogénie du psoriasis. Le ciblage thérapeutique de POMP pourrait potentiellement avoir une action anti-proliférative et anti-inflammatoire (via notamment l’inhibition de NF-κB) et représente donc une perspective thérapeutique intéressante dans le psoriasis. / Proteasomes play a major role in non-lysosomal intracellular proteolysis and thereby are involved in the regulation of all intracellular biological pathways as well as in the production of antigenic peptides presented to the immune system. The biosynthesis of the 20S proteasome (proteolytic core) is a complex process that requires several chaperone proteins that facilitate the assembly of the subunits to form "hemi-proteasomes" that then combine in pairs to create the 20S proteasome. Proteasome Maturation Protein (POMP) is a key chaperone for proteasome assembly: its overexpression induces an increase in proteolytic activities of the proteasome and an increase in expression of several of its subunits. This overexpression also increases the longevity of the yeast Saccharomyces cerevisiae and increases its capabilities of resistance against oxidative stress. The proteolytic activity and expression of the proteasome subunits are increased in psoriatic epidermis. Our previous findings suggested a post-transcriptional regulation of this expression. We hypothesized that POMP was involved in the increased biosynthesis of proteasome in psoriatic skin. The objective of the thesis project was to study proteasome assembly and POMP expression in psoriatic skin and during keratinocyte differentiation. The expression of proteasomes and POMP has been evaluated at the protein and mRNA levels by immunohistochemistry, Western blots and RT-PCR. Proteasome assembly was studied by electrophoresis under non-denaturing conditions, from lysates of psoriatic skin or keratinocytes. POMP expression was modulated by RNA interference in HaCaT cell lines. We observed that proteasomes 26S and 20S (and their proteolytic activity) were increased in the lesional psoriatic skin, as well as the proteasome regulators PA28β, PA28γ, PA200 and the 19S subunit RPT4. These results suggest that all the major proteasomal forms are increased in psoriatic skin. POMP is overexpressed in psoriatic epidermis and associated with precursors of the proteasome. During the differentiation of HaCaT cells, the assembly of 20S and 26S proteasomes and POMP expression increases at early time (first 3 days), while in later times 26S proteasomes disassemble. This disassembly profile (marked by the decrease of the 26S proteasome, the increase of the 20S proteasome complex and the accumulation of its 19S regulator) is very similar to that observed during oxidative stress. The strong inhibition of POMP expression has an anti-proliferative and pro-apoptotic effect via inhibition of proteasome assembly in the HaCaT keratinocyte cell line. However a moderate inhibition decreases the expression of differentiation markers keratin 10 and involucrin during calcium-induced differentiation of these cells. All these results suggest that the increase of the assembly, the activity and amount of proteasomes in psoriatic epidermis are linked (at least partially) to the overexpression of POMP and that deregulation of POMP could alter keratinocyte proliferation and differentiation. Our work is therefore in favor of the involvement of POMP and assembly of the proteasome in the pathogenesis of psoriasis. Therapeutic targeting of POMP could potentially have anti-proliferative and anti-inflammatory effects (particularly through the inhibition of NF-κB) and therefore represents an interesting therapeutic perspective in psoriasis.

Les prions sont des protéines qui suite à des changements de conformation acquièrent un caractère infectieux. Ils sont à l’origine de traits dominants, héritables de façon non-Mendélienne, chez les mammifères, les champignons filamenteux et les levures. Le mauvais repliement et l’agrégation des protéines sont à l’origine de plus de 40 maladies, parmi lesquelles on retrouve des maladies neurodégénératives telles que les maladies d’Alzheimer, de Parkinson et de Huntington. Il a été montré que les formes agrégées des protéines supposées responsables de ces maladies (i.e. peptide amyloïde-β, tau, α-synucléine, huntingtine) se propagent de cellule en cellule à la manière des prions. La levure Saccharomyces cerevisiae possède plusieurs prions qui sont autant d’excellents modèles biologiques pour la compréhension des mécanismes de formation et de propagation des prions.[PSI+] et [URE3], issus respectivement de la conversion sous forme prion du terminateur de la traduction Sup35p et d’un régulateur du métabolisme azoté Ure2p, sont à ce jour les deux prions les mieux documentés chez la levure. Les chaperons moléculaires et leurs co-chaperons modulent la formation, la réplication et la propagation des prions chez la levure. Cependant, l’élimination ou la dégradation de ces prions sont encore mal connus. Notre laboratoire a montré que le protéasome 26S est capable de dégrader les formes soluble et fibrillaire de Sup35p. Dans la première partie de ma thèse, nous avons étudié le rôle du protéasome 26S dans la dégradation des formes soluble et fibrillaire d’Ure2p. Nous avons montré que, comme pour Sup35p, le protéasome 26S dégrade Ure2p soluble en générant des peptides amyloïdes issus du domaine prion N-terminal ainsi qu’un fragment C-terminal résistant à la protéolyse. Nous avons montré que le domaine prion déstructuré est nécessaire pour la reconnaissance et la dégradation par le protéasome. Contrairement à ce qui avait été observé pour Sup35p, Ure2p sous sa forme fibrillaire est totalement résistante à la dégradation protéasomale. Nous suggérons que la variabilité structurale aux seins des particules de prions dans un contexte cellulaire dicte leurs interactions avec les machineries protéolytiques, et plus particulièrement avec le protéasome.Les prions de levure ont principalement été étudiés dans un contexte de cellules en division active. Cependant, dans la nature, la plupart des cellules sont retrouvées dans un état quiescent post-mitotique. Nous n’avons que très peu d’informations sur le devenir des particules de prions lorsque les cellules entrent dans un état quiescent. De même les conséquences physiologiques des prions sur la survie à long terme des levures sont très peu documentées. Dans la seconde partie de ma thèse, nous avons utilisé le prion [PSI+] comme modèle pour répondre à ces questions. Différentes conformations des agrégats de Sup35p conduisent à des souches phénotypiquement distinctes du prion [PSI+]. Nous avons constaté que les agrégats de Sup35p subissent des changements ultra-structuraux et fonctionnels au cours des différentes phases de croissance cellulaire. Ainsi, nous avons observés des changements importants dans la distribution de taille et dans l’infectiosité des polymères de Sup35p résistants au SDS formant les briques élémentaires du prion [PSI+]. Ces changements interviennent sans affecter les informations structurales spécifiques à chaque souche de prion [PSI+]. De façon remarquable, bien que [PSI+] n’affecte pas le taux de croissance des levures, ce prion semble prolonger significativement la durée de vie des levures. Cet effet bénéfique semble pouvoir se fixer de façon efficace et permanente dans les cellules et persister même après élimination de [PSI+]. La fixation génétique de caractéristiques épigénétiques induites par [PSI+] ont été déjà observées et l’ensemble de ces résultats suggère que [PSI+] (et éventuellement d’autres prions) peut jouer le rôle de capaciteurs évolutifs transitoires. / “Proteinaceous infectious particles”, or prions, are self-perpetuating alternate conformations of proteins that are responsible for heritable non-Mendelian traits in mammals, filamentous fungi and yeast. On a more general note, protein misfolding and aggregation is at the origin of over forty protein folding disorders including devastating neurodegenerative diseases such as Alzheimer’s, Parkinson’s or Huntington’s diseases. The aggregated proteins responsible for these diseases (i.e. amyloid-β peptide/tau, α-synuclein and huntingtin) were shown to propagate from cell to cell in a prion-like manner. The yeast Saccharomyces cerevisiae hosts many prion or prion-like proteins, unrelated in sequence and function, which proved to be excellent models for understanding the dynamics of prion aggregation and distribution upon cell division.Sup35p and Ure2p which cause the [PSI+] and [URE3] heritable traits, respectively, stand out as the most studied and best characterized yeast prions to date. A plethora of cellular factors, mostly belonging to various molecular chaperone families, were shown to affect yeast prion formation and propagation. Clearance of protein aggregates and prion particles is however poorly understood and documented. Our laboratory showed that the 26S proteasome degrades both the soluble and prion-associated fibrillar forms of Sup35p. In the first part of my thesis, we investigated the role of the 26S proteasome in the degradation of the soluble and fibrillar forms of Ure2p. We found that, as with Sup35p, the 26S proteasome is able to degrade the soluble native Ure2p, generating an array of amyloidogenic N-terminal peptides and a C-terminal fragment which is resistant to proteolysis. The N-terminal prion domain was shown to act as a degron required for proteasomal engagement and degradation. In contrast to Sup35p, fibrillar Ure2p resisted proteasomal degradation. We expect the structural variability within prion assemblies in a cellular context to dictate their interaction with proteolytic machineries in general and the proteasome in particular.The biology of yeast prions has been mostly explored in the context of logarithmically dividing cells. In nature however, most cells are generally in a post-mitotic non-dividing quiescent state. Yet little is known about the fate and properties of prion particles upon yeast cells entry into the stationary or quiescent states and the physiological consequences of harboring these prions throughout the lifespan of yeast cells. In the second part of my thesis, we addressed this issue using the [PSI+] prion as a model. Structurally different conformers of Sup35p aggregates can lead to distinct [PSI+] strains with different prion phenotypes. We found that Sup35p prion particles undergo growth phase-dependent ultrastructural and functional changes. Indeed, the size distributions of SDS-resistant core-prion particles significantly change during growth without affecting the structural information specific to each prion strain. The infectious properties of Sup35p prion particles undergo dramatic growth phase-dependent changes. Importantly, we found that while [PSI+] has little to no effects on the growth rates of yeasts, it robustly prolongs their chronological lifespan. Furthermore, this beneficial effect can then be permanently and efficiently fixed in the cells even when [PSI+] is subsequently lost. Similar genetic fixation of [PSI+]-induced epigenetic characteristics were previously observed and suggested [PSI+] (and possibly other prions) can act as transient evolutionary capacitators.

AABBSSTTRRAACCTT Phosducin (Pdc) is a highly conserved acidic phosphoprotein, which plays an important role in the regulation of G-protein signalization in intact retina. It binds to Gβγ dimer of heterotrimeric G-protein transducin thereby decreases the pool of available transducin resulting in modulation of signal. Function of phosducin is negatively regulated by its phosphorylation followed by interaction with the 14-3-3 protein. Besides this established way of regulation, we were interested in other putative interaction partners of phosducin, like SUG1 and CRX. SUG1 is a subunit of 26S proteasome with a large scale of biological functions, especially a degradation of many transription factors. Its role in regulation of phosducin is still unclear, but is probably involved in targeting of phosducin to 26S proteasome for its degradation. Subsequently, we prepared four different expression constructs of full-length protein in order to find the best expression and purification strategy. These results suggest that all purified fusion proteins of SUG1 form stable and soluble high molecular weight oligomers. This behaviour was confirmed by dynamic light scattering and analytical ultracentrifugation measurements. In addition, this observation is consistent with previous studies of its bacterial counterpart, PAN...

The plant hormone cytokinin plays essential roles in many aspects of growth and development. The cytokinin signal is transmitted by a multistep phosphorelay to the members of two functionally antagonistic classes of Arabidopsis response regulators (ARRs): the type-B ARRs (response activators) and type-A ARRs (negative-feedback regulators). Previous studies have shown that mutations in AXR1, encoding a subunit of the E1 enzyme in the related to ubiquitin (RUB) modification pathway, leads to decreased cytokinin sensitivity. This research shows that the cytokinin resistance of axr1 seedlings is suppressed by loss-of-function of type-A ARRs and that the cytokinin resistance caused by ectopic expression of ARR5, a type-A ARR family member, is enhanced in axr1 background. Based on the established role of the RUB pathway in ubiquitin-dependent proteolysis, these data suggested that AXR1 promotes the cytokinin response by facilitating type-A ARR degradation. Indeed, both genetic (axr1 mutants) and chemical (MLN4924) suppression of RUB E1 increased ARR5 stability, suggesting that the ubiquitin ligase that promotes ARR5 proteolysis requires RUB modification for optimal activity. In addition, ARR1, a type-B ARR family member, also accumulated in the axr1 mutant background, suggesting that AXR1 regulates primary cytokinin signaling at multiple levels.

The proteasome is a major non-lysosomal proteolytic complex present in eukaryotic cells and has a central role in regulating many protein levels. The complex has been shown to participate in various intracellular pathways including cell cycle regulation or quality control of newly synthesized proteins and many other key pathways. This amazing range of substrates would not be possible without the help of regulators that are able to bind to the 20S proteasome and modulate its activity. Among those, the PA700 or 19S regulator and the PA28 family are the best characterized in higher eukaryotes. The 19S regulatory particle is involved in the recognition of ubiquitinated proteins, targeted for degradation by the proteasome. The PA28 (also termed 11S REG) family is composed of two members the PA28αβ and PA28γ. The function of PA28αβ is related to the adaptive immune response with a proposed contribution in MHC class I peptide presentation whereas the biological role PA28γ remains unknown. The main objectives of the laboratory, and subsequently of this thesis are to use Drosophila melanogaster model system and its advantages to better understand the precise contribution of these different activators in the regulation of the proteasome. Using genomic resources, a unique Drosophila PA28 member was identified, characterized and was shown to be a proteasome regulator with all the properties of PA28γ. Through site-directed mutagenesis we identified a functional nuclear localization signal in the homolog-specific insert region. Study of the promoter region revealed that transcription of Drosophila PA28γ (dPA28γ) gene is under control of DREF, a transcription factor involved in the regulation of genes related to DNA synthesis and cell proliferation. To confirm that dPA28γ has a role in cell cycle progression, the effect of removing dPA28γ from S2 cells was tested using RNA interference. Drosophila cells depleted of dPA28γ showed partial arrest in G1/S cell cycle transition confirming a conserved function between Drosophila and mammalian forms of PA28γ. Finally, characterization of the Dictyostelium regulator, an evolutionarily distant member of the PA28γ, was carried out using fluorogenic degradation assays. We are currently knocking-out the gene in order to determine the biological function of the activator. A second part of my work consisted in the generation of a Drosophila assay used to identify in vivo substrates of the 19S regulator, an assay system that has been originally engineered by Dantuma and coworkers in human cell lines. This was achieved by cloning of GFP behind a series of modified ubiquitins that create substrates degraded through different pathways involving the proteasome pathways. The last project of my thesis concerns the characterization of the mechanism for upregulation of proteasomal gene mRNA after MG132 (proteasome inhibitor) treatment. So far, we found that the 5´-UTR of the genes is responsible for this induction. We are now looking for the precise motif involved in this regulation.

Lichens are defined as symbiotic associations composed of a fungal partner, the mycobiont, and one or more photosynthetic partners, the photobiont (1). A currently employed method for the identification of photobionts is the culture of photobiont from the lichen, but this method employs a labor intensive and long cultivation period, thus identification has been neglected. Out of the approximatelyl4,000 lichen described, only about 4% oflichen photobionts have been identified to species (1). In this study we investigated the feasibility of developing a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) identification system for rapidly identifying lichen algae of the genus Trebouxia (In this study we consider Pseudo-Trebouxia part of the genus Trebouxia). DNA was isolated and purified from cultures of each Trebouxia species. A 1300 hp fragment of the 5' region of the nuclear-encoded large subunit (26S) ribosomal RNA genes was amplified by PCR (2). This 5'region of the 26S region is considered to be a byper-variable region because it differs amongst Trebouxia (2) making it a good candidate for RFLP. The sequences were then analyzed with restriction analysis software to determine restriction maps and individual virtual RFLP patterns. Patterns were constructed using the program SPR Opt (SNP and PCR-RFLP Optimization) allowing each Trebouxia species to be identified by a distinctive restriction pattern. We were unable to validate the key due to contamination of materials, which lead to inconclusive data. Future experiments aim to validate the key by comparing the virtual RFLP patterns to the actual patterns obtained for each type culture of each species.

O proteassoma 26S é uma estrutura em forma de barril com um núcleo catalítico 20S que é flanqueado por tampas 19S em ambos os lados. Nosso grupo tem demonstrado que o proteassoma 26S é crítico para o desenvolvimento e sobrevivência do Schistosoma mansoni, sendo que mais de 95% dos casais de vermes tratados com o inibidor de proteassoma MG132 apresentaram alteração na postura dos ovos e viabilidade. O tampão 19S é o complexo regulador e funciona no desdobramento e na desubiquitinação das proteínas, antes de sua entrada no complexo 20S, usando enzimas desubiquitinadoras (DUBS). Recentemente, foi demonstrado que a inibição das DUBS, UCHL5 e USP14, que estão reversivelmente ligadas à partícula 19S do complexo 26S, chamada b-AP15, resulta em autofagia, seguida de morte celular, pois leva uma variedade de células de mamíferos ao estresse oxidativo. O objetivo geral deste trabalho foi avaliar o efeito da droga sobre o desenvolvimento reprodutivo de S. mansoni e como possível alvo terapêutico, tendo como objetivos específicos determinar os seus efeitos: na inibição da oviposição in vitro de casais de parasitas Schistosoma mansoni através da contagem dos ovos e expressão do gene p14; avaliar se os órgãos reprodutores dos parasitas sofreram alguma modificação estrutural; se a substância utilizada leva a autofagia; se o tratamento dos casais de parasita com a droga leva a apoptose, ocasionada pela ativação de caspase-3. Este estudo é o primeiro a documentar o papel da droga b-AP15 como um agente esquistossomicida, pois desencadeia alterações ultraestruturais em casais de vermes de S. mansoni. Os seguintes métodos foram utilizados para analisar as alterações: Microscopia Eletrônica de Transmissão (MET), Varredura (MEV) e Confocal; ensaio quantitativo colorimétrico baseado no brometo de 3- (4,5-dimetiltiazol-2-il) -2,5-difenil tetrazólio (MTT), atividade do proteassoma através do substrato Suc-Leu-Le-Al-Tyr-AMC específico para atividade de quimotripsina-like; western blotting; Reação de Polimerase em Cadeia em tempo real e TUNEL. Foram testadas várias concentrações do fármaco (0,2; 0,4; 0,8; 1,6; 3,2 até 50 µM), sendo que a partir de 1,6 µM de b-AP15 ocorreu a inibição da produção de ovos dos casais de parasitas tratados in vitro por 24 h, não havendo alteração da viabilidade, mas mostrou alterações a partir da dosagem de 3,2 µM. A partir de 1,6 µM ocorreram alterações celulares e tegumentares e vermes adultos tratados com 50 µM estavam mortos. O Western blotting mostrou acúmulo de proteínas poliubiquitinadas de alto peso molecular na presença de 1,6µM, havendo mudanças na atividade quimotripsina-like do proteassoma 20S. Os resultados mostraram que o b-AP15 altera a oviposição, a viabilidade e leva à morte de casais de parasitas, reforçando a hipótese de que o sistema ubiquitina/ proteassoma e que as enzimas deubiquitinadoras ligadas a partícula 19S são essenciais para a biologia de S. mansoni. / The 26S proteasome is a barrel structure with a catalytic core 20S that is flanked by 19S caps on both sides. Our group has been showed that 26S proteasomes are critical for Schistosoma mansoni development and survival, being that more than 95% of worms pairs treated with the proteasome inhibitor MG132 showed alteration on egg laying and viability. The 19S cap is the regulatory complex and functions in unfolding and deubiquitinating the proteins before their entry into the 20S complex using constitutive deubiquitinating enzymes (DUBS). Recently, it has been demonstrated that inhibition of the DUBS, UCHL5 and USP14, which are reversibly bound to the 19S particle of the 26S complex, called b-AP15 , results in cell death because it leads to autophagy, followed by cell death in a variety of mammalian cells. The general objective of this work was to evaluate the effect of the drug on the reproductive development of S. mansoni and as a possible therapeutic target. The specific objectives to determine its effects: in inhibiting the in vitro oviposition of couples of Schistosoma mansoni parasites by counting eggs and p14 gene expression; evaluate whether the reproductive organs of the parasites have undergone some structural modification; if the substance used leads to autophagy; if the treatment of the parasite couples with the drug leads to apoptosis, caused by the activation of caspase-3. This study is the first to document the role of the b-AP15 drug as a schistosomicidal agent, as it triggers ultrastructural changes in couples of S. mansoni worms. The following methods were used to analyze the changes: Transmission Electron Microscopy (MET), Scanning (SEM) and Confocal; Colorimetric assay based on 3- (4,5- dimethylthiazol-2-yl) -2,5-diphenyl tetrazolium bromide (MTT), proteasome activity through Suc-Leu-Le-Al-Tyr-AMC substrate specific for chymotrypsin-like activity; western blotting; Polymerase chain reaction in real time and TUNEL. Various concentrations of the drug (0.2, 0.4, 0.8, 1.6, 3.2 to 50 ?M) were tested. 1.6 ?M b-AP15 occurred the inhibition of egg production of couples of parasites treated in vitro for 24 h, with no change in viability, but showed changes from the dosage of 3.2 ?M. Cell and tegumentary changes occurred in 1.6 ?M and adult worms treated with 50 ?M were dead. Western blotting showed accumulation of high molecular weight polyubiquitinated proteins in the presence of 1.6?M, with changes in the chymotrypsin-like activity of the 20S proteasome. The results showed that b-AP15 alters the oviposition, viability and leads to the death of couples of parasites, reinforcing the hypothesis that the ubiquitin / proteasome system and the deubiquitinating enzymes bound to the 19S particle are essential for the biology of S. mansoni.

O proteassoma 26S é uma estrutura em forma de barril com um núcleo catalítico 20S que é flanqueado por tampas 19S em ambos os lados. Nosso grupo tem demonstrado que o proteassoma 26S é crítico para o desenvolvimento e sobrevivência do Schistosoma mansoni, sendo que mais de 95% dos casais de vermes tratados com o inibidor de proteassoma MG132 apresentaram alteração na postura dos ovos e viabilidade. O tampão 19S é o complexo regulador e funciona no desdobramento e na desubiquitinação das proteínas, antes de sua entrada no complexo 20S, usando enzimas desubiquitinadoras (DUBS). Recentemente, foi demonstrado que a inibição das DUBS, UCHL5 e USP14, que estão reversivelmente ligadas à partícula 19S do complexo 26S, chamada b-AP15, resulta em autofagia, seguida de morte celular, pois leva uma variedade de células de mamíferos ao estresse oxidativo. O objetivo geral deste trabalho foi avaliar o efeito da droga sobre o desenvolvimento reprodutivo de S. mansoni e como possível alvo terapêutico, tendo como objetivos específicos determinar os seus efeitos: na inibição da oviposição in vitro de casais de parasitas Schistosoma mansoni através da contagem dos ovos e expressão do gene p14; avaliar se os órgãos reprodutores dos parasitas sofreram alguma modificação estrutural; se a substância utilizada leva a autofagia; se o tratamento dos casais de parasita com a droga leva a apoptose, ocasionada pela ativação de caspase-3. Este estudo é o primeiro a documentar o papel da droga b-AP15 como um agente esquistossomicida, pois desencadeia alterações ultraestruturais em casais de vermes de S. mansoni. Os seguintes métodos foram utilizados para analisar as alterações: Microscopia Eletrônica de Transmissão (MET), Varredura (MEV) e Confocal; ensaio quantitativo colorimétrico baseado no brometo de 3- (4,5-dimetiltiazol-2-il) -2,5-difenil tetrazólio (MTT), atividade do proteassoma através do substrato Suc-Leu-Le-Al-Tyr-AMC específico para atividade de quimotripsina-like; western blotting; Reação de Polimerase em Cadeia em tempo real e TUNEL. Foram testadas várias concentrações do fármaco (0,2; 0,4; 0,8; 1,6; 3,2 até 50 µM), sendo que a partir de 1,6 µM de b-AP15 ocorreu a inibição da produção de ovos dos casais de parasitas tratados in vitro por 24 h, não havendo alteração da viabilidade, mas mostrou alterações a partir da dosagem de 3,2 µM. A partir de 1,6 µM ocorreram alterações celulares e tegumentares e vermes adultos tratados com 50 µM estavam mortos. O Western blotting mostrou acúmulo de proteínas poliubiquitinadas de alto peso molecular na presença de 1,6µM, havendo mudanças na atividade quimotripsina-like do proteassoma 20S. Os resultados mostraram que o b-AP15 altera a oviposição, a viabilidade e leva à morte de casais de parasitas, reforçando a hipótese de que o sistema ubiquitina/ proteassoma e que as enzimas deubiquitinadoras ligadas a partícula 19S são essenciais para a biologia de S. mansoni. / The 26S proteasome is a barrel structure with a catalytic core 20S that is flanked by 19S caps on both sides. Our group has been showed that 26S proteasomes are critical for Schistosoma mansoni development and survival, being that more than 95% of worms pairs treated with the proteasome inhibitor MG132 showed alteration on egg laying and viability. The 19S cap is the regulatory complex and functions in unfolding and deubiquitinating the proteins before their entry into the 20S complex using constitutive deubiquitinating enzymes (DUBS). Recently, it has been demonstrated that inhibition of the DUBS, UCHL5 and USP14, which are reversibly bound to the 19S particle of the 26S complex, called b-AP15 , results in cell death because it leads to autophagy, followed by cell death in a variety of mammalian cells. The general objective of this work was to evaluate the effect of the drug on the reproductive development of S. mansoni and as a possible therapeutic target. The specific objectives to determine its effects: in inhibiting the in vitro oviposition of couples of Schistosoma mansoni parasites by counting eggs and p14 gene expression; evaluate whether the reproductive organs of the parasites have undergone some structural modification; if the substance used leads to autophagy; if the treatment of the parasite couples with the drug leads to apoptosis, caused by the activation of caspase-3. This study is the first to document the role of the b-AP15 drug as a schistosomicidal agent, as it triggers ultrastructural changes in couples of S. mansoni worms. The following methods were used to analyze the changes: Transmission Electron Microscopy (MET), Scanning (SEM) and Confocal; Colorimetric assay based on 3- (4,5- dimethylthiazol-2-yl) -2,5-diphenyl tetrazolium bromide (MTT), proteasome activity through Suc-Leu-Le-Al-Tyr-AMC substrate specific for chymotrypsin-like activity; western blotting; Polymerase chain reaction in real time and TUNEL. Various concentrations of the drug (0.2, 0.4, 0.8, 1.6, 3.2 to 50 ?M) were tested. 1.6 ?M b-AP15 occurred the inhibition of egg production of couples of parasites treated in vitro for 24 h, with no change in viability, but showed changes from the dosage of 3.2 ?M. Cell and tegumentary changes occurred in 1.6 ?M and adult worms treated with 50 ?M were dead. Western blotting showed accumulation of high molecular weight polyubiquitinated proteins in the presence of 1.6?M, with changes in the chymotrypsin-like activity of the 20S proteasome. The results showed that b-AP15 alters the oviposition, viability and leads to the death of couples of parasites, reinforcing the hypothesis that the ubiquitin / proteasome system and the deubiquitinating enzymes bound to the 19S particle are essential for the biology of S. mansoni.

In total, 84 yeast strains (originated from water, plants, fruits and soil) were selected for MALDI-TOF biotyping. All strains were cultivated on malt agar and YPD medium. Samples for biotyping were processed according to methods of Bruker Daltonik GmbH company, Institute of Chemistry of SAS and combination of these methods. Single strains were identified based on the analysis of intracellular ribozomal proteins using MALDI-TOF mass spectrometry. In case of ambiguous results, the DNA was isolated and the D1/D2 26S rRNA domain sequencing was performed. The strain identification was carried out by comparing its mass spectra with spectra of sequenced strains using MALDI Biotyper 3.0 software. The mutual similarity of strains was considered by score value, which was the result of the analysis. In total, 18 strains from 84 were previously sequenced and used as model strains for comparison with unknown isolates. Altogether 51 strains were definitely taxonomically categorized into 18 phylogenetic groups at the species level. The MALDI-TOF biotyping was repeated for overall 6 strains because of ambiguity of results. The taxonomic classification of 15 strains was not clearly determined and, therefore, these strains were suggested for D1/D2 26S rRNA domain sequencing. It was not possible to identify one strain, based on the results of sequencing, therefore, the DNA isolation was repeated. In the case of 8 strains, the results were identical with originally designed taxonomic classification. Conversely, the remaining 6 strains were identified as species. For 20 selected strains the basic characteristics were determined using microbiological methods. The shape of colonies growing on solid medium and appearance of cultures in liquid medium was assessed. Furthermore, the radial growth constant and the presence of urease were determined. Finally the microscopic observation of cells and the fermentation test for carbohydrate substrates were performed.

Das Ubiquitin-Proteasom-System gewährleistet in eukaryontischen Zellen den regulierten Abbau der meisten intrazellulären Proteine und ist mit der Generierung von T-Zell-Epitopen grundlegend an der Immunantwort beteiligt. Wegen der Komplexität des Systems stand ein in vitro Verfahren zur 26S proteasomalen Prozessierung eines ubiquitinierten Modellsubstrates erstmals im Jahr 2000 zur Verfügung. In der vorliegenden Arbeit gelang es, diese von Thrower et al. publizierte Methode hinsichtlich einer größeren Proteinquantität zu optimieren und auf die in vitro Ubiquitinierung und Degradation von Proteinen anzuwenden, deren proteasomaler Abbau unter dem Aspekt der Epitopgenerierung immunologisch von besonderer Relevanz ist. Die biochemische und massenspektrometrische Analyse der 26S proteasomalen Degradation von penta-ubiquitinierten Derivaten des tumorassoziierten Glykoproteins Mucin1 zeigte erstens, dass die Prozessierung der Substrate zu einem 26S proteasomalen gating-Effekt führt, der von Substratbindung und -deubiquitinierung sowie von ATP-Hydrolyse abhängig ist. Zweitens ließ sich als Folge der Substratprozessierung eine Instabilisierung des 26S Proteasoms beobachten, die auf einen Assoziations-Dissoziations-Zyklus hindeutet. Drittens ergab die Untersuchung zum Einfluss des Proteasom-Aktivators PA28 auf den Abbau eines Mucin1-Polyepitops, dass PA28 eine erhöhte Quantität an 26S proteasomal generiertem Epitop verursacht. Viertens war unter Zuhilfenahme des Inhibitors clasto-Lactacystin und anhand der ubiquitinierten Mucin1-Derivate erstmals für ubiquitinierte Substrate nachweisbar, dass die proteasomale katalytische Untereinheit beta5 für die Proteindegradation durch den 26S-Komplex nicht essentiell ist. Das in der vorliegenden Arbeit etablierte Abbausystem hat zudem sein Potential unter Beweis gestellt, als ein in vivo-nahes in vitro Testsystem zu fungieren, um die Wirksamkeit von Proteasom-Inhibitoren sowie die Prozessierbarkeit von Polyepitop-Vakzinen zu bewerten. / The ubiquitin-proteasome pathway plays a major role in cellular protein degradation. By generating T-cell epitopes it contributes essentially to the immune response. The complexity of the system impeded the establishment of an in vitro approach that would make a detailed analysis of the protein degradation by the 26S proteasome possible. Finally, in 2000 Thrower et al. published an in vitro method enabling the synthesis and degradation of an ubiquitinated model substrate by the 26S proteasome. In the study presented here the approach of Thrower et al. was improved by scaling up and by the synthesis of substrates the degradation of which by the 26S proteasome is of great importance immunologically, mainly in regard to the generation of T-cell epitopes. In vitro synthesised penta-ubiquitinated versions of the tumor-associated glycoprotein mucin1 were processed by purified 26S proteasomes. The analysis of substrate degradation and product generation by biochemistry and mass spectrometry showed firstly, that substrate processing initiated a 26S proteasomal gating effect which depends on substrate binding to the proteasome, deubiquitination and proteasomal ATP-hydrolysis. Secondly, a disassembly of the 26S proteasome was observed following substrate processing which suggests a regulated association-dissociation cycle of the proteasome. Thirdly, supplementation of the degradation approach with the proteasome activator PA28 led to a higher quantity of epitope generated by the 26S proteasome out of a mucin1 polyepitope string. Fourthly, use of the proteasome inhibitor clasto-Lactacystin revealed, for the first time, that the catalytic proteasome subunit beta5 is not essential for the degradation of ubiquitinated protein substrates by the 26S proteasome. In addition, the in vitro approach established in the presented study has shown its potential to function as an in vivo-like system which helps to assess proteasome inhibitors and potential polyepitope vaccines.