Degradation of membrane proteins in the vacuole/lysosome is dependent on their prior sorting into the multivesicular body (MVB) pathway. This sorting process involves incorporation of proteins into vesicles that are formed by budding of the limiting membrane of the endosome into the lumen of the organelle. The MVB sorting process on the whole is highly conserved from yeast to human, and depends on the Vps27/Hrs, ESCRT-I, -II, and -III protein complexes functioning sequentially on the endosomal membrane, as well as on additional factors, such as the ubiquitinating enzyme Rsp5/Nedd4. It has now been established that ubiquitin serves as a sorting signal for many cargoes into the MVB pathway. In this thesis work, we provide evidence that Bro1 is not required for protein ubiquitination or early steps of endocytosis, but functions at the late endosome level as an integral component of the MVB pathway. Similarly to its human homologue Alix, Bro1 interacts with components of the ESCRT-I and ESCRT-III complexes. The putative role of Bro1/Alix in bridging an interaction between ESCRT-I and –III might be important to strengthen an association of these protein complexes to allow efficient sorting of cargo proteins. Deficiency in Bro1 results in recycling of the endocytosed Gap1 permease back to the plasma membrane, a process coupled to deubiquitination of the permease. This recycling is a non-classical phenotype for cells impaired in MVB pathway thus suggesting Bro1 to have a particular role in this sorting process. Furthermore, the conserved C-terminal proline-rich domain (PRD) of Bro1 is specifically important for MVB sorting of cargo proteins that are subject to ubiquitination. We show Bro1 (via its PRD) to play a highly important role in recruitment of the deubiquitinating enzyme Doa4 to the endosome. Consistent with this, Bro1 is required for deubiquitination of cargo proteins, a step occurring just before cargo incorporation into the endosomal vesicles, and similarly to Doa4, for ubiquitin recycling. In contrast to previous interpretations, we show that Doa4 has a direct role in sorting of ubiquitinated cargo proteins into the MVB pathway. We propose that Doa4 – via its association to Bro1 - achieves this role by catalyzing deubiquitination of cargo proteins and/or some components of the MVB sorting machinery. We further show Bro1 to interact with the ubiquitin ligase Rsp5, which, in addition to being required for cargo protein ubiquitination at the plasma membrane, apparently contributes to multiple steps of endocytosis and MVB sorting. Also the Bro1-Rsp5 interaction is dependent on the C-terminal PRD region of Bro1. We propose that this interaction is conserved. A role for ubiquitin in regulation of the MVB sorting machinery is emerging: the function of factors recognizing and sorting ubiquitinated cargo proteins in the MVB pathway is suggested to be coupled to their cycling between ubiquitinated and deubiquitinated stages. A growing body of evidence indicates that ubiquitin ligases of the Rsp5/Nedd4 family play a central role in this regulation. We speculate the Bro1/Alix protein, through its ability to simultaneously interact with factors of the MVB sorting machinery and with ubiquitinating and deubiquitinating enzymes to play a central role in the successive rounds of ubiquitination and deubiquitination of specific factors along the MVB pathway.

The maturation of early endosomes into multivesicular bodies (MVBs) and subsequent trafficking to lysosomes is an important event for the control and silencing of endocytosed membrane receptors. The endosomal-sorting complex required for transport (ESCRT) proteins appear to play a key role in this event. Phosphatidylinositol lipids including PtdIns(3,5)P2 have been implicated in the MVB-lysosomal pathway and an ESCRT-III component CHMP3 binds to this lipid in vitro. The purpose of this thesis was to investigate the link between ESCRT proteins, PtdIns(3,5)P2 and endo-lysosomal trafficking. Firstly, a protein expressed by Salmonella, which is a phosphatase that acts on PtdIns(3,5)P2, was investigated as a potential tool for manipulating cellular PtdIns(3,5)P2 levels. Our results suggest that it is potentially a useful tool for this purpose and that expression of SopB perturbs endosome to lysosome trafficking. These findings provide further evidence for a role of PtdIns(3,5)P2 in endo-lysosomal trafficking.

Ubiquitination is a post-translational modification tht mediates sorting of integral membrane proteins to lysosomes for their degradation. ESCRTs (Endosomal Sorting Complex Required For Transport) bind and sequester ubiquitinated membrane proteins and direct them into multivesicular bodies (MVBs). ESCRTs themselves become covalently ubiquitinated, simply by virtue of non-covalently binding Ub. However, it is unclear whether this regulates a critical aspect of ESCRT function. In yeast, many MVB cargo proteins are ubiquitinated by the HECT-type Ub-ligase Rsp5, sometimes via the action of Rsp5 adaptor proteins. While many Rsp5 targets are modified by polyubiquitination, it remains unclear whether polyubiquitination is a necessary signal for their incorporation into MVBs. Despite years of research, these and related questions have been difficult to resolve because it is technically quite challenging to control the level of a given protein's ubiquitination. The aim of this research was to develop a novel technique, which can render proteins resistant to ubiquitination. The technique involved the fusion of the Ub-peptidase to a protein of interest via a flexible linker, essentially creating a "DUb module". The intent of this module would be to cleave any Ub form the target protein, essentially immunizing it from the effects of ubiquitination. This novel method was used in combination with several conventional methods to examine the role of ubiquitination within the endocytic pathway and in particular focus on the questions of what type of ubiquitin signal was sufficient for sorting into MVB vesicles and whether ubiquitination of ESCRTs was required for their sorting activity. We found that a single Ub was sufficient for membrane protein entry into MVBs in the absence of ESCRT ubiquitination.

A degradação lisossomal de proteínas de membrana endocitadas ocorre por meio do direcionamento destas proteínas para vesículas intralumenais (ILVs), formadas no lúmen dos corpos multivesiculares (MVBs), e subsequente fusão dos MVBs com lisossomos. Apesar de sua importância na degradação de proteínas transmembrana, os MVBs possuem outra importante função, a de produzir e liberar vesículas extracelulares (EVs). Neste processo os MVBs não se fundem com lisossomos, mas sim com a membrana plasmática o que resulta na liberação das vesículas residentes no interior dos MVBs para o meio extracelular. Diversas proteínas participam do direcionamento de cargas para os MVBs. Os estudos que delinearam a via de tráfego mediada por AP1 concentraram-se nos complexos contendo a subunidade ?1 que medeia o transporte de proteínas entre a rede trans-Golgi (TGN) e endossomos. Contudo, o genoma humano codifica uma segunda isoforma desta subunidade, denominada ?2, e evidências presentes na literatura e também observadas por nosso grupo indicam que AP1?2 pode regular uma via de tráfego distinta da via classicamente atribuída a AP1. Utilizando ensaios de uptake de EGF em condições onde foi realizado o KD de ?1 ou ?2, foi observado que o silenciamento de ?2 prejudica a degradação de EGF internalizado por seu receptor. Efeito também observado para o próprio receptor de EGF (EGFR) em ensaios de biotinilação da superfície celular. Demonstrando que a degradação lisossomal do complexo EGF-EGFR pela via canônica dos MVBs requer o complexo AP1?2, mas não AP1?1. Em conjunto com este estudo também foi analisado o mecanismo molecular de direcionamento da proteína Nef do HIV-1 para os MVBs associados a liberação de EVs. A proteína Nef do HIV é determinante na modulação do ambiente intracelular favorecendo a replicação do vírus e progressão à AIDS. Nef é ativamente secretado em EVs e sua liberação pode levar a apoptose de células vizinhas aceptoras dessas vesículas. Nef também medeia a redução dos níveis de CD4 e moléculas de MHC-I em EVs. Ainda não é conhecido o mecanismo molecular utilizado por Nef para ser exportado em EVs, mas sabe-se que Nef interage fisicamente com a proteína II acessória da maquinaria ESCRT, Alix, importante no processo de formação das ILVs e seleção das cargas que serão internalizadas nos MVBs. EVs coletadas de células HeLa e linfócitos T CD4+ silenciados para Alix demostraram reduções significativas na liberação de Nef. Estes resultados indicam que Nef requer Alix para sua eficiente liberação em EVs. / Lysosomal degradation of endocytosed membrane proteins occurs through the targeting of these proteins to intraluminal vesicles (ILVs), formed in the multivesicular bodies (MVBs) lumen, and the subsequent fusion of MVBs with lysosomes. Despite its importance in the degradation of transmembrane proteins, MVBs have another important function, the production and release of extracellular vesicles (EVs). In this process, the MVBs do not fuse with lysosomes, but fuse with the plasma membrane resulting in the release of these vesicles that reside within MVBs to the extracellular environment. Several proteins regulate the targeting of cargo to MVBs. Studies that delineated the functions of AP1 in protein trafficking, focused on complexes containing the ?1 subunit, which mediates transport between trans-Golgi network (TGN) and endosomes. However, the human genome encodes a second isoform of this subunit, named ?2. Evidences from the literature, as well as results from our research group, indicate that AP1?2 regulates transport pathways that are distinct from the pathways classically attributed to AP1. By performing EGF-uptake assays under ?1 or ?2 knockdown (KD) conditions, it was observed that ?2 is required for degradation of internalized EGF, effect also observed for the EGF receptor (EGFR) using cell surface biotinylation assays. These results demonstrate that the lysosomal degradation of the EGFEGFR complexes via the canonical MVBs pathway requires the AP1?2 complex, but not AP1?1. In parallel with this study, we also analyzed the molecular mechanism of HIV-1 Nef targeting to MVBs associated with the EVs release. Nef is an important determinant in the modulation of the intracellular environment for efficient HIV replication and progression to AIDS. Nef is actively secreted via EVs and its release may lead to apoptosis of bystander acceptor cells. Moreover, Nef reduces the levels of CD4 and MHC-I molecules in EVs. Despite the importance of Nef release in EVs, the molecular mechanism used by Nef to be exported via EVs is unknown. Nef physically interacts with the ESCRT machinery accessory protein Alix, an important player in the process of ILVs formation and cargo selection. EVs released from HeLa cells and CD4+ T lymphocytes under Alix KD conditions demonstrated a significant IV reduction in Nef release via EVs. These results indicate that Nef requires Alix for its efficient release in EVs.

A degradação lisossomal de proteínas de membrana endocitadas ocorre por meio do direcionamento destas proteínas para vesículas intralumenais (ILVs), formadas no lúmen dos corpos multivesiculares (MVBs), e subsequente fusão dos MVBs com lisossomos. Apesar de sua importância na degradação de proteínas transmembrana, os MVBs possuem outra importante função, a de produzir e liberar vesículas extracelulares (EVs). Neste processo os MVBs não se fundem com lisossomos, mas sim com a membrana plasmática o que resulta na liberação das vesículas residentes no interior dos MVBs para o meio extracelular. Diversas proteínas participam do direcionamento de cargas para os MVBs. Os estudos que delinearam a via de tráfego mediada por AP1 concentraram-se nos complexos contendo a subunidade ?1 que medeia o transporte de proteínas entre a rede trans-Golgi (TGN) e endossomos. Contudo, o genoma humano codifica uma segunda isoforma desta subunidade, denominada ?2, e evidências presentes na literatura e também observadas por nosso grupo indicam que AP1?2 pode regular uma via de tráfego distinta da via classicamente atribuída a AP1. Utilizando ensaios de uptake de EGF em condições onde foi realizado o KD de ?1 ou ?2, foi observado que o silenciamento de ?2 prejudica a degradação de EGF internalizado por seu receptor. Efeito também observado para o próprio receptor de EGF (EGFR) em ensaios de biotinilação da superfície celular. Demonstrando que a degradação lisossomal do complexo EGF-EGFR pela via canônica dos MVBs requer o complexo AP1?2, mas não AP1?1. Em conjunto com este estudo também foi analisado o mecanismo molecular de direcionamento da proteína Nef do HIV-1 para os MVBs associados a liberação de EVs. A proteína Nef do HIV é determinante na modulação do ambiente intracelular favorecendo a replicação do vírus e progressão à AIDS. Nef é ativamente secretado em EVs e sua liberação pode levar a apoptose de células vizinhas aceptoras dessas vesículas. Nef também medeia a redução dos níveis de CD4 e moléculas de MHC-I em EVs. Ainda não é conhecido o mecanismo molecular utilizado por Nef para ser exportado em EVs, mas sabe-se que Nef interage fisicamente com a proteína II acessória da maquinaria ESCRT, Alix, importante no processo de formação das ILVs e seleção das cargas que serão internalizadas nos MVBs. EVs coletadas de células HeLa e linfócitos T CD4+ silenciados para Alix demostraram reduções significativas na liberação de Nef. Estes resultados indicam que Nef requer Alix para sua eficiente liberação em EVs. / Lysosomal degradation of endocytosed membrane proteins occurs through the targeting of these proteins to intraluminal vesicles (ILVs), formed in the multivesicular bodies (MVBs) lumen, and the subsequent fusion of MVBs with lysosomes. Despite its importance in the degradation of transmembrane proteins, MVBs have another important function, the production and release of extracellular vesicles (EVs). In this process, the MVBs do not fuse with lysosomes, but fuse with the plasma membrane resulting in the release of these vesicles that reside within MVBs to the extracellular environment. Several proteins regulate the targeting of cargo to MVBs. Studies that delineated the functions of AP1 in protein trafficking, focused on complexes containing the ?1 subunit, which mediates transport between trans-Golgi network (TGN) and endosomes. However, the human genome encodes a second isoform of this subunit, named ?2. Evidences from the literature, as well as results from our research group, indicate that AP1?2 regulates transport pathways that are distinct from the pathways classically attributed to AP1. By performing EGF-uptake assays under ?1 or ?2 knockdown (KD) conditions, it was observed that ?2 is required for degradation of internalized EGF, effect also observed for the EGF receptor (EGFR) using cell surface biotinylation assays. These results demonstrate that the lysosomal degradation of the EGFEGFR complexes via the canonical MVBs pathway requires the AP1?2 complex, but not AP1?1. In parallel with this study, we also analyzed the molecular mechanism of HIV-1 Nef targeting to MVBs associated with the EVs release. Nef is an important determinant in the modulation of the intracellular environment for efficient HIV replication and progression to AIDS. Nef is actively secreted via EVs and its release may lead to apoptosis of bystander acceptor cells. Moreover, Nef reduces the levels of CD4 and MHC-I molecules in EVs. Despite the importance of Nef release in EVs, the molecular mechanism used by Nef to be exported via EVs is unknown. Nef physically interacts with the ESCRT machinery accessory protein Alix, an important player in the process of ILVs formation and cargo selection. EVs released from HeLa cells and CD4+ T lymphocytes under Alix KD conditions demonstrated a significant IV reduction in Nef release via EVs. These results indicate that Nef requires Alix for its efficient release in EVs.

No description available.

CE TRAVAIL A PORTE SUR L'ETUDE DE LA PROTEINE MULTIMODULAIRE ALIX CHEZ DICTYOSTELIUM DISCOIDEUM, UN ORGANISME EUCARYOTE Où DEVELOPPEMENT MULTICELLULAIRE ET PROGRAMME DE MORT CELLULAIRE SONT ETROITEMENT LIES. L'INVALIDATION D'ALX MONTRE QUE CETTE PROTEINE EST ESSENTIELLE A LA DIFFERENCIATION CELLULAIRE ET A LA MORPHOGENESE AU COURS DU DEVELOPPEMENT MULTICELLULAIRE MAIS PAS AU PROGRAMME DE MORT. UNE APPROCHE STRUCTURE/FONCTION A REVELE QUE LE DOMAINE SUSCEPTIBLE DE PARTICIPER A DES TORSADES D'HELICES DE CETTE PROTEINE EST INDISPENSABLE A SA FONCTION DEVELOPPEMENTALE. DE PLUS, SA DISTRIBUTION CELLULAIRE INDIQUE QU'ALIX EST LOCALISE DANS LA VOIE ENDOCYTAIRE EN ASSOCIATION AVEC LA MACHINERIE ESCRT (ENDOSOMAL SORTING COMPLEX REQUIRED FOR TRANSPORT), IMPLIQUEE DANS LE TRI ET LA FORMATION DU CORPS MULTIVESICULAIRE (MVB). CEPENDANT, L'INVALIDATION DES GENES CODANTS POUR DIFFERENTS COMPOSANTS DES COMPLEXES ESCRT INDUIT DES PHENOTYPES DISSEMBLABLES. CECI SUGGERE QUE SOIT CES PROTEINES ONT DES ROLES DANS D'AUTRES PROCESSUS CELLULAIRES SOIT QUE CETTE VOIE N'EST PAS PERTURBEE DE LA MEME FAÇON DANS LES DIFFERENTS MUTANTS.

Degradation of membrane proteins in the vacuole/lysosome is dependent on their prior sorting into the multivesicular body (MVB) pathway. This sorting process involves incorporation of proteins into vesicles that are formed by budding of the limiting membrane of the endosome into the lumen of the organelle. The MVB sorting process on the whole is highly conserved from yeast to human, and depends on the Vps27/Hrs, ESCRT-I, -II, and -III protein complexes functioning sequentially on the endosomal membrane, as well as on additional factors, such as the ubiquitinating enzyme Rsp5/Nedd4. It has now been established that ubiquitin serves as a sorting signal for many cargoes into the MVB pathway. <p>In this thesis work, we provide evidence that Bro1 is not required for protein ubiquitination or early steps of endocytosis, but functions at the late endosome level as an integral component of the MVB pathway. Similarly to its human homologue Alix, Bro1 interacts with components of the ESCRT-I and ESCRT-III complexes. The putative role of Bro1/Alix in bridging an interaction between ESCRT-I and –III might be important to strengthen an association of these protein complexes to allow efficient sorting of cargo proteins. Deficiency in Bro1 results in recycling of the endocytosed Gap1 permease back to the plasma membrane, a process coupled to deubiquitination of the permease. This recycling is a non-classical phenotype for cells impaired in MVB pathway thus suggesting Bro1 to have a particular role in this sorting process. Furthermore, the conserved C-terminal proline-rich domain (PRD) of Bro1 is specifically important for MVB sorting of cargo proteins that are subject to ubiquitination. We show Bro1 (via its PRD) to play a highly important role in recruitment of the deubiquitinating enzyme Doa4 to the endosome. Consistent with this, Bro1 is required for deubiquitination of cargo proteins, a step occurring just before cargo incorporation into the endosomal vesicles, and similarly to Doa4, for ubiquitin recycling. In contrast to previous interpretations, we show that Doa4 has a direct role in sorting of ubiquitinated cargo proteins into the MVB pathway. We propose that Doa4 – via its association to Bro1 - achieves this role by catalyzing deubiquitination of cargo proteins and/or some components of the MVB sorting machinery. We further show Bro1 to interact with the ubiquitin ligase Rsp5, which, in addition to being required for cargo protein ubiquitination at the plasma membrane, apparently contributes to multiple steps of endocytosis and MVB sorting. Also the Bro1-Rsp5 interaction is dependent on the C-terminal PRD region of Bro1. We propose that this interaction is conserved. <p>A role for ubiquitin in regulation of the MVB sorting machinery is emerging: the function of factors recognizing and sorting ubiquitinated cargo proteins in the MVB pathway is suggested to be coupled to their cycling between ubiquitinated and deubiquitinated stages. A growing body of evidence indicates that ubiquitin ligases of the Rsp5/Nedd4 family play a central role in this regulation. We speculate the Bro1/Alix protein, through its ability to simultaneously interact with factors of the MVB sorting machinery and with ubiquitinating and deubiquitinating enzymes to play a central role in the successive rounds of ubiquitination and deubiquitination of specific factors along the MVB pathway. <p> / Doctorat en sciences, Spécialisation biologie moléculaire / info:eu-repo/semantics/nonPublished

The high specific energy consumption from all existing seawater desalination methods has heightened the motivation for having more efficient and greener desalination processes to meet the future goals of sustainable seawater desalination. One of the promising thermally-driven desalination methods is the direct-contact spray evaporation and condensation (DCSEC) where the excess enthalpy between feed and equilibrium states of evaporator chambers is exploited with reasonably high flashing efficiency. Further improvements in energy efficacy of DCSEC are boosted by firstly the incorporation of micro/nano-bubbles (M/NB) where micro or nano size subcooled vapor are embedded in the sprayed liquid droplets of evaporator, thereby lowering the temperature brine in evaporator and minimizing the thermal equilibrium effect of brine. The presence of subcooled bubbles increased the available surface area for heat transfer. Secondly, the concept of an evaporator-condenser pair of DCSEC could be extended to a multi-stage arrangement where the latent heat of vapor condensing on the water droplets sprayed within the condenser is recovered. From the experiments, the effect of incorporating the (M/NB) in the DCSEC at optimum feed flow rate yields more than 34% increase in distillate production at feed temperatures greater 47oC and the cooling inlet temperature set at 35oC. The other salient improvement found from the experiments is the increase in performance ratio (PR) up to 3.3 for a 6-stage configuration. This quantum jump in the PR is attributed to the heat recovery effect by as much as 70% of the total heat input. Arising from the DCSEC design, the implicit benefits are the low capital and operational cost, i.e., low CAPEX and OPEX. The former savings is attributed zero physical interfaces such as tube-based heat exchangers or membranes, whilst the latter savings is contributed by significant lesser use of chemicals in the pre-treatment of seawater feed. Lastly, the accompanied benefit is the robustness of the DCSEC processes where it could within stand high salinity of the brine, typically as high as 200,000 ppm.

Regression analysis is very popular among researchers in various fields but almost all the researchers use the classical methods which assume that X is nonstochastic and the error is normally distributed. However, in real life problems, X is generally stochastic and error can be nonnormal. Maximum likelihood (ML) estimation technique which is known to have optimal features, is very problematic in situations when the distribution of X (marginal part) or error (conditional part) is nonnormal. Modified maximum likelihood (MML) technique which is asymptotically giving the estimators equivalent to the ML estimators, gives us the opportunity to conduct the estimation and the hypothesis testing procedures under nonnormal marginal and conditional distributions. In this study we show that MML estimators are highly efficient and robust. Moreover, the test statistics based on the MML estimators are much more powerful and robust compared to the test statistics based on least squares (LS) estimators which are mostly used in literature. Theoretically, MML estimators are asymptotically minimum variance bound (MVB) estimators but simulation results show that they are highly efficient even for small sample sizes. In this thesis, Weibull and Generalized Logistic distributions are used for illustration and the results given are based on these distributions. As a future study, MML technique can be utilized for other types of distributions and the procedures based on bivariate data can be extended to multivariate data.