Thesis (Ph. D.)--University of Washington, 1999. / Vita. Includes bibliographical references (leaves 82-90).

Thesis (M.SC.) - Carleton University, 2007. / Includes bibliographical references (p. 92-103). Also available in electronic format on the Internet.

Thesis (Ph. D.)--University of Chicago, Dept. of Neurobiology, Pharmacology and Physiology, December 2002. / Includes bibliographical references. Also available on the Internet.

Thesis (Ph. D.)--West Virginia University, 2005. / Title from document title page. Document formatted into pages; contains vi, 124 p. : ill. Vita. Includes abstract. Includes bibliographical references.

Thesis (Ph. D.)--University of California, San Diego and San Diego State University, 2006. / Vita. Includes bibliographical references (leaves 90-106).

Thesis (Ph. D.)--University of Missouri-Columbia, 2000. / Typescript. Vita. Includes bibliographical references (leaves 130-155). Also available on the Internet.

The eukaryotic endoplasmic reticulum (ER) is a multifunctional organelle, primarily responsible for the folding and maturation of secretory proteins, as well as lipid metabolism, calcium homeostasis, ubiquitin-dependent signalling and cell fate decisions. ER-associated degradation (ERAD) oversees protein folding and delivers misfolded proteins for degradation by the proteasome via ubiquitin conjugation mediated by RING-type E3 ubiquitin ligases. An intact ERAD is crucial to cellular homeostasis, as unresolved protein imbalances cause ER stress that ultimately lead to apoptosis. The human ER accommodates at least 25 E3s, however our understanding is mostly limited to Hrd1 and AMFR/gp78, both of which have a defined function in ERAD. To understand the contribution of ER E3s to cellular and organelle homeostasis, this study used mass spectrometry of purified E3 complexes to identify cofactors and build interaction networks of ER-resident E3s. These findings will form the foundation for investigating the biological roles of these ubiquitin ligases. Transcriptional analysis highlighted the centrality of Hrd1 among all ER-resident E3s in response to protein misfolding in the ER. Additionally, the contribution of individual Hrd1 complex components to resolving proteotoxic stress was assessed using a misfolded antibody subunit (IgM heavy chain), rather than conventional pharmacological treatments. The ERAD components essential for substrate degradation and survival under proteotoxic stress were identified, highlighting the pivotal role of Hrd1, its cofactor SEL1L and the Derlin family members. Finally, it was demonstrated that autophagy induction in response to proteasome inhibition is key to relieve the burden of protein misfolding in the ER, as it sustained the survival of cells defective for ERAD. Importantly, this study proposes a potential involvement of Hrd1 in signalling from the ER to autophagy, suggesting potential crosstalk between the ERAD and autophagic pathways.

Submitted by Marco Antônio de Ramos Chagas (mchagas@ufv.br) on 2016-10-06T17:03:40Z No. of bitstreams: 1 texto completo.pdf: 247331 bytes, checksum: ae57be7c82f87c29d3a84555f4712a48 (MD5) / Made available in DSpace on 2016-10-06T17:03:40Z (GMT). No. of bitstreams: 1 texto completo.pdf: 247331 bytes, checksum: ae57be7c82f87c29d3a84555f4712a48 (MD5) Previous issue date: 2004-07-30 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / O dobramento de proteínas secretórias no lúmen do retículo endoplasmático é altamente facilitado por chaperones moleculares. A proteína BiP é um dos chaperones mais bem caracterizados residentes do RE. BiP tem demonstrado ser um modulador multifuncional de vários processos que ocorrem no retículo endoplasmático. Essa proteína auxilia no dobramento e enovelamento das cadeias polipeptídicas nascentes e exerce papel no controle de qualidade do RE, reconhecendo e direcionando proteínas incorretamente dobradas para degradação. Também funciona como um sensor da via de resposta a proteínas incorretamente enovelada (UPR), regulando indiretamente a atividade da cinase eIF-2 e a sua própria expressão. As proteínas de reserva são sintetizadas em ribossomos associados à membrana do retículo endoplasmático, sendo co-traducionalmente translocadas para o lúmen do RE, onde podem permanecer associadas em corpos protéicos ou ser transportadas, via complexo de Golgi, para vacúolos de proteínas de reserva. Evidências na literatura indicam que BiP associa-se com proteínas de reserva in vitro, e seu acúmulo nas sementes está coordenado com a síntese de proteínas de reserva. Nesta investigação, foram analisados o nível de BiP nos diferentes estádios de desenvolvimento de sementes de soja e a correlação entre as concentrações de BiP e de proteínas de reserva de sementes. Para isso, utilizaram-se sementes de soja das variedades CAC-1 e CC3 com diferentes teores de proteína, determinados pelo método Kjeldahl. BiP acumulou-se predominantemente nos estádios iniciais de desenvolvimento das sementes, coincidindo com a síntese ativa de proteínas de reserva. O acúmulo temporal de BiP foi significativamente superior nas sementes CC3 que apresentam maior teor protéico. A capacidade de BiP associar-se a proteínas de reserva foi funcionalmente avaliada por meio do sistema duplo híbrido. Este ensaio demonstrou que BiP interage eficientemente com a subunidade de - conglicinina em leveduras. A partir desses resultados, surgiu a hipótese de que um aumento da expressão de BiP poderia resultar em elevação do teor de proteína das sementes. O efeito da superexpressão da proteína BiP no teor de proteína total foi diretamente avaliado em sementes de Nicotiana tabacum transgênicas (geração T4) superexpressando o gene BiP da soja. Embora o nível de BiP nas sementes transgênicas, detectado por immunoblotting e quantificado por densitometria, tenha sido maior nas linhagens transgênicas senso em relação às controle e anti-senso, a superprodução constitutiva de BiP não foi correlacionada diretamente com o aumento do teor protéico, determinado pelo método Kjeldahl. Esses resultados sugerem que a capacidade de processamento do retículo endoplasmático em tabaco durante o desenvolvimento da semente não constitui o fator limitante do processo de síntese e acúmulo de proteínas de reserva. / The folding of secretory proteins within the lumen of the endoplasmic reticulum (ER) is greatly facilitated by molecular chaperones. The binding protein BiP is one of the best characterized ER-resident molecular chaperones. In mammalian cells, BiP has been demonstrated to serve as a multifunctional modulator of various ER-supported processes including regulation of eIF-2 kinase and mRNA translation, regulation of BiP expression, and the catalysis of protein folding, as well as, potentially, the targeting of misfolded proteins for degradation. The storage proteins from soybean are synthesized in ER membrane-bound polyribosomes and through a Golgi-mediated translocation are deposited into specialized vacuoles, designated protein bodies. We have previously demonstrated that BiP associated detectably with storage proteins in vitro and the efficiency of BiP synthesis correlated with the accumulation of seed storage protein, such that soybean cultivars that exhibited a greater content of storage proteins also accumulated higher levels of BiP. In this investigation, we further analyzed the synthesis of soybean BiP during seed development and the naturally occurring correlation between the content of BiP and seed storage proteins. Immunoblottings of total protein extracts from CAC1 (normal protein content) and CC3 (high protein content) cultivars demonstrated that, during the seed development, BiP accumulates to high levels at developmental stages that coincide with the onset of active storage protein synthesis. We further characterized the association of BiP and β-conglycinin storage proteins though the two-hybrid system. In order to understand whether an increase in BiP levels would promote a concomitant increase in seed storage protein accumulation, we obtained tobacco transgenic seeds-overexpressing a soybean BiP gene. The effect of BiP overexpression on total protein accumulation was directly evaluated in Nicotiana tabacum homozygous transgenic seeds (T3 generation). The BiP protein levels detected in the transgenic seeds were significantly higher than those of wild type and antisense BiP-transformed seeds. Nevertheless, total protein from seeds-overexpressing BiP, as determined by Kjeldahl, did not differ significantly from that of wild type and antisense seeds. These results suggest, although do not prove, that the capacity of ER processing during seed development does not constitute a rate limiting process for storage protein synthesis and accumulation. / Dissertação importada do Alexandria

A imunodeficiência comum variável (CVID) é caracterizada por hipogamaglobulinemia. Anteriormente identificou-se uma paciente com CVID que apresenta nível aumentado de estresse de retículo endoplasmático (ER), secundário a desregulação da via UPR. No presente trabalho, estendemos esta análise para outros pacientes e avaliamos o perfil de maturação de seus linfócitos B. Métodos: Western-blot, RT-PCR, Q-PCR, Citometria de Fluxo e cultura de células B ex vivo e imortalizadas. Resultados: A análise de 16 pacientes com CVID e 9 indivíduos saudáveis revelou três pacientes com porcentagens aumentadas de linfócitos B imaturos no sangue periférico. A análise da expressão de RNAm para BiP e XBP-1 em linfócitos B destes pacientes, após estímulo com LPS in vitro, identificou que os linfócitos B de um deles apresenta estresse de RE. Conclusão: Identificamos um subgrupo de pacientes com CVID que apresentam linfócitos B imaturos no sangue periférico. Um membro deste subgrupo apresenta estresse aumentado de ER. / Common Variable Immunodeficiency (CVID) is characterized by hypogammaglobulinemia. Previously a CVID patient was identified with increased levels of Endoplasmic Reticulum (ER) stress due to dysregulation of the UPR. In the present study these analyses were performed in other patients and healthy donors. Maturation markers of B lymphocytes were also characterized in these individuals. Methods: Western-blot, RT-PCR, Q-PCR, Flow cytometry and culturing of ex vivo and immortalized B cells. Results: The analysis of 16 CVID patients and 9 healthy donors revealed three patients that present higher percentage of immature B cells in peripheral blood. Analysis of expression of BiP and XBP1 induced by LPS treatment of B lymphocytes from these patients revealed that one patient present increased levels of ER stress.

Nonalcoholic fatty liver disease (NAFLD) is categorized into nonalcoholic fatty liver (NAFL) and nonalcoholic steatohepatitis (NASH) and has emerged as a risk factor for more critical clinical conditions. However, the underlying mechanisms of NAFLD pathogenesis are not fully understood. In this study, expression of proteins associated with endoplasmic reticulum (ER) stress, apoptosis and autophagy were analyzed in normal, NAFL and NASH human livers by western blotting. Levels of some ER stress-transducing transcription factors, including cleaved activating transcription factor 6, were higher in NASH than in the normal tissues. However, the expression of a majority of the ER chaperones and foldases analyzed, including glucose-regulated protein 78 and ER protein 44, was lower in NASH than in the normal tissues. Levels of apoptosis markers, such as cleaved poly (ADP-ribose) polymerase, were also lower in NASH tissues, in which expression of some B-cell lymphoma-2 family proteins was up-or down-regulated compared to the normal tissues. The level of the autophagy substrate p62 was not different in NASH and normal tissues, although some autophagy regulators were up-or down-regulated in the NASH tissues compared to the normal tissues. Levels of most of the proteins analyzed in NAFL tissues were either similar to those in one of the other two types, NASH and normal, or were somewhere in between. Together, these findings suggest that regulation of certain important tissues processes involved in protein quality control and cell survival were broadly compromised in the NAFLD tissues.