Thesis (Ph.D.). / Written for the Dept. of Experimental Surgery. Title from title page of PDF (viewed 2008/01/12). Includes bibliographical references.

Thesis (M.Sc.). / Written for the Dept. of Human Genetics. Title from title page of PDF (viewed 2009/06/18). Includes bibliographical references.

Thesis (Ph. D.) -- University of Texas Southwestern Medical Center at Dallas, 2004. / Vita. Bibliography: 100-110.

Thesis (M.Sc.)--University of Alberta, 2010. / A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfillment of the requirements for the degree of Department of Physiology. Title from pdf file main screen (viewed on March 20, 2010). Includes bibliographical references.

Thesis (M.Sc.). / Written for the Dept. of Surgery, Division of Surgical Research. Title from title page of PDF (viewed 2009/06/30). Includes bibliographical references.

Thesis (M.S.)--University of Delaware, 2005. / Principal faculty advisor: Norman J. Karin, Dept. of Biological Sciences. Includes bibliographical references.

Thesis (Ph.D.)--McMaster University, 2003. / Advisor: David W. Andrews. Includes bibliographical references (leaves 122-141) Also available via World Wide Web.

Orientador: Prof. Dr. Alexandre Hiroaki Kihara / Dissertação (mestrado) - Universidade Federal do ABC, Programa de Pós-Graduação em Neurociência e Cognição, 2016. / Anóxia é uma das maiores causas de morbidade e mortalidade neonatal, especialmente em neonatos pré-maturos, constituindo um importante problema de saúde pública devido às sequelas neurológicas permanentes em pacientes. A privação de oxigênio dispara uma série de cascatas, culminando em morte celular em regiões cerebrais mais vulneráveis, como o hipocampo. Neste processo de morte celular causada pela privação de oxigênio, o cálcio citosólico possui um papel crucial. Receptores intracelulares de inositol 1,4,5-trifosfato (IP3Rs) são importantes reguladores de níveis de deste cálcio, no entanto, não se sabe sobre sua função na anóxia. O objetivo deste estudo é analisar se os IP3Rs do tipo 1 (IP3R1) participam no processo de morte no hipocampo de ratos após a anóxia neonatal. A análise quantitativa de real-time PCR revelou uma diminuição da expressão gênica de IP3R1 24 horas após a anóxia neonatal. Na análise da distribuição de células IP3R1-positivas foi observada uma densidade de IP3R1 na região de CA1 em ambos os grupos, porém, não se observou diferença entre os grupos controle e anóxia. Interessantemente os animais anóxia apresentaram uma alta colocalização de IP3R1 e marcador de núcleo (DAPI), sugerindo que a anóxia causa uma translocação de IP3R1 para o núcleo nas células hipocampais. Além disso, o padrão de marcação mostrou diferentes tamanhos de clusters dos receptores, indicando uma organização diferente entre os grupos. Foi injetado 2-APB, um bloqueador de IP3R1, ou veículo, no hipocampo de forma bilateral após a anóxia. Foi utilizado metodologias de marcação de células degeneradas e foi visto que no grupo 2APB houve uma diminuição do número de células FJC-positivas e TUNEL-positivas em comparação ao grupo veículo anóxia. Porém, não foi observado nenhuma diferença de marcação entre os grupos na imunofluorescência de caspase-3 ativada. Não foi detectada nenhuma diferença entre os grupos no teste de labirinto de Barnes. No teste de campo aberto, observou-se que o grupo 2APB apresentam maiores níveis de ansiedade. Desta forma, este estudo pode contribuir com novas perspectivas na investigação de mecanismos de neurodegeneração ativadas pela privação de oxigênio. / Anoxia is one of the most prevalent causes of neonatal morbidity and mortality, especially in preterm neonates, constituting an important public health problem due to permanent neurological sequelae observed in patients. Oxygen deprivation triggers a series of simultaneous cascades, culminating in cell death mainly located in more vulnerable metabolic brain regions, such as the hippocampus. In the process of cell death by oxygen deprivation, cytosolic calcium plays crucial roles. Intracellular inositol 1,4,5-trisphosphate receptors (IP3Rs) are important regulators of cytosolic calcium levels, although the role of these receptors in neonatal anoxia is completely unknown. This study focused on the functional role of inositol 1,4,5-trisphosphate receptor type 1 (IP3R1) in rat hippocampus after neonatal anoxia. Quantitative real-time PCR analysis revealed a decrease of IP3R1 gene expression 24 hours after neonatal anoxia. Distribution analysis of IP3R-positive cells was performed and we observed higher IP3R1 pixels quantity in CA1 of both groups; however, we were not able to observe alterations between control and anoxia animals. Interestingly, we observed that anoxia animals present a higher colocalization of IP3R1 and nucleus marker (DAPI), suggesting that neonatal anoxia may cause IP3R1 translocation to the nucleus in hippocampal cells. Furthermore, puncta-labelling pattern showed different cluster sizes, larger in control group, indicating different organization between groups. We injected 2-APB, an IP3R1 blocker, or vehicle in hippocampus bilaterally after anoxia. Labelling techniques of degenerate cells was performed and we observed that 2APB group decrease the number of FJC-positive cells compared to vehicle anoxia group. In contrast, TUNEL labelling and active caspase-3 immunofluorescence showed no difference between groups. Barnes maze test showed no differences between 2APB group and anoxia vehicle group. On the other hand, the open field test showed that 2APB group presents higher anxiety levels than vehicle group. In this way, this study may contribute to new perspectives in the investigation of neurodegenerative mechanisms triggered by oxygen deprivation.

When unfolded proteins accumulate in the endoplasmic reticulum (ER), the unfolded protein response (UPR) increases ER protein folding capacity to restore protein folding homeostasis. Unfolded proteins activate UPR signalling across the ER membrane to the nucleus by promoting oligomerisation of IRE1, a conserved transmembrane ER stress receptor. Despite significant research, the mechanism of coupling ER stress to IRE1 oligomerisation and activation has remained contested. There are two proposed mechanisms by which IRE1 may sense accumulating unfolded proteins. In the direct binding mechanism, unfolded proteins are able to bind directly to IRE1 to drive its oligomerisation. In the chaperone inhibition mechanism, unfolded proteins compete for the repressive BiP bound to IRE1 leaving IRE1 free to oligomerise. Currently, these two mechanisms respectively lack compelling in vivo and in vitro evidence required to assess their validity. The work presented here first describes in vivo experiments that identify a role of the ER co-chaperone ERdj4 as an IRE1 repressor that promotes a complex between the luminal Hsp70 BiP and the luminal stress-sensing domain of IRE1α (IRE1LD). This is then built on by a series of in vitro experiments showing that ERdj4 catalyses formation of a repressive BiP-IRE1LD complex and that this complex can be disrupted by the presence of competing unfolded protein substrates to restore IRE1LD to its default, dimeric, and active state. The identification of ERdj4 and the in vitro reconstitution of chaperone inhibition establish BiP and its J-domain co-chaperones as key regulators of the UPR. This thesis also utilises the power of Cas9-CRISPR technology to introduce specific mutations into the endogenous IRE1α locus and to screen for derepressing IRE1α mutations. Via this methodology, two predicted unstructured regions of IRE1 are found to be important for IRE1 repression. Finally, this thesis challenges recent in vitro findings concerning the direct binding mechanism.

The biosynthesis of integral membrane proteins (IMPs) is an essential cellular process. IMPs comprise roughly 20-30% of the protein coding genes of all organisms, nearly all of which are inserted and assembled at the endoplasmic reticulum (ER). The defining structural feature of IMPs is one or more transmembrane domains (TMDs). TMDs are typically stretches of predominately hydrophobic amino acids that span the lipid bilayer of biological membranes as an alpha helix. TMDs are remarkably diverse in terms of their topological and biophysical properties. In order to accommodate this diversity, the cell has evolved different sets of machinery that cater to particular subsets of proteins. Our knowledge of how the TMDs of IMPs are selectively recognized, chaperoned into the lipid bilayer, and assembled remains incomplete. This thesis is broadly interested in investigating how TMDs are correctly inserted and assembled at the ER. To address this the biosynthesis of multi-pass IMPs was first considered. Multi-pass IMPs contain two to more than twenty TMDs, with TMDs that vary dramatically in terms of their biophysical properties such as hydrophobicity, length, and helical propensity. The beta-1 adrenergic receptor (β1-AR), a member of the G-protein-coupled receptor (GPCR) family was established as a model substrate in an in vitro system where the insertion and folding of its TMDs could be interrogated. Assembly of β1-AR is not a straightforward process, and current models of insertion fail to explain how the known translocation machinery correctly identifies, inserts, and assembles β1-AR TMDs. An in vivo screen in mammalian cells was therefore conducted to identify additional factors which may be important for multi-pass IMP assembly. The ER membrane protein complex (EMC), a well conserved ER-resident complex of unknown biochemical function, was identified as a promising hit potentially involved in this assembly process. The complexity of working with multi-pass IMPs in an in vitro system prompted the investigation of a simpler class of proteins. Tail-anchored proteins (TA) are characterized by a single C-terminal hydrophobic domain that anchors them into membranes. Though structurally simpler compared to multi-pass IMPs, the TMDs of TA proteins are similarly diverse. We found that known TA insertion pathways fail to engage low-to-moderately hydrophobic TMDs. Instead, these are chaperoned in the cytosol by calmodulin (CaM). Transient release from CaM allows substrates to sample the ER, where resident machinery mediates the insertion reaction. The EMC was shown to be necessary for the insertion of these substrates both in vivo and in vitro. Purified EMC in synthetic liposomes catalysed insertion of its TA substrates in a fully reconstituted system to near-native levels. Therefore, the EMC was rigorously established as a TMD insertase. This key functional insight may explain its critical role in the assembly of multi- pass IMPs – which is now amenable to biochemical dissection.