Thesis (Ph. D.)--Cornell University, May, 1998. / Vita. Includes bibliographical references (leaves 146-154).

The present study was undertaken to establish the mechanism(s) involved in the endocytic pathway, in particular, early endosome processing and delivery to the lysosomes. Two models for endosome processing have previously been proposed in the literature, namely the maturation and vesicular traffic models. The general consensus has been an early phase of intermingling of the endocytic contents markers within early endosomes that mature to form non-fusogenic late endosomes (maturation model). This maturation phase is followed by a segregation phase where intermingling of contents between vesicles no longer takes place. To establish the mechanism(s) involved in early endosome processing and delivery to lysosomes, a kinetic analysis was made using results from cellular fluid-phase uptake assays. This unique approach offers an alternative view to previous studies on the mechanisms in operation during endocytic processing. The results and conclusions made could thus confirm or disprove previously proposed mechanisms.

No description available.

Sandhoff disease (SD), a fatal lysosomal storage disease, results from a deficiency of the β-subunit of the β-hexosaminidase A and B enzymes. This deficiency leads to severe accumulation of GM2 gangliosides in lysosomes within the central nervous system (CNS) resulting in mass neuronal apoptosis. The mouse model of SD shows progressive neurodegeneration that closely resembles Sandhoff and Tay Sachs disease (TSD) in humans. SD and TSD consist of infantile, juvenile, and late-onset forms. These diseases can present with a multiplicity of symptoms including cognitive and speech impairments, ataxia, and lower motor neuron disease. Late-onset SD and TSD show motor neuron disease in over 40% of patients. In this study, we explore the role of endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) in the spinal cord during the development and progression of disease in Sandhoff mice. Using immunocytochemistry and western blotting, we analyzed the expression level and localization of several ER stress and cellular apoptosis markers within the cervical, thoracic, and lumbar regions of the spinal cord of Sandhoff mice. Our results revealed significant upregulation of several ER stress markers in motor neurons that appeared to coincide with significant lysosomal accumulations. In addition, we observed sequential and age-dependent expression changes in ATF6 and CHOP and their prominent nuclear localization within anterior horn motor neurons. Markers of apoptosis, caspases and PARP also appeared to be activated in the spinal cords of Sandhoff mice starting as early as 60 days. Interestingly, we noted more than 50% reduction in neuronal numbers in all regions of the spinal cord of Sandhoff mice between ages 80 and 120 days. Overall, this study provides strong evidence for the role of chronic ER stress and UPR activation in the spine pathophysiology of SD. / Thesis / Master of Science (MSc) / Lysosomal storage diseases are a rare group of inherited neurological disorders that are often fatal at a young age. Two diseases that fall within this category, Sandhoff and Tay Sachs disease, are similar in their cause and symptoms. Current research lacks a complete understanding of the mechanism behind these disorders making the development of new therapeutics challenging. This research highlights a group of cells in the spine that are vulnerable in these diseases. These cells show physical and functional changes in their structure as the diseases progress. We provide evidence of a new stress pathway which appears to be strongly implicated in the development and progression of these diseases. We also show an association between this pathway and the death of these vulnerable cells leading to the symptoms exhibited by patients. These findings expand our current knowledge of these disorders and open new avenues for therapeutic interventions.

Diss. (sammanfattning) Linköping : Univ., 2003. / Härtill 5 uppsatser.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

No abstract available. / Thesis (M.Sc.)-University of Natal, Pietermaritzburg, 1996.

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

Fat facets in Mouse (FAM) or mUSP9x is a deubiquitylating enzyme of the USP class. Knockdown of FAM protein levels in mouse pre-implantation embryos by antisense oligonucleotides is known to prevent embryos from progressing to the blastocyst stage indicating an important role for FAM in early mammalian development. In mammals, the Fam gene is located on the X-chromosome. In mice, the Y homologue, Dffry or usp9y, is expressed exclusively in the testes and maps to the Sxrb deletion (Brown et al., 1998). Sxrb is associated with an early post-natal blockage of spermatogonial proliferation and differentiation leading to absence of germ cells (Bishop et al., 1988; Mardon et al., 1989). The human Y homologue of Fam is closely associated with oligozoospermia (Sargent et al., 1999; Sun et al., 1999) and the human X homologue has been linked to the failure of oocytes to pass through the first meitoc prophase in Turner syndrome (Cockwell et al., 1991; Speed, 1986) Despite these associations, the substrates and precise role of Fam and its homologues in these processes have not yet been defined. Due to the complex nature of Fam expression and the lack of data tying FAM to specific cellular functions, much attention has been paid in identifying interacting partners and cellular targets of FAM activity to aid in the definition of its role in the cell and development. Three common molecular biology techniques were applied here in an attempt to further characterise known interactions of FAM, including interactions with the cell adhesion molecule β-catenin and the protein trafficking pathway proteins epsin-1 and itch. The aim of these investigations was to generate FAM mutants that could abolish individual interactions, enabling investigation of individual interactions in cellular function and development. These experiments failed to identify the amino acids of FAM that were critical for its interactions with β-catenin, epsin-1, or itch. Experiments aimed at characterising a novel ubiquitin-like domain located in the N-terminal half of the FAM protein, did however identify novel interactions of FAM with the three Golgi associated adaptor proteins GGA1, GGA2, and GGA3. Further investigations prompted by this interaction, examined the role of FAM in the trafficking of proteins from the Golgi apparatus. Cellular FAM protein levels were altered either by exogenous expression of FAM protein or knockdown of endogenous FAM using FAM specific shRNA triggers. The cellular protein levels and extent of post-translational modification of eleven lysosomal proteins were monitored in each case. It was found that increased FAM protein levels resulted in decreased cellular protein levels of five of the eleven lysosomal proteins studied. In contrast, a reduction in FAM protein levels was found to result in an increase in the cellular protein levels of eight of the eleven lysosomal proteins. This study provides the first evidence of a deubiquitylating enzyme that is able to interact with the GGA proteins. It is also the first to describe a deubiquitylating enzyme that can affect the biosynthesis of lysosomal proteins and provides valuable new insight into the cellular function of FAM/USP9X. / Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Sciences, 2008

Notre travail concerne la transfection génique. Celle-ci nécessite l'envoi au noyau cellulaire d'ADN exogène, c’est-à-dire, d'une grosse molécule hydrophile, qui, normalement ne peut pénétrer dans la cellule que par endocytose. Or, un tel processus conduit habituellement à une dégradation de la molécule, en grande partie dans les lysosomes. Dans notre introduction, après un bref rappel sur l'endocytose, nous présenterons les moyens qui permettent à l'ADN exogène d'échapper à une hydrolyse intracellulaire et d'atteindre le noyau sous une forme fonctionnelle. Les résultats que nous avons présentés nous apportent des informations sur l’influence de l’injection hydrodynamique sur l’endocytose par le foie. Rappelons que notre travail avait deux buts principaux : contribuer à l’élucidation du mécanisme de la transfection hydrodynamique et rechercher si l’injection hydrodynamique pouvait présenter des avantages pour introduire des protéines dans les cellules hépatiques.