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

The liver is a vital organ, playing numerous critical roles in the body. The liver's ability to perform essential functions is disturbed by injuries that are often associated with many complications such as calcification. Although many reports in the literature document observations of liver calcification, the mechanisms regulating this phenomenon remain unclear. Herein, we aim to investigate the cellular and molecular events that occur during pathological calcification of the liver. / To study the mechanisms of calcification, assessments included histological-staining, immunolabeling, and biochemical and electron microscopy analyses. The findings suggest that calcification may result from hydroxyapatite precipitation in necrotic or apoptotic hepatocytes. Similarly, calcification may be associated with differentiated myofibroblasts expressing bone matrix proteins downstream of TGFbeta signalling. / To identify specific protein regulators linked to the various stages in calcification, and to assess the protein composition of the tissue, a proteomic analysis was used. This analysis identified IQGAP1, an effector of the Rho-GTPases and a master regulator of cell adhesion and migration. IQGAP1 is strongly expressed in myofibroblasts, suggesting that IQGAP1 may be implicated in myofibroblasts migrating towards calcification. Studies on IQGAP1 interactions with its binding partners reveal that it is part of a protein complex that includes beta-catenin, an adhesion protein, and Rac1, a cytoskeletal regulator. These results suggest that IQGAP1 may play an important role in myofibroblast migration upon liver injury. / Having identified that activin and TGFbeta signalling are activated in myofibroblasts, we hypothesised that they may stimulate myofibroblast differentiation and proliferation. Studies using a C3H/10T1/2 cell model reveal that both activin and TGFbeta stimulate differentiation, but only activin induces cell proliferation in a Smad-independent fashion, which requires activation of the ERK/MAPK pathway. / In summary, this work provides new mechanistic insights on the global regulation of liver calcification. The various phases of this work collectively cover the central role of myofibroblasts in liver injury: association with calcification, rapid proliferation, differentiation to an activated form, and migration toward the injured area. The findings allow us to better understand the mechanisms by which liver myofibroblasts are regulated in a specific pathological context.

Thesis (Ph.D. in Cell Biology, Stem Cells, and Development) -- University of Colorado Denver, 2008. / Typescript. Includes bibliographical references (leaves 105-116).

Although much progress has been made in understanding the events that lead to successful cell division, many details of this process remain a mystery. This dissertation presents findings which help to explain events that occur in the latest stages of cytokinesis, with an emphasis on the role of centrosome proteins. The first chapter introduces the novel centrosome protein centriolin. We show that this protein is localized specifically to the subdistal appendages of the maternal centriole in interphase, and it localizes to the midbody during cytokinesis. Disruption of this protein results in a unique cytokinesis defect in which cleavage furrow formation and ingression appear normal, but the cells remain connected by a thin intracellular bridge for extended periods of time. These results lead us to the conclusion that centriolin has an important function in cytokinesis. The second chapter describes our attempt to identify centriolin interacting partners. A yeast two hybrid screen was performed, and the results of this screen revealed an interaction between centriolin and proteins involved in vesicle target specificity and fusion. Further studies of these proteins revealed a novel localization to the midbody in cycling cells and a novel function in the final stages of cytokinesis, similar to centriolin. The third chapter discusses my attempts to clone and characterize a novel GTPase Activating Protein (GAP), which was also discovered in the screen for centriolin interacting proteins.

No description available.

Thesis (Ph. D.)--University of Alabama at Birmingham, 2008. / Title from first page of PDF file (viewed June 10, 2008). Includes bibliographical references.

The KIL gene of vaccinia virus encodes for a host range protein; in the absence of which, the virus is unable to grow in certain cell lines (RK-13 and some human cell lines). KIL function can be complemented in RK-13 cells by the cowpox host range gene product CP77 despite a lack of homology between the two proteins except for ankyrin repeats. We investigated the role of ankyrin repeats ofthe K1L gene in the host-range restriction of growth in RK-13 cells. The growth of a recombinant vaccinia virus, with the K1L gene mutated in the most conserved ankyrin repeat, was severely impaired as evidenced by lack of plaque formation and reduction in viral titers. Infection of RK-I3 cells with the mutant recombinant vaccinia virus resulted in total shutdown of both cellular and viral protein synthesis early in infection, indicating that the host restriction mediated by the ankyrin repeat is due to a translational block. A comparison of the cellular localization of the K1L wild type and mutated forms showed no difference, as both localized exclusively in the cytoplasm of RK-I3 cells. We also investigated the interaction of the vaccinia virus K1L protein with cellular proteins in RK-13 cells and co-immunoprecipitated a 90 kDa protein identified as the rabbit homologue of human ACAP2, a GTPase-activating protein with ankyrin repeats. Our result suggests the importance of ankyrin repeat for host-range function of K1L in RK-13 cells and identifies ACAP2 as a cellular protein which may be interacting with K1L.

Human immunodeficiency virus type 1 (HIV-1) is a member of the lentivirus subfamily of retroviruses. HIV-1 expresses multiple genes from a single provirus by alternative splicing. Early in viral expression, fully spliced 2-kb viral RNA is exported from the nucleus and encodes the viral regulatory protein, Rev, which is essential for nuclear transport of partially spliced and unspliced genomic-length RNA. Rev binds to an RNA structural element called the Rev response element (RRE) and mediates nuclear export through the leucine-rich nuclear export signal (NES) pathway. The human Rev Interacting Protein (hRIP) interacts specifically with the Rev NES. Rev NES mutants that are unable to export Rev-dependent RNAs are also unable to bind to hRIP. The hRIP cDNA encodes a 562 amino acid protein containing an N-terminal zinc finger with homology to Arf GAP domains, a central serine and threonine rich region, and C-terminal phenylalanine-glycine (FG) repeats characteristic of nucleoporins. To identify an hRIP ortholog in a genetically tractable organism, we performed database searches using the N-terminal zinc finger of hRIP. Using this approach, we identified a novel gene in Schizosaccharomyces pombe. Alignment of the entire reading frame of the putative ortholog with hRIP indicates similarity with the serine/threonine rich region and with the FG repeats, suggesting that S. pombecould be a good model system to study the cellular function of hRIP. We find that the S. pombe ORF is an essential gene, which encodes a 483 amino acid protein that is also able to interact with the NES of HIV-1 Rev. Based on being an essential gene, and the presence of a putative Arf GAP domain, the ORF was named an Arf GAP essential for viability, agv1+. We show that Agv1 is not directly involved in the nuclear export of poly(A+) RNA or 5S rRNA, nuclear export of leucine-rich NES-containing proteins, or nuclear import of nuclear localization signal (NLS)-containing proteins. However, Agv1 does appear to play a role in the cytoplasmic localization of 5S rRNA. We demonstrate that loss of Agv1 alters the localization of endoplasmic reticulum (ER) membrane and Golgi membrane resident proteins, accumulates intracellular membrane, and blocks processing of carboxypeptidase Y. Furthermore, the S. cerevisiae ADP-ribosylation factor (Arf) GTPase activating protein (GAP) Glo3, but not a catalytically inactive Glo3 mutant [R59K], is able to partially compensate for the loss of Agv1 function in temperature sensitive strains, indicating that Agv1 is an S. pombe Arf GAP with some functional features similar to S. cerevisiae Glo3.

No description available.

Rho GTPases and their regulators such as guanosine exchange factors (GEFs) and GTPase activating proteins (GAPs) represent an important class of molecules controlling dendritic spine plasticity. Although they are typically described as cytoskeletal modulators, roles for the GTPases in endocytosis and cell polarity establishment have also been defined. The neuronal proteins a1- and a2-chimaerins belong to a group of Rac and Cdc42 GAPs that inactivate these GTPases; in addition to a GAP domain, the a-chimaerins share a phosphokinase C (PKC)-like C1 domain but have distinct N-terminal domains (NTDs). My project has explored the importance of specific domains of a1-chimaerin both in induction of a morphological cellular protrusion collapse phenotype ('circularisation') and in interactions with partner proteins that may help to explain the phenotype. The results described in my thesis show that a1-chimaerin possesses a previously undescribed C-terminal domain (CTD) that is indispensable for the ability of the protein to induce collapse of protrusions, and consequent circularisation, in various cell types; moreover, an intact CTD is also important for association of a1-chimaerin with its known effector EphA4, and potentially with other undefined membrane proteins, in a C1-domain- dependent manner. In addition, my results show that a1-chimaerin associates via its NTD with the Src kinase Fyn, and via its C1 domain with the NR2A subunit of the NMDA receptor. Further experiments explored a1-chimaerin effects on EphA4 and NMDA receptor cell surface expression, as well as binding to other putative partners - including the adaptor protein p35 and the polarity protein PAR6. Finally, I have shown that inhibition of a pathway involving the Rho-associated coiled-coil containing protein kinase (ROCK) reverts circularisation induced by a1- chimaerin, and that a blocking peptide based on the CTD may be employed to partially counteract the phenotype. These results uncover a novel domain in a1-chimaerin that may have a crucial importance for the induction of cellular process collapse by a1-chimaerin with a potential relevance to the EphA4-induced dendritic spine retraction, EphA4 receptor endocytosis, and cell surface expression of NR2A-containing NMDA receptors. This suggests a model of a multi-protein signalling complex involving a1-chimaerin that coordinates cellular process remodelling, and that is likely to be important both for adult neuronal circuit plasticity and for neurodegenerative diseases.