In eukaryotes, the endoplasmic reticulum is the site where folding of secretory proteins and the assembly of multimeric cell surface receptors take place. These processes are mediated by molecule chaperones that include the ER membrane bound chaperone calnexin and the sequence related calreticulin. Using a PCR strategy, a homologue for the mammalian calnexin/calreticulin family, CNE1, was isolated in S. cerevisiae. The CNE1 gene product, Cne!p, is an integral membrane glycoprotein of the ER. Disruption of the CNE1 gene did not lead to inviable cells or to gross effects on the levels of secreted wild type proteins. However, in CNE1 disrupted cells, there was an increase in the cell-surface expression of a normally intracellularly retained temperature sensitive mutant of the $ alpha$-pheromone receptor, Ste2-3p. In addition, an increase in the secretion of heterologously expressed mammalian $ alpha sb1$-antitrypsin was also observed in CNE1 disrupted cells. In order to study calnexin function in another genetically manipulable organism, a Schizosaccharomyces pombe calnexin homologue was sought. Using a similar PCR strategy, a S. pombe calnexin homologue, $cnx1 sp+$, was identified. The $cnx1 sp+$ gene product, Cnx1p, was shown to be a calcium binding type I integral membrane glycoprotein. Unlike the sequence related S. cerevisiae CNE1 gene, the $cnx1 sp+$ gene was essential for cell viability. Full length Cnx1p was able to complement the $cnx1 sp+$ gene disruption but full length mammalian calnexin could not. The ER lumenal domain of Cnx1p, which was secreted from cells, was capable of complementing the $cnx1::ura4 sp+$ lethal phenotype. Both wild type PI M1 (Val 213) $ alpha sb1$-antitrypsin and the ER retained PI Z variant were expressed in S. pombe cells. As in mammalian cells, wild type $ alpha sb1$-antitrypsin was normally secreted whereas the PI Z variant was retained intracellularly. Rescue of the secretion defective phenotype of the PI Z variant occurred in

Mitochondria are dynamic organelles known primarily for their roles in oxidative metabolism and programmed cell death. Both of these processes are regulated by the mitochondrial matrix calcium concentration. Little is known about how mitochondrial calcium is regulated: no plant mitochondrial Ca2+-ATPase pumps or no mitochondrial Ca2+ channels have been identified to date. In addition, little is known concerning any physical interactions between mitochondria and endoplasmic reticulum (ER), an important cellular calcium store, and how these modulate cellular calcium fluxes. In this work stable transgenic Arabidopsis lines expressing fluorescent marker proteins were generated to allow visualisation of mitochondria and the ER in the same cells, and to measure mitochondrial calcium fluxes using aequorin. According to my results, there is a physical association between mitochondria and ER and this association cannot be disrupted by chemical treatments (latrunculin B, methyl viologen and antimycin A). As part of this work I identified an Arabidopsis gene, Mitochondrial Calcium Uptake 1 (MCU1), which encodes a protein with features that suggest a role in mitochondrial calcium dynamics. Fluorescent protein fusions of this protein demonstrated that it localizes to mitochondria. An Arabidopsis T-DNA line was identified with an insertion in MCU1. However, little effect of the insertion on transcript abundance of MCU1 was observed.

Access to abstract permanently restricted. / Access to thesis permanently restricted. / Department of Biology

Ankylosing spondylitis (AS) is associated with HLA-B*2704 and B*2705 but not with HLA-B*2706 and B*2709. Genome wide studies recently identified ERAP1 as an important genetic association in AS and could be the missing link in the pathogenesis of AS. I studied the implications of the two known actions of ERAP1 on AS pathogenesis. For assessing the peptide trimming function, surface HLA-B27 and MHC-I free heavy chain (FHC) expression on peripheral blood mononuclear cells of AS patients were studied. Subsequently, in an in vitro system of C1R cells expressing different AS-associated and AS-neutral HLA-B27 subtypes, I studied the effect of ERAP1 suppression on HLA-B27 and FHC expression. To assess the cytokine receptor shedding function, I studied serum cytokine receptor level variation with ERAP1 polymorphisms and its relationship to disease activity in AS patients. Finally, I studied the effect of variants of ERAP1 and other members of the antigen presentation machinery on radiographic severity in AS patients. AS patients with the major allele of the ERAP1 rs27044 polymorphism had higher FHC expression on monocytes. In C1R cells ERAP1 suppression led to an increase in intracellular FHC (IC-FHC) and B27-peptide complexes identified by a special MARB4 antibody, but only in C1R cells expressing the AS-associated subtypes HLA-B*2704 and B*2705. ERAP1 variants had no effect on serum cytokine receptor levels. Baseline radiographic severity was associated with ERAP1 polymorphism in univariate analysis only. LMP2 variants were associated with baseline radiographic severity in multivariate analysis. ERAP1 affects peptide presentation and FHC formation by HLA-B27 and could be the missing link in the pathogenesis of AS. ERAP1 through its differential HLA-B27 subtype interaction could explain why certain subtypes of HLA-B27 are associated with AS while others are not. Larger studies are required to look closely at the effect of ERAP1 on radiographic severity and progression in AS.

Ankylosing spondylitis (AS) is associated with HLA-B*2704 and B*2705 but not with HLA-B*2706 and B*2709. Genome wide studies recently identified ERAP1 as an important genetic association in AS and could be the missing link in the pathogenesis of AS. I studied the implications of the two known actions of ERAP1 on AS pathogenesis. For assessing the peptide trimming function, surface HLA-B27 and MHC-I free heavy chain (FHC) expression on peripheral blood mononuclear cells of AS patients were studied. Subsequently, in an in vitro system of C1R cells expressing different AS-associated and AS-neutral HLA-B27 subtypes, I studied the effect of ERAP1 suppression on HLA-B27 and FHC expression. To assess the cytokine receptor shedding function, I studied serum cytokine receptor level variation with ERAP1 polymorphisms and its relationship to disease activity in AS patients. Finally, I studied the effect of variants of ERAP1 and other members of the antigen presentation machinery on radiographic severity in AS patients. AS patients with the major allele of the ERAP1 rs27044 polymorphism had higher FHC expression on monocytes. In C1R cells ERAP1 suppression led to an increase in intracellular FHC (IC-FHC) and B27-peptide complexes identified by a special MARB4 antibody, but only in C1R cells expressing the AS-associated subtypes HLA-B*2704 and B*2705. ERAP1 variants had no effect on serum cytokine receptor levels. Baseline radiographic severity was associated with ERAP1 polymorphism in univariate analysis only. LMP2 variants were associated with baseline radiographic severity in multivariate analysis. ERAP1 affects peptide presentation and FHC formation by HLA-B27 and could be the missing link in the pathogenesis of AS. ERAP1 through its differential HLA-B27 subtype interaction could explain why certain subtypes of HLA-B27 are associated with AS while others are not. Larger studies are required to look closely at the effect of ERAP1 on radiographic severity and progression in AS.

Endoplasmic reticulum (ER) stress is a homeostatic mechanism, which is utilized by cells to adapt to inter- and intra-cellular changes. There is a burgeoning literature showing that the human endogenous retroviral envelope glycoprotein, Syncyin-1, oxidative stress and reactive oxygen species participate in the pathogenesis of multiple sclerosis (MS). I investigated the contribution of Syncytin-1-induced ER stress in MS and its animal model, experiment autoimmune encephalomyelitis (EAE). The prototypic ER stress biomarker, XBP-1 spliced variant (XBP-1/S), was increased in cerebral white matter of MS patients compared to non-MS controls and was correlated with Syncytin-1 expression. Syncytin-1 over-expression caused glia cytotoxicity but was mitigated by the ROS scavenger, crocin. Treatment with crocin on day 7 post-EAE induction ameliorated EAE disease severity in mice by reducing EAE pathology. Herein, I demonstrate that crocin attenuates Syncytin-1-induced ER stress in astrocytes while also diminishing disease severity in EAE in conjunction with suppression of neuroinflammation.

Thesis (Ph. D.)--Oregon State University, 2008. / Printout. Includes bibliographical references (leaves 128-139). Also available on the World Wide Web.

Thesis (M.S.)--University of Texas at El Paso, 2008. / Title from title screen. Vita. CD-ROM. Includes bibliographical references. Also available online.

Thesis (Ph.D.)--University of Tennessee Health Science Center, 2008. / Title from title page screen (viewed on February 9, 2009). Research advisor: Linda M. Hendershot, Ph.D. Document formatted into pages (ix, 90 p. : ill.). Vita. Abstract. Includes bibliographical references (p. 67-82).

Thesis (Ph.D.)--Flinders University, School of Medicine, Dept. of Medical Biochemistry. / Typescript bound. Includes bibliographical references: (leaves 211-230) Also available online.