Thesis (M.S. in biochemistry)--Washington State University, December 2007. / Includes bibliographical references (p. 36-47).

Thesis (Ph. D.)--University of Rhode Island, 2006. / Includes bibliographical references (leaves 138-154).

Thesis (Ph. D.)--University of Oregon, 2006. / Typescript. Includes vita and abstract. Includes bibliographical references (leaves 115-121). Also available for download via the World Wide Web; free to University of Oregon users.

In this thesis I present complex experimental data on the physiological and molecular adaptations during diapause development and overwintering in a linden bug, Pyrrhocoris apterus (Heteroptera, Pyrrhocoridae). I focus on adjustments of the enzymatic complement, which is involved in the biosynthesis of cryoprotectants, and heat shock proteins, which are expressed in response to temperature stress.

Ph.D. / Heat shock proteins (HSP) (in particular hsp70) are increasingly synthesised during and following exposure to stressful insults, playing an important role in protection and adaptation. Protective effects of HSP concerning infection and immunity include self/non-self discrimination, enhancement of the immune response, immune protection, thermotolerance and cytoprotection from inflammatory mediators (reactive oxygen species and cytokines). Considering the general protective role of hsp70 and its specific immunological functions, including antigen processing and presentation, variation in hsp 70 genes may contribute towards differential coping with stress and disease susceptibility. In humans, three members of the hsp70 gene family, hspl0-1, hsp70-2 and hsp70-hom, were mapped to the MHC class Ill region approximately 280 kbp centromeric to the TNFa gene and 92 kbp telomeric to the C2 gene. Polymorphisms in MHC-Iocalized hsp70 genes have been implicated in susceptibility to a number of diseases, independently or in combination with class II polymorphisms due to linkage disequilibrium (LD). MHC alleles are most often associated with immunosuppressive diseases. Tuberculosis (TB) has a strong immunological basis, involving cell-mediated immunity with human leukocyte antigen (HLA) variants implicated in its susceptibility/resistance. In the light of the above, the role of hsp70 polymorphism in TB susceptibility, alone or in combination with MHC class II alleles, was investigated through the following objectives: 1) Typing of hsp70 gene polymorphism (hsp70-1, hsp70-2 and hsp70-hom) in controls and TB cases from the Cape Coloured population of South Africa 2) Comparison between Cape Coloureds and Caucasoid populations with regard to hsp70 allele and genotype distribution 3) Studying linkage disequilibrium between members of MHC class II (HLADRB1) and Ill (hsp70) alleles in the Cape Coloureds 4) Simulation of MHC class II and Ill haplotypes in this particular population Hsp70 polymorphism was studied in controls (n=106) and TB cases (n=107) from the complex hybrid Cape Coloured population inhabiting the Western Cape region of South Africa - a population showing increased susceptibility to TB. PCR-RFLP and PAGE analysis were used to determine the hsp70 allele frequencies and genotype distribution of the individuals studied, while linkage disequilibrium between MHC class II and Ill, and within class Ill alleles, was investigated using the software "Graphical Overview of Linkage Disequilibruim" (GOLD). Haplotypes comprising MHC class II and Ill alleles were simulated using the software PHASE.

The need to monitor fresh water ecosystems for pollution is increasing, as is the need to develop a biomarker sensitive to a range of environmental insults. Recently, heat shock proteins have been identified as possible biomarkers of environmental contamination. However, evaluation as to their use as a biomarker of metal contamination in fish species endemic to Southern Africa is limited. The purpose of this study was to identify what members of the 70 kDa family of heat shock proteins (Hsp70) were present in the liver of Oreochromis mossambicus, and if their accumulation was altered after short-term (96 hour) exposure to aqueous copper. In addition to copper exposure, the effect of acclimation media was also examined. Tissue-level analysis was done by means of histological examination so as to determine if alterations in the accumulation of the Hsp70 family had a marked effect on the structural integrity of the liver. Specimens of Oreochromis mossambicus acclimated in either aged tap water or borehole water were placed in flow-through systems and exposed to either 10% or 20% of the LC50 value of cupric chloride for a duration of 96 hours. Control groups were run in conjunction with the exposure groups so as to set control values by which to compare. Heat shock protein analysis was done by Western blotting after separation of hepatic proteins by SDS-PAGE. For the purpose of histological analysis, representative samples were randomly selected. Analysis of the hepatic heat shock protein 70 family identified the presence of three (3) members, each of a different molecular weight. These included members of 70 kDa (Hsp70), 74 kDa (Hsp74) and 76 kDa (Hsp76). In addition to these findings, it was found that Oreochromis mossambicus accumulated high levels of particular members of the heat shock protein 70 family under unstressed conditions, affording the fish adaptability to environmental extremes. Furthermore, individuals acclimated in aged tap water showed decreased Hsp76 accumulation after exposure to sub-lethal copper concentrations, whereas those individuals acclimated in borehole water retained relatively high levels of Hsp76. Additionally, it was shown that the hepatic structure deteriorated in those individuals acclimated to the aged tap water after copper exposure, with observed increases in vacuolation, number of macrophage centres present and the occurrence of intracellular golden-brown granules. However, there was little change from the already-altered hepatic structure of those individuals acclimated in borehole water, with conspicuous golden-brown granules the most obvious histopathological condition present. Histological examination therefore proved to supplement the heat shock protein results obtained. This study thus concluded that a decrease in the accumulation of the Hsp70 family resulted in a negative organismal response, initiating deleterious alterations in the hepatic structure. Additionally, this study concluded that past water quality has a marked effect on a given biomarker response, and should be taken into careful consideration when conducting biomarker studies. / Prof. J.H.J. Van Vuren

Sequences coding for three related 16 kd heat shock proteins (hsps) of the nematode Caenorhabditis elegans were isolated and characterized. The extensive accumulation of hsp16 mRNA during heat stress facilitated the identification of two cDNAs, CEHS48 and CEHS41, which encoded hsp16 variants. These plasmids were selected by their ability to hybridize to mRNA which directed the synthesis of hspl6 in vitro, and were further characterized by sequence analysis. Two-dimensional gel electrophoresis of hspl6 synthesized in vitro from mRNA selected by hybridization to either of the cDNAs under conditions of low stringency revealed the existence of at least five electrophoretic variants with significantly different isoelectric points. The above cDNAs were used as specific probes to isolate recombinant bacteriophage containing C. elegans genomic DNA. Overlapping phage clones were used to define a region of approximately 30 kilobases. The genes coding for hsp16-48, previously identified by cDNA cloning, and for another 16 kd hsp designated hspl6-l were characterized by DNA sequencing. These two genes were arranged in a head-to-head orientation. Both the coding and flanking regions of these genes were located within a 1.9 kb region which was duplicated exactly to form a perfect 3.8 kb inverted repeat structure. This structure ended in unusual G + C-rich sequences 24 bp in length. The identity of the two arms of the inverted repeat at the nucleotide sequence level implied that the duplication event may have occurred relatively recently in evolution. Alternatively, gene conversion between the two modules could have maintained homology between the two gene pairs. Comparison of the hsp16-48 gene with its corresponding cDNA revealed the presence of a single, short intron. An intron of comparable length and in an analogous position was also found in the hsp16-1 gene. The introns separated variable and conserved regions within the amino acid sequences of the encoded heat shock proteins. A domain of approximatey 80 amino acids is contained within the conserved second exon and is homologous to a similar region in the small hsps of Drosophila, Xenopus, soybean and man as well as the a-crystallin protein of the vertebrate lens. Each hsp16 gene contained a TATA box upstream of the start of transcription. Promoter sequences, which have been shown to be required for heat inducibility in various systems, were located upstream of either TATA box Northern blot analysis showed that the hsp16-48 and hsp16-1 genes are expressed at levels approximately 20 - 40 fold lower than two closely related genes, hsp16-41 and hsp16-2, upon temperature elevation. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

Autism is a behavioral syndrome characterized by a severe impairment of reciprocal social relations, and of verbal and nonverbal communications. Many different etiologic factors such as viral infection and genetic predisposition have been proposed to explain the development of this disorder. Immune abnormalities, such as a decreased lymphoblastic response to T-cell mitogen, defective antibody responses to rubella vaccine, and decreased numbers of T lymphocytes, also have been identified in a subpopulation of patients with autism, which implies that the development of autism in some cases may be due to autoimmune mechanisms. Recent evidence suggests that immune response to the heat-shock proteins 60 and 70 is associated with several autoimmune diseases, including juvenile arthritis, type 1 diabetes, and multiple sclerosis. Therefore, in this study, the plasmas of patients with autism were examined by enzyme linked immunosorbent assay (ELISA) for antibodies to the heat-shock proteins 60 and 70. The autistic subjects were found to have increased levels of antibodies against heat-shock protein 70 as compared to that of age-matched controls (p=O. 0148). However, levels of antibodies to heat shock protein 60 in the autistic subjects showed considerable individual variation and no significant difference was found. Abnormal immune reactions to myelin basic protein have also been found in autistic subjects. Since epitopes on myelin basic protein have been shown to crossreact with determinants on heat-shock protein 60, the similarity between anti-myelin basic protein monoclonal antibodies and antiheat- shock protein 60 antibodies in the autistic subjects was also studied. The results showed no crossreactivity between these two antibodies. In conclusion, the data from the study of antibodies against heat-shock protein 70 suggest an elevated immune response to heat-shock protein 70 in autistic subjects. This result implies that autism could be an autoimmune disease.

Indiana University-Purdue University Indianapolis (IUPUI) / The ubiquitin-proteasome system is responsible for the targeted degradation of proteins within the cell. The 26S proteasome, which is the protease of this system, is a high molecular weight complex consisting of 33 subunits that arrange to form two smaller complexes the 19S regulatory particle (RP) and the 20S core particle (CP). The 19S RP can bind one or both ends of the 20S CP and is responsible for recognizing the ubiquitinated substrates. After recognition, the 19S RP will subsequently deubiquitinate, unfold, and translocate the substrates into the proteolytic 20S CP. The 20S CP consists of seven unique alpha and seven unique beta subunits that arrange into four stacked rings, with two alpha rings capping two beta rings. Assembly of the alpha(1-7)beta(1-7)beta(1-7)alpha(1-7) structure begins with the formation of an alpha ring and proceeds through specific assembly intermediates. This process is assisted by assembly chaperone proteins that promote on pathway interactions to efficiently construct the 20S CP. In this dissertation, three new findings are described which further characterize the proteasome assembly pathway. First, novel non-canonical complexes comprised of proteasome subunit alpha4 were identified in vivo, revealing proteasome subunits can assemble into complexes outside of the proteasome. Second, Hsp70 proteins, Ssa1/2, were shown to assist in the assembly of 20S CPs, adding to the growing list of proteins guiding proteasome assembly. Third, a novel complex was identified which is believed to represent a new proteasome assembly intermediate.

No description available.