Bacterial polyketides possess an enormous range of chemical diversity and biological function. Many polyketides such as tetracycline, epothilone, and rapamycin have been developed into key clinical pharmaceuticals in a broad range of therapeutic areas. Sequencing of bacterial genomes has shown that there are many more polyketide biosynthetic pathways than there are polyketides isolated from standard cultivation techniques. These genetically encoded polyketide natural products from cultivatable and uncultivatable bacteria represent one of the greatest remaining untapped reservoirs of new natural product diversity. To access this untapped diversity of polyketide products, a general method for heterologous expression of these pathways is needed. Heterologous expression has proven to be a valuable asset in the discovery, production, engineering, and characterization of bacterial secondary metabolites and the complex enzymology involved in their biosynthesis. Herein we discuss the development and investigation of two unique heterologous expression platforms utilizing host strains of Myxococcus xanthus and Escherichia coli. Using our developed heterologous hosts, we were able to produce the Streptomyces rimosus polyketide oxytetracycline. Through production of oxytetracycline in E .coli we have identified the potential of alternative transcription factors as regulators of secondary metabolism. Further investigation and development of alternative transcription factors as regulators of secondary metabolism in heterologous hosts could benefit the development of robust general methodology for the heterologous expression of polyketides.

Background: Excess plugging of small airways is associated with premature death in chronic obstructive pulmonary disease (COPD). Over-expression of beta-epithelial sodium channel (β-ENaC) in airway epithelia in mice resulted in plugging of small airways while cystic fibrosis transmembrane regulator (CFTR) negatively regulated ENaC activity in cell models. Purpose: To test the hypothesis that accumulation of mucus exudates observed with the progression of COPD is related to excess airway epithelial sodium re-absorption as a result of over-expression of β-ENaC and reduced expression of CFTR by small airway epithelia. Methods: Small airway epithelial samples from frozen lungs from patients at different levels of COPD severity were isolated by laser capture microdissection (LCM). β-ENaC, CFTR, and β-actin (control) gene expression was determined by qRT-PCR and compared to expression in entire airways and lung parenchyma surrounding these airways. β-ENaC protein as well as epithelial mucin expression and mucus plugging were localized and quantified after immunohistochemical and periodic acid Schiff staining, respectively. Results: β-ENaC mRNA expression had a strong positive correlation with that of CFTR (p<O.0001) in airway epithelia and surrounding lung parenchyma (p=O.Ol) but not whole airways. β-ENaC mRNA and protein expression were positively correlated (p=O.4O, p=O.O5) and protein expression significantly increased with GOLD stage of COPD severity. Epithelial mucin expression positively correlated with β-ENaC (p=O.38, p=O.O5) and CFTR (p=OAO, p=O.O4.) mRNA and with mucus plugging (p=O. 43 , ptO.OOO2). CFTR mRNA also correlated positively with mucus plugging (p=O. 48 , p=O.O2). Conclusions: Strong positive correlations between β-ENaC and CFTR mRNA expression that are limited to the lung parenchyma and epithelium suggest a novel mechanism of mRNA regulation. This differs from their functional relationship where an inverse relationship between CFTR expression and β-ENaC activity has been reported. Positive correlations of epithelial mucin or mucus plugging with CFTR mRNA but not β-ENaC protein expression in the small airway epithelium suggest that CFTR may regulate mucin at this site independently of β-ENaC protein. The relationship between β-ENaC mRNA andepithelial mucin expression could be due to strong correlations between β-ENaC and CFTR mRNA expression but β-ENaC’s relationship with COPD GOLD stage suggests it may nevertheless play a role in COPD.

There is an inherent conflict over land use between humans and wildlife. Human activities can alter habitat, creating pressure on North American large carnivore populations. Traditional wildlife techniques can be slow to show population declines, especially in long lived species with slow reproduction rates and high mortality of young, such as grizzly bears (Ursus arctos), which leads to delayed information for land managers trying to find the balance between human use of land and preservation of wildlife. Concern about population health of grizzlies in Western Alberta, Canada has lead to investigation of the impacts of current land use within grizzly bear habitat. The objective of this work was to develop a protein microarray that could detect patterns of physiological stress in a rapid manner with small samples of grizzly bear tissue. Sampling from four regions in the foothills of the Rocky Mountains in Alberta resulted in the capture of 133 bears. During the developmental phase, proteins involved with mitochondrial function were found, using two dimensional gel electrophoresis, to be altered in situations of increased stress. Limited cross-reactivity was found when evaluating grizzly bear stress protein expression using commercially available protein microarrays. The protein microarray developed in this thesis consists of 31commercial antibodies validated for grizzly bears. These antibodies recognize proteins associated with different aspects of the stress response, including the hypothalamic-pituitary-adrenal axis, apoptosis/cell cycle, cellular stress, and oxidative stress and inflammation. Skin was selected as the tissue for evaluation of protein expression. Strong correlations were found between many of the proteins within functional categories. Model selection for the protein categories revealed variation that corresponded with region, serum markers of stress (total cortisol and hsp60), growth, the density of roads in the habitat and the amount of anthropogenic change in the bears home range. Regional trends of expression found bears in Swan Hills and bears from North highway 16 having elevated expression of the proteins measured by the microarray. The protein microarray was thus able to detect expression patterns reflecting physiological and environmental markers. The array shows great promise for future use in detection of potential distress in wildlife populations due to alterations of their habitat.

Coral reefs are one of the world s most valuable ecosystems but are declining at an accelerating rate. Common stressors impacting coral health include elevated temperatures, changes in light intensity, sedimentation, and increased exposure to pollutants. Traditionally, physiological responses have been measured to assess coral health but usually do not identify the stressor or the underlying mechanisms causing a response. In addition, coral may be stressed beyond recovery by the time a physiological response is observed. Changes in gene expression are key elements of the stress response, usually occur before physiological damage is evident, and can be directly related to the causative agent of stress. My research focuses on detecting sublethal responses to stress in Scleractinian coral using genetic biomarkers and gene expression profiling. Through the application of molecular technology, I have developed a coral stress gene microarray to investigate the responses of coral to various stressors. Results from controlled laboratory exposures provide evidence for unique gene expression profiles associated with specific stressors. Results from field studies reveal the feasibility of using array technology to investigate changes in gene expression of natural coral populations across time and between sites. For example, the array has been used to detect stress in coral populations related to seasonal events, such as precipitation as well as point source stress, such as xenobiotics. The temporal and spatial regulation of specific genes within a genome determines the metabolic activity of an organism and can be used to identify changes in cellular responses to various stimuli. These cellular events precede population-level changes and could be useful biomarkers if linked to specific physiological or ecological events. This research is important because it identifies stress at a sub-lethal level and can aid resource managers in decision making by prioritizing the stressors impacting coral reefs.

The parallel gene expression profiles of Brucella melitensis and the host have not been elaborated. In this study, I analyze and discuss the transcriptional profiles of B. melitensis invasive-associated genes, the expression profile of intracellular B. melitensis and B. melitensis-infected non-phagocytic cells in the first 12 h post-infection (PI), and the in vivo temporal global transcriptome of both B. melitensis and the infected bovine host in the first 4 h PI. The initial study found that B. melitensis at late-log phase of growth were more invasive in non-phagocytic cells than at early-log or stationary growth phase. Microarray-based studies identified 454 Brucella genes differentially expressed between the most and the least invasive growth phases. Additionally, B. melitensis strains with transposon interrupted in loci BMEII0380 (acrA) and BMEI1538 (hypothetical protein) were found to be deficient in internalization compare with the wild-type strain. A second experiment was designed with the goal of characterizing host and pathogen transcriptome in parallel. For detecting intracellular Brucella gene expression, a combined protocol consisting of a linear amplification of sense-stranded RNA biased to pathogen transcripts to the previously enriched host:pathogen RNA mixed sample, was developed. RNA samples were hybridized on human and Brucella cDNA microarrays, which analysis revealed a common down-regulation transcriptional profile at 4 h PI that was reverse at 12 h PI. The integrity of B. melitensis virB operon and the expression of host MAPK1 were confirmed as critical for early B. melitensis intracellular survival and replication in non-phagocytic cells. Finally, a temporal morphological and molecular characterization of the initial B. melitensis:bovine host interaction using a calf ileal loop model was performed. B. melitensis was isolated from intestinal Peyer’s patches as soon as 15 min and from systemic blood after 30 min postintra luminal inoculation. Microarray results revealed a common transcriptional profile in Brucella, but two different transcriptional profiles were identified in the host in the first 4 h PI. The importance of differentially expressed biological processes, pathways and individual genes in the initial Brucella pathogenesis is discussed.

The chromosomal location of endocrine genes was established, and relationships between expression of specific endocrine genes and measures of testis function in normal and poor semen quality stallions was assessed. Consensus primer sequences for glucocorticoid receptor (GR) and luteinizing hormone receptor (LHR) were used to screen the CHORI-241 equine bacterial artificial chromosome (BAC) library. The identity of PCR-positive BAC clones was confirmed by sequencing. Verified BACs were mapped to horse metaphase chromosome spreads by fluorescence in situ hybridization (FISH). The BACs containing the GR and LHR were localized by FISH to ECA 14q16-q21 and ECA15q22-q23, respectively. In addition to FISH mapping, the 5000rad horse x hamster radiation hybrid (RH) panel was screened in duplicate. Two-point linkage analysis placed GR 0 cR from LEX047, while LHR was 36.67 cR from TKY011 on ECA14 and ECA15, respectively. Total testicular parenchymal weight, mean daily sperm production (DSP) per gram parenchyma and mean apoptotic rate (406.05 ± 24.33g vs. 180.01 ± 34.41g, 15.29 ± 0.87 vs. 10.24 ± 1.10, 6.70 ± 0.88 vs. 14.25 ± 1.11, respectively) differed (P<0.05) between normal (n=8) and poor semen quality (n=5) stallions. Also, plasma estradiol and inhibin concentrations were higher (P<0.05) in normal stallions than in poor semen quality stallions. Testicular expression of estrogen receptor beta (ER beta), &#946;B inhibin, prolactin receptor (PRLR), growth hormone receptor (GHR) and insulin-like growth factor I receptor (IGF-IR) mRNAs were all lower (P<0.05) in poor semen quality stallions than in normal stallions. The BACs and primers developed in this study will facilitate future investigations of GR and LHR gene structure in the horse as well as providing a resource for physiological investigation of these two genes that are primary regulators of stress responsiveness and fertility. These data add important endocrine genes to the horse cytogenetic map. Also, important hormonal and gene expression changes have been identified in poor semen quality stallions for further investigation.

Texte remanié de: Th. doct.--Droit--Aix-Marseille 3, 2001. Titre de soutenance : Le rôle des volontés individuelles dans la génèse des délibérations collectives. / Bibliogr. p. 583-598. Index.

Diss.--Saarbrücken--Universität des Saarlandes, 2000. / Bibliogr. p. 275-295.

Thesis (M. Med. Sc.)--University of Hong Kong, 2009. / Includes bibliographical references (p. 77-97).

Thesis (Ph. D.)--University of Washington, 2001. / Vita. Includes bibliographical references (p. 132-140).