Human endogenous retroviruses (HERVs) constitute about 8% of the human genome. There are around 4000 pol-containing retroviral integrations in the human genome, which makes it impractical to measure each of them separately. Therefore we developed a set of degenerate real time PCRs to detect major groups bearing sequence similarities to gammaretroviruses, one of the largest groups of human endogenous retrovirus, and betaretroviruses, some of which have integrated into the human genome most recently and which remain the most intact. It was found that, although both gammaretroviral and betaretroviral RNAs were broadly expressed in various healthy tissues including reproductive tissues and brain, a differential expression pattern was observed. My work further revealed that HERVE and HERVW, two gammaretroviral sequences, were ubiquitously and highly expressed in pathologic and normal female reproductive tissues with tissue specific patterns. Expression of HERVE was higher in endometriotic tissue than in normal endometrium. HERVE and HERVW RNAs were higher in normal ovarian tissue than in ovarian cancer. Besides these tissue- and neoplasia-related differences, there were wide differences in HERV expression among individuals. Next, a selective pattern of HERVW upregulation was demonstrated in SK-N-DZ, a neuroblastoma cell line, upon re-oxygenation after a period of hypoxia or with 5-azacytidine, a demethylating agent. Furthermore, broad and high expressions of gammaretrovirus-like transcripts in different brain areas analyzed were identified. The expression levels were variable among different donors. In conclusion a ubiquitous HERV expression was observed in tissues and cell lines, with various patterns. At this stage the data are not sufficient to conclude whether HERV has any physiological or pathological roles in humans. However, their differential expression patterns are compatible with functional roles of HERV in humans.

Background: Angiogenesis is recognised as a critical step in tumour progression. The angiogenic switch is activated by various trigger signals, such as hypoxia, low pH, and genetic mutations. Renal cell carcinoma (RCC) is often an aggressive tumour, and advanced disease has limited treatment options and bad prognosis. This study was focused on markers of angiogenesis in RCC: endoglin (CD105) and CD31 assessing microvessel density (MVD), and carbonic anhydrase (CA) IX and hypoxia-inducible factor (HIF)-2α expressed at hypoxia. Upregulation of HIF is also associated with inactivation of the von Hippel-Lindau (VHL) tumour suppressor gene, which is common in conventional/clear cell (c)RCC. Method: A tumour bank containing 308 tumours from patients operated 1982-2003 was used. The tumours were well characterised regarding tumour type, TNM stage, nuclear grade, tumour size, and patient survival. The tumours were prepared in tissue microarrays and fresh frozen in whole sections. To analyse the expression of endoglin, CD31, CA IX, and HIF-2α mRNA, immunohistochemistry and real-time PCR were used. Results: There was a higher endoglin expression in cRCC than in papillary (p)RCC and chromophobe (ch)RCC, and a higher CD31 expression in cRCC than in pRCC. MVD correlated inversely to TNM stage and nuclear grade in cRCC. There was also an inverse correlation between tumour diameter and CD31 expression in cRCCs. Patients with cRCC with high MVD had a more favourable prognosis than patients with lower MVD. Endoglin and CD31 were not independent prognostic factors. The CA IX expression was higher in cRCC than in pRCC and chRCC. Patients with cRCC expressing low CA IX had a significantly less favourable prognosis compared with those with higher expression. CA IX is an independent prognostic factor. There was a higher HIF-2α mRNA expression in cRCC than in pRCC and chRCC. In cRCC, there was a significant inverse correlation between HIF-2α mRNA expression, and TNM stage and nuclear grade. There was also an inverse correlation between HIF-2α mRNA expression and tumour size among patients with cRCC. HIF-2α was not an independent prognostic factor. Conclusion: In these studies, the factors related to hypoxia and vascularity were all inversely correlated to tumour aggressiveness in cRCC. MVD, CA IX, and HIF-2α expression were also higher in cRCC than in pRCC and chRCC. The relationship between angiogenesis, vascularity, and hypoxia is ambiguous. A line of reasoning including mutations increasing angiogenesis in advanced disease may also be applied to RCC. Measurements of individual angiogenic factors seem to provide prognostic information, and can potentially be combined in patient monitoring and treatment.

The interactions between Medicago truncatula and Phytophthora medicaginis were examined using skl, a mutant blocked in ethylene perception, and a range of wild accessions of this plant species. P. medicaginis infection of M. truncatula plants resulted in compatible responses, whereas the mutant genotype was found to be hyper-susceptible to the pathogen. Phytophthora reproduction and colonization rates of Medicago tissues supported this conclusion. Infection of skl with different pathogens reinforced this observation. Ethylene production in infected A17 and skl roots showed reduced ethylene evolution in the mutant and suggested that a positive feedback loop, known as autocatalytic ethylene production, amplified the ethylene signal. To complement the study, expression analyses of defense response genes in this interaction were studied by real time RTPCR of Phytophthora-infected and mock-infected roots. The genes analyzed were PAL, CHS, IFR, ACC oxidase, GST, and PR10. The sequences needed for the analysis were found through the scrutiny of the M. truncatula EST database employing phylogenetics and bio-informatics tools. In A17 all the genes studied were up-regulated, although the specific gene expression patterns differed. The comparison of gene expression between A17 and skl genotypes allowed the differentiation between ethylene-dependent and ethylene-independent responses. Discrete results showed that ACC oxidase homologues were downregulated in the ethylene perception mutant, corroborating the ethylene observations. However, the expression of genes involved in the phenylpropanoid metabolism was increased in skl relative to A17, suggestive of an antagonism between the ethylene perception pathway and the regulation of the phenylpropanoid pathway. This result implied that Medicago phytoalexins accumulate in the disease interaction, but raised questions about their role in resistance to Phytophthora infection. This study establishes a link between mechanisms that regulate symbiotic infection and the regulation of disease resistance to Oomycete pathogens, especially P. medicaginis. The results served to identify a series of Phytophthora-induced genes, which remain pathogen-responsive even in the absence of a functional ethylene perception pathway. While it is possible that the products of these genes are involved in resistance to P. medicaginis, the present results demonstrate that ethylene perception is required for resistance.

Includes vita. / Title from PDF of title page. / Document formatted into pages; contains 329 pages. / Thesis (Ph.D.)--University of South Florida, 2003. / Includes bibliographical references. / Text (Electronic thesis) in PDF format. / ABSTRACT: River discharge leads to extensive phytoplankton blooms often observed in ocean color satellite images to extend far into the open ocean as high chlorophyll plumes. We investigated diversity, distribution and ecology of phytoplankton populations in the Mississippi River plume, both spatially and in the water column using molecular tools. A method was developed for the quantification of diatom/pelagophyte rbcL (large subunit of Ribulose-1,5-bisphosphate Carboxylase/Oxygenase) mRNA using quantitative PCR and applied to cultures and in the plume. The vertical structure of phytoplankton species in the Mississippi River plume was described by flow cytometry, pigments, rbcL mRNA and rbcL cDNA libraries. High productivity in the plume was associated with a large population of Synechococcus and elevated levels of cellular form IA rbcL mRNA. / ABSTRACT: rbcL cDNA libraries indicated two vertically separated clades of Prochlorococcus (high-light and low-light adapted) in addition to a diverse group of prymnesiophytes and a microdiverse clade of prasinophytes, which may have dominated the SCM (Subsurface Chlorophyll Maximum). In situ sampling and satellite image analysis were used to estimate that the plume accounted for 41% and 13% of all surface water column ix productivity in the oligotrophic Gulf of Mexico, while covering less than 3% of its area. Coastally the plume is dominated by diatoms, which are replaced by a bloom of Synechococcus as the plume moves offshore. Diatoms as indicated by pigments and rbcL clone libraries again dominated the offshore, least productive plume. 15N uptake measurements indicated that rapid recycling of ammonium despite higher levels of nitrate primarily drives production in the offshore plume. / ABSTRACT: rbcL mRNA levels and photosynthetic capacity displayed strong diel patters in three out of four time series sampled during the GRIST (Geochemical Rate/mRNA Integrated Study). In addition it was demonstrated that transcriptional regulation of the global nitrogen regulatory protein NtcA in Synechococcus WH7803 may involve a small cis-encoded anti-sense mRNA. Methods for the generation of large insert BAC (Bacterial Artificial Chromosome) from cultures and the environment were refined. Partial sequencing and genomic comparison of an ntcA containing BAC clone obtained from Synechococcus WH7803 indicated that ntcA is not part of a larger nitrogen assimilation operon in cyanobacteria. / System requirements: World Wide Web browser and PDF reader. / Mode of access: World Wide Web.

In tomato (Solanum lycopersicum), race-specific resistance against the fungal wilt pathogen Verticillium dahliae race 1 (Vd1) is established in the stem. However, the molecular factors and mechanisms leading to this resistance response are still unknown. In this study, Craigella resistant (CR) and susceptible (CS) tomato plants were successfully infected with Vd1 and this was verified by fungal quantification and symptom score assays. Previous microarray results showed interesting patterns of defence gene expression that correlated with biological phenomena. Plant defence genes code for proteins that are responsible for or associated with the plant resistance response. Through RT-PCR, this thesis set out to confirm these microarray observations and also to generate expression data for genes in which sensitivity was an issue in the microarray. The standard RT-PCR data confirmed a number of the microarray results, but some conflicts remained. From the defence genes investigated, there was agreement between the microarray data and the RT-PCR data for pre-mRNA processing factor 8, class IV chitinase, cyclin-dependent kinase inhibitor and IMP dehydrogenase/GMP reductase. Partial agreement was observed for genes coding for ethylene response factor 2, phenylalanine ammonia lyase and P6 protein. However, there was total disagreement for 14-3-3, beta-glucanase, P1a, RNA-binding protein, calcium-binding protein and S-Adenosyl-L-methionine: hydroxide adenosyltransferase. Real-time RT-PCR was attempted to clarify the remaining issues but further discrepancies arose, particularly in the Ve resistance genes. To resolve these discrepancies, two approaches were designed: (1) one based on the use of a universal internal control and (2) another based on restriction enzyme digestion. In general, the results were more consistent with standard RT-PCR. Overall, this study showed that standardization of a system involving vascular pathogens, leading to reproducible analysis, was possible but only with proper controls and additional validation. Standard RT-PCR appeared to offer a more accurate picture of the expression of defence genes in the tomato-Verticillium pathosystem. The defence gene expression results confirmed in this study remain as potential insights into the molecular mechanisms for Verticillium resistance in tomato plants.

The factors influencing senescence in white clover (Trifolium repens L.) are of considerable importance to the pastoral sector of New Zealand’s economy. The plant hormones, ethylene and the cytokinins, have been implicated as having opposing influences on senescence. This project focused on the cytokinins. The rate limiting step in cytokinin biosynthesis is catalysed by isopentenyl transferase (IPT) and the primary enzyme in the degradation of cytokinins is cytokinin oxidase/dehydrogenase (CKX). Both IPT and CKX genes are present as multi-gene families. A reduction in the level of active cytokinins either via a decrease in IPT expression, or an increase in CKX expression, or both, would implicate the cytokinins in developmental leaf senescence in white clover. White clover grows in a sequential pattern with leaves at all stages of development making it a good model for studying leaf development and senescence. A decrease in leaf chlorophyll is used as a marker for the onset of senescence. A micro-scale chlorophyll analysis was developed using the NanoDrop™ thus allowing tissue from the same leaflet to be used for gene expression and chlorophyll measurements. The pattern of chlorophyll changes was similar to that shown by Hunter et al.(1999) and Yoo et al.(2003) in white clover stolons used for ethylene research. Reverse transcriptase PCR (RT-PCR) and BLAST analysis was used to identify five putative IPT genes and seven putative CKX genes from white clover. RT-PCR demonstrated the expression of seven of these genes (TrIPT1. TrIPT13, TrIPT15 TrCKX1, TrCKX2, TrCKX6). Analysis with quantitative real-time PCR showed expression of TrCKX2 increased markedly during leaf expansion and was consistently high during senescence, suggesting a potential role for CKX in facilitating the progression of senescence.

The developmental aspect of drug transport across the blood-brain barrier (BBB) was investigated. Microdialysis was used to study unbound morphine BBB transport at different ages in sheep. An in vitro study was performed to find differentially expressed genes in brain capillary-rich fractions of the brain in rats of different ages. Microdialysis and brain-to-plasma ratios were used to study the contribution of breast cancer resistance protein (Bcrp) to the transport of nitrofurantoin (NTF) across the BBB of rats during development as well as in adult rats and mice. A method of analysing morphine and its metabolites in plasma and microdialysis samples was developed and validated. The in vivo recovery of deuterated morphine, used as a calibrator in microdialysis experiments, was not affected by the presence of morphine in the tissue. A net influx of morphine was observed in premature lambs and adult sheep, in contrast to the efflux seen in other species. This influx decreased with age, indicating that the morphine transport across the BBB changes with age. In contrast, the transport of the morphine metabolite morphine-3-glucuronide (M3G) did not change with age. Microarray data indicated that several active transporters are differentially expressed with age. Moreover, the mRNA expression levels of Abcg2 (Bcrp) and Slc22a8 (organic anion transporter 3) changed with age when quantified using real-time polymerase chain reaction. In contrast, the expression of Abcb1 (P-glycoprotein) and occludin (a tight junction protein) did not change with age. In rats, the brain distribution of NTF decreased with age due to increased protein binding in plasma. The concentration ratio of unbound NTF across the BBB was low in the adult rat, due to intra-brain metabolism and/or efflux by other transporters. Bcrp did not appear to have a significant contribution in the developing rat or in knock-out mice compared to wild-type controls with regard to NTF BBB transport. In conclusion, in vitro studies showed that the expression levels of some genes changed with age, presumably affecting subsequent drug distribution to the brain. Further, in vivo studies showed that distribution across the BBB changed with age for morphine but not for M3G or NTF.

Members of the important disease causing bacterial generas, Chlamydia and Chlamydophila, are characterised by a complex developmental cycle which is comprehensively described by microscopy. The inability to use standard genetic techniques for this obligate intracellular bacterium, however, means that significant gaps in our understanding of the molecular mechanisms used to control growth and development of Chlamydia still exist. The current study investigated the function of bacterial guanosine triphosphatases (GTPases), components of the organism's limited signal transduction arsenal, in regulatory control of the chlamydial development cycle. Initial analysis of the gene transcription of chlamydial GTPases and other predicted signal transduction genes using real time RT-PCR, in a Chlamydophila pneumoniae A-03 tryptophan depletion model of persistence, revealed significant differential expression of genes in response to the addition of interferon gamma (IFN-γ). Predicted chlamydial GTPase encoding genes, ychF, yhbZ and yphC, associated with ribosome function amongst other processes were strongly up-regulated, while hflX was down-regulated in the persistent cultures. Analysis of an additional model of Cp. pneumoniae persistence, induced by limitation of host cell iron, revealed that ychF, yhbZ and yphC were also up-regulated in the persistent cultures. This study provided the most comprehensive analysis of Cp. pneumoniae gene transcription to date and suggest that chlamydial GTPases serve a role in generation of the persistent chlamydial phenotype. Cloning and expression of Cp. pneumoniae and Cp. abortus yhbZ, including demonstration of in vitro GTPase activity, indicates that this chlamydial gene encodes a member of the universally conserved and essential bacterial Obg subfamily of GTPases. Evidence is building that members of this latter family of bacterial GTPases are important regulators of bacterial growth and morphological differentiation in developmentally complex bacteria. Over-expression of chlamydial YhbZ subfamily GTPases in Escherichia coli revealed inhibition of bacterial growth and disruption of cell division and chromosome functions leading to the generation of elongated cells with limited chromosome segregation, as described for Obg subfamily members from E. coli and other bacteria. Although more analysis is required, we suggest a novel mechanism of chlamydial Obg GTPase regulation involving sensing of host cell GTP/GDP pools to control secondary differentiation of reticulate bodies (RBs) back to elementary bodies (EBs). Analysis of the chlamydial complement of bacterial GTPases was extended to HflX, a previously uncharacterised and only predicted GTPase conserved in bacteria. HflX sequence analysis revealed conservation of G motifs responsible for nucleotide binding and hydrolysis (G1, G3, G4) and protein interaction (G2), although the latter was unique to HflX subfamily GTPases. Recombinant Cp. pneumoniae HflX displays GTPase activity with nucleotide specificity for GTP. We tested Cp. pneumoniae HflX function by over-expression in E. coli which led to inhibition of growth in E. coli and elongation of cells with normal chromosome partitioning. This phenotype was the probable result of disruption of a stage in cell division subsequent to chromosome segregation. This present study provides the first evidence to show that bacterial HflX is a GTPase and suggests a regulatory role in bacterial cell cycle control.

Chlamydial infections have been associated with several chronic human diseases, including trachoma, pelvic inflammatory disease, chronic obstructive pulmonary disease and atherosclerotic cardiovascular disease. In Chlamydia-associated disease, the organisms are believed to exist in an atypical, persistent phase that is not well understood at the genetic level. The research presented in this thesis investigated chlamydial gene expression in in vitro cell culture models of persistence. The first set of studies analysed a continuous-infection model of persistence that has been recently developed for two C. pneumoniae isolates (TW-183 and CM-1). The spontaneous establishment and unique cyclical nature of continuous infections could be particularly relevant to in vivo events. An initial analysis using a semi-quantitative reverse transcriptase PCR (sqRT-PCR) approach provided evidence of differential gene expression in C. pneumoniae TW-183 continuous infections relative to acute control infections. Using a subsequently established fully quantitative real-time reverse transcriptase PCR (rtRT-PCR) assay, up-regulated expression profiles were confirmed for five genes (CPn0483, nlpD, ompA, pmp1 and porB) in the continuous C. pneumoniae TW-183 infections. The omcB, pmp1 and porB genes, all of which encode membrane proteins, showed similar patterns of expression over both the acute and continuous time courses tested. Gene expression data for a second C. pneumoniae isolate, CM-1, revealed similar overall expression trends to those seen for C. pneumoniae TW-183 but also supported previous observations of different growth characteristics between the two isolates in the continuous-infection model. The rtRT-PCR assay was further optimised for use in gene expression studies of the gamma interferon (IFN-γ)-mediated model of C. pneumoniae A-03 persistence, in which altered growth and morphological traits typical of chlamydial persistence have been well characterised. Meanwhile, chlamydial genes such as euo, ftsK and hctB were emerging from the literature as reliable genetic markers of persistence. Therefore, a preliminary rtRT-PCR analysis of marker gene expression was used to assess the likely extent of persistence in individual IFN-γ-treated C. pneumoniae A-03 infections from a series of experiments that had been prepared for this persistence model. In this way, an appropriate pair of duplicate experiments was selected for further studies based on strong genetic evidence of persistence in IFN-γ-treated samples at 48 h post-infection (PI) in those experiments. Using rtRT-PCR, 14 genes of interest from the related peptidoglycan, aminosugars and lipopolysaccharide (LPS) biosynthetic pathways were analysed in the validated experiments of the IFN-γ-mediated C. pneumoniae A-03 persistence model. Selective up- and down-regulated expression trends were associated with IFN-γ-treatment at 48 h PI for genes encoding products that are located at specific enzymatic points in these pathways. Most strikingly, the expression of glmU, the product of which controls the amount of an essential precursor metabolite that enters both peptidoglycan and LPS biosynthesis, was strongly and reproducibly down-regulated in the 48-h PI IFN-γ-treated samples. This expression profile may contribute to a reduced rate of peptidoglycan biosynthesis in this persistence model and may therefore be related to the inhibited cell division and RB-to-EB differentiation that characterise chlamydial persistence. While most other genes in these pathways showed unchanged expression associated with IFN-γ treatment, murA and kdsB (from peptidoglycan and LPS biosynthesis, respectively) were selectively up-regulated in the 48-h PI IFN-γ-treated samples. Taken together, these data supported the concept of a persistence stimulon in C. pneumoniae that is regulated at key points in various metabolic pathways. In addition to the analysis of biosynthetic genes, the up-regulated gene set from continuous C. pneumoniae TW-183 infections was also analysed in the validated IFN-γ-mediated C. pneumoniae A-03 persistence experiments. The data revealed similarities and differences in gene expression patterns between these two in vitro persistence models. Furthermore, the profiles obtained for genes such as pmp1 and porB provided insights into the widely predicted phenomenon of late developmental gene shut-down during chlamydial persistence. A final investigation into an analogous IFN-γ-mediated persistence system for C. trachomatis serovar L2 focussed on one up-regulated (murA) and one down-regulated (glmU) gene from the validated IFN-γ-mediated persistent C. pneumoniae A-03 data set. Both genes were significantly down-regulated in persistent C. trachomatis, adding to a growing body of evidence for key differences among chlamydial species in their persistent gene expression patterns. This project has contributed significantly to our understanding of the molecular basis of the important persistent phase of chlamydial development.

Extended spectrum β-lactamases or ESBLs, which are derived from non-ESBL precursors by point mutation of β-lactamase genes (bla), are spreading rapidly all over the world and have caused considerable problems in the treatment of infections caused by bacteria which harbour them. The mechanism of this resistance is not fully understood and a better understanding of these mechanisms might significantly impact on choosing proper diagnostic and treatment strategies. Previous work on SHV β-lactamase gene, blaSHV, has shown that only Klebsiella pneumoniae strains which contain plasmid-borne blaSHV are able to mutate to phenotypically ESBL-positive strains and there was also evidence of an increase in blaSHV copy number. Therefore, it was hypothesised that although specific point mutation is essential for acquisition of ESBL activity, it is not yet enough, and blaSHV copy number amplification is also essential for an ESBL-positive phenotype, with homologous recombination being the likely mechanism of blaSHV copy number expansion. In this study, we investigated the mutation rate of non-ESBL expressing K. pneumoniae isolates to an ESBL-positive status by using the MSS-maximum likelihood method. Our data showed that blaSHV mutation rate of a non-ESBL expressing isolate is lower than the mutation rate of the other single base changes on the chromosome, even with a plasmid-borne blaSHV gene. On the other hand, mutation rate from a low MIC ESBL-positive (≤ 8 µg/mL for cefotaxime) to high MIC ESBL-positive (≥16 µg/mL for cefotaxime) is very high. This is because only gene copy number increase is needed which is probably mediated by homologous recombination that typically takes place at a much higher frequencies than point mutations. Using a subinhibitory concentration of novobiocin, as a homologous recombination inhibitor, revealed that this is the case.