The detection and quantitation of substances using analytical techniques is an important area in many fields. Accuracy,dependability, reproducibility, specificity and stability of the techniques used are all important. The first three parameters are largely operator dependent, however, specificity and stability of the components of the tests also play a part. The specificity of analysis may be determined chemically or biologically by using enzymes, immunological reagents or receptors of some sort. However, biological molecules are often unstable in purified, isolated forms and must be stabilised in some way to retain activity. The work reported here attempts to increase the knowledge of enzyme stabilisation, using the enzyme alcohol oxidase as a test enzyme. This enzyme was used in : (i) A manual assay for ethanol determination. (ii) An automated assay using both soluble and immobilised enzyme in segmented flow and flow injection analysis. (iii) A dry phase stabilised enzyme based test for ethanol in saliva. During the course of the work a method for stabilising the enzyme was discovered. This has been applied to a number of other enzyme systems, successfully stabilising them in most cases. This work forms the basis of a patent application for enzyme stabilisation. A novel detection system allowing semi-quantitative results to be obtained, using stabilised dry phase technology has also been discovered. A second patent application has been filed and the system has been applied to analytes measured by oxidase enzymes. The final area of investigation was to develop an enzymic assay for diacetyl. This substance is a contaminant in beer and as such requires accurate detection at low levels. The purification and characterisation of diacetyl reductase from a new source, (chicken liver) enabled various assay formats to be investigated. These included dye linked assays and enzyme amplified recycling assays to determine diacetyl in aqueous samples.

This work has studied several aspects of the physiology of the animal-colonizing species Campylobacterjejuni, Helicobacter pylori. Wolinella succinogenes and Arcobacter butzleri. C. jejuni and H pylori were found to be obligate microaerophiles and W.succinogenes an anaerobe. A. butzleri was found to be an aerobe able to grow anaerobically. Comparative analyses of the responses of C. jejuni, H pylori and W. succinogenes to various oxygen concentrations were investigated using transcriptomics and genes differentially expressed at higher oxygen concentrations were identified. At the time of this study no microarrays were available for A. butzleri. These comparative studies provided a better understanding of bacterial adaptation to and interaction with their environment. Several enzymes involved in oxireduction processes, including disulfide reductases, were upregulated under oxidative stress. Disulfide reductases of host-colonizing bacteria are involved in the expression of virulence factors, resistance to drugs, and elimination of toxic compounds. CXXC and CXXC-derived motifs are present in the active sites of disulfide reductases and are essential for the catalysis of these redox reactions. Large-scale genome analyses of 281 prokaryotes identified CXXC and CXXC-derived motifs in each microorganism. The total number of these motifs showed correlations with genome size and oxygen tolerance of the prokaryotes. Specific bioinformatic analyses served to identify putative disulfide reductases in the four Campylobacterales species. The project investigated the involvement of these enzymes in resistance to the antibiotic metronidazole, cadmium detoxication and pathogenesis. The activities of disulfide reductases were modulated by the presence of metronidazole, and its reduction was inhibited by the presence of disulfide reductase substrates. In addition, proteins involved in oxireduction of the low redox potential ferredoxin were downregulated in metronidazole resistant strains, suggesting that ferredoxin is involved in the resistant phenotype. Cellular processes and pathways regulated under cadmium stress included fatty acid biosynthesis, protein biosynthesis, chemotaxis and mobility, the tricarboxylic acid cycle, protein modification, redox processes and heat shock response. Notably, the data provided evidence for a role of oxireduction processes in the development of metronidazole resistance and the detoxication of cadmium. Furthermore, a method was developed to identify thiol disulfide oxidoreductases in the four Campylobacterales. The results suggested that H pylori contained a novel disulfide bond formation system. Investigation of their potential involvement in virulence or colonization indicated that the putative thiol disulfide oxidoreductases HP0231 and HP0595 are related to the colonization efficiency ofH pylori. Finally, the only known disulfide reduction system in Campylobacterales, the thioredoxin system, was investigated in more detail. Phylogenetic analyses of the thioredoxin reductases TrxB1 and TrxB2 of the four bacteria were performed. The phylogenetic features of the TrxB2 suggested a special role for this enzyme in the physiology of these bacteria; thus, the enzyme was investigated further in H pylori. TrxB2 was found to be an NADPH reductase, possibly involved in important oxireduction processes within the cell.

Diss. (sammanfattning) Umeå : Univ., 2006. / Härtill 3 uppsatser.

by Ng Wai-Leung. / Thesis submitted in: August 1997. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1998. / Includes bibliographical references (leaves 135-144). / Title page --- p.i / Thesis Committee --- p.ii / Abstract --- p.iii / Acknowledgments --- p.v / Abbreviations --- p.vi / Table of contents --- p.vii / List of figures --- p.x / List of tables --- p.xiii / Chapter Chapter 1. --- General introduction --- p.1 / Chapter Chapter 2. --- Literature review / Chapter 2.1 --- Biosynthesis of deoxyribonucleotide triphosphates --- p.3 / Chapter 2.2 --- Ribonucleotide reductase --- p.6 / Chapter 2.2.1 --- Class I ribonucleotide reductase --- p.6 / Chapter 2.2.2 --- Class II ribonucleotide reductase --- p.13 / Chapter 2.2.3 --- Class III ribonucleotide reductase --- p.14 / Chapter 2.3 --- Proposed mechanism for ribonucleotide reduction --- p.17 / Chapter 2.4 --- Allosteric control of ribonucleotide reductase --- p.21 / Chapter 2.4.1 --- Allosteric control of class I ribonucleotide reductase --- p.21 / Chapter 2.4.2 --- Allosteric control of class II and class III ribonucleotide reductases --- p.23 / Chapter 2.5 --- Evolution of ribonucleotide reductase --- p.25 / Chapter 2.6 --- Central metabolism pathways of enteric bacteria --- p.28 / Chapter 2.7 --- Regulation of gene expression by oxygen in enteric bacteria --- p.33 / Chapter 2.7.1 --- Regulation of gene expression by Fnr --- p.33 / Chapter 2.7.2 --- Regulation of gene expression by AcrAB --- p.39 / Chapter 2.7.3 --- Regulation of gene expression by NarXL and NarQP --- p.42 / Chapter 2.7.4 --- Other aspects in anaerobic gene expression --- p.45 / Chapter 2.7.5 --- Relationship between NrdD and anaerobic metabolism --- p.45 / Chapter 2.8 --- Objectives --- p.46 / Chapter Chapter 3. --- Molecular cloning and sequencing of nrdDG operon from Salmonella typhimurium / Chapter 3.1 --- Introduction --- p.47 / Chapter 3.2 --- Material and methods --- p.48 / Chapter 3.2.1 --- Bacterial strains and bacteriophages --- p.48 / Chapter 3.2.2 --- Culture media --- p.48 / Chapter 3.2.3 --- Preparation of lambda lysate and phage DNA --- p.48 / Chapter 3.2.3.1 --- Plating out pf lambda phage and preparation of plate lysate --- p.48 / Chapter 3.2.3.2 --- Preparation of lambda lysate stock --- p.49 / Chapter 3.2.3.3 --- Preparation of lambda phage DNA --- p.50 / Chapter 3.2.4 --- Long distance polymerase chain reaction (LD-PCR) of nrdDG gene fragment --- p.51 / Chapter 3.2.5 --- Restriction enzyme digestion of LD-PCR products and subcloning of restriction fragments --- p.52 / Chapter 3.2.6 --- Confirmation of recombinants by colony-PCR --- p.53 / Chapter 3.2.7 --- Preparation of plasmid DNA by alkaline lysis using Wizard´ёØ Plus Miniprep DNA Purification System (Promega) --- p.54 / Chapter 3.2.8 --- DNA cycle sequencing by using dye-labeled dideoxy chain terminator and data collection --- p.55 / Chapter 3.2.9 --- Computer software for analyzing and manipulating DNA sequences --- p.57 / Chapter 3.3 --- Results --- p.59 / Chapter 3.3.1 --- Preparation of lambda DNA --- p.59 / Chapter 3.3.2 --- Long distance PCR amplification of nrdDG from lambda DNA --- p.59 / Chapter 3.3.3 --- Restriction digestion of LD-PCR products --- p.61 / Chapter 3.3.4 --- Subcloning of LD-PCR restriction fragments --- p.61 / Chapter 3.3.5 --- Miniprep of plasmid DNA from recombinants and verification of nrdDG identities --- p.64 / Chapter 3.3.6 --- Nucleotide sequence of nrdDG --- p.66 / Chapter 3.4 --- Discussions --- p.72 / Chapter 3.4.1 --- Sequence analysis of S. typhimurium nrdDG --- p.72 / Chapter 3.4.2 --- Experimental design --- p.79 / Chapter Chapter 4. --- Transcriptional regulation of anaerobic ribonucleotide reductase in Salmonella typhimurium in aerobic and anaerobic environments / Chapter 4.1 --- Introduction --- p.84 / Chapter 4.2 --- Materials and methods --- p.86 / Chapter 4.2.1 --- Bacteria and bacteriophages strains / Chapter 4.2.2 --- Culture media --- p.86 / Chapter 4.2.3 --- Construction and characterization of oxrA mutant --- p.87 / Chapter 4.2.3.1 --- Preparation of P22 lysate of TN2336 --- p.87 / Chapter 4.2.3.2 --- P22 transduction for construction of oxrA mutant --- p.87 / Chapter 4.2.3.3 --- Characterization of oxrA mutant --- p.87 / Chapter 4.2.4 --- Extraction of bacterial RNA by hot phenol method --- p.88 / Chapter 4.2.5 --- Formaldehyde gel electrophoresis of RNA --- p.88 / Chapter 4.2.6 --- Reverse transcriptase polymerase chain reaction (RT-PCR) of nrdD transcript --- p.89 / Chapter 4.2.7 --- Transfer of DNA/RNA to solid support --- p.90 / Chapter 4.2.7.1 --- Transfer of DNA to solid support by Southern blotting --- p.90 / Chapter 4.2.7.2 --- Transfer of RNA to solid support by Northern blotting --- p.91 / Chapter 4.2.7.3 --- RNA Dot blot --- p.91 / Chapter 4.2.8 --- Preparation of radioactive-labeled probes for hybridization --- p.92 / Chapter 4.2.8.1 --- Synthesis of radioactive-labeled probes by labeling --- p.92 / Chapter 4.2.8.2 --- Preparation of RNA probe by in vitro transcription --- p.93 / Chapter 4.2.9 --- Hybridization and membrane washing conditions --- p.95 / Chapter 4.2.10 --- Normalization of samples by 16S ribosomal RNA (rRNA) --- p.95 / Chapter 4.3 --- Results --- p.97 / Chapter 4.3.1 --- Preparation of RNA --- p.97 / Chapter 4.3.2 --- RT-PCR of nrdD transcript --- p.97 / Chapter 4.3.3 --- Northern blot analysis of nrdD transcript --- p.103 / Chapter 4.3.4 --- Dot blot hybridization analysis of nrdD expression in an oxrA mutant --- p.103 / Chapter 4.4 --- Discussions --- p.107 / Chapter 4.4.1 --- Expression of nrdD of S. typhimurium in aerobic and anaerobic environments --- p.107 / Chapter 4.4.2 --- Experimental design --- p.110 / Chapter Chapter 5. --- Characterization of nrdD::Tn10 mutant of S. typhimurium / Chapter 5.1 --- Introduction --- p.112 / Chapter 5.2 --- Materials and methods --- p.112 / Chapter 5.2.1 --- Bacteria and bacteriophages strains --- p.113 / Chapter 5.2.2 --- Transduction of zzz-3875::Tn10 to S. typhimurium --- p.113 / Chapter 5.2.3 --- Characterization of zzz-3875::Tn10 by Southern hybridization --- p.113 / Chapter 5.2.3.1 --- Preparation of genomic DNA from S. typhimurium --- p.113 / Chapter 5.2.3.2 --- Restriction enzyme digestion of genomic DNA and Southern hybridization --- p.114 / Chapter 5.2.4 --- Characterization of growth pattern of nrdD::Tn10 mutant --- p.115 / Chapter 5.3 --- Results --- p.116 / Chapter 5.3.1 --- Characterization of zzz-3 875: :Tn7 0 in S. typhimurium --- p.116 / Chapter 5.3.2 --- Characterization of growth pattern of nrdD mutant --- p.120 / Chapter 5.4 --- Discussions --- p.125 / Chapter Chapter 6. --- General Discussions / Chapter 6.1 --- General discussions --- p.131 / Chapter 6.2 --- Further studies --- p.134 / References --- p.135

No description available.

Thesis (Ph. D.)--University of Washington, 1996. / Vita. Includes bibliographical references (leaves [122]-147).

The detrimental effects of oxidative stress caused by the accumulation of Reactive Oxygen Species (ROS) have been acknowledged as major factors in aging, senescence and several neurodegenerative diseases and conditions such as Parkinson’s disease and stroke (ischemia/reperfusion). Mammalian models are extremely susceptible to these stresses that follow the restoration of oxygen after anoxia; however, some organisms including the freshwater turtle Trachemys scripta can withstand several bouts of anoxia and repeated reoxygenation without any apparent pathology. T. scripta thus provides us with an alternate vertebrate model in which we can investigate physiological mechanisms of neuroprotection without the damaging effects that come with oxidative stress. The major objective of this study was to investigate the protective mechanisms in the turtle brain under conditions of anoxia and oxidative stress. Specifically, the focus is on the Methionine Sulfoxide Reductase system (Msr), an antioxidant and cellular repair system, and how it is regulated to protect the brain against such stressors. Previous studies in my lab have demonstrated that Msr mRNA and protein levels are differentially upregulated during anoxia and reoxygenation. To investigate the regulation of Msr, FOXO3a was directly induced by transfecting a human FOXO3a plasmid into turtle brain cell cultures, as FOXO3a has been shown to regulate MsrA levels in other animal models. Pharmacological manipulation of FOXO3a was also performed using the green tea extract Epigallocatechin gallate (EGCG) as it has been shown to increase expression of FOXO3a during oxidative stress conditions in other models. I found that an induction of human FOXO3a increased FOXO3a levels and showed protection against cell death during oxidative stress. Furthermore, treatment of cells with EGCG increased expression of FOXO3a only when the cells were exposed to oxidative stress and decreased cell death. Induction of FOXO3a and EGCG treatment did not increase MsrA levels, however MsrB3 levels were upregulated under both treatments but only in the presence of oxidative stress. These results suggest that MsrA and MsrB3 protect the cells from oxidative stress damage through different molecular pathways and that EGCG may be a therapeutic target to treat diseases related to damage by oxidative stress. / Includes bibliography. / Dissertation (Ph.D.)--Florida Atlantic University, 2018. / FAU Electronic Theses and Dissertations Collection

The oxidation of methionine (Met) into methionine sulfoxide (met-(o)) leads to deleterious modifications to a variety of cellular constituents. These deleterious alterations can be reversed by enzymes known as methionine sulfoxide reductases (Msr). The Msr (MsrA and MsrB) family of enzymes have been studied extensively for their biological roles in reducing oxidized Met residues back into functional Met. A wide range of studies have focused on Msr both in vivo and in vitro using a variety of model organisms. More specifically, studies have noted numerous processes affected by the overexpression, under expression, and silencing of MsrA and MsrB. Collectively, the results of these studies have shown that Msr is involved in lifespan and the management of oxidative stress. More recent evidence is emerging that supports existing biological functions of Msr and theorizes the involvement of Msr in numerous biological pathways. / Includes bibliography. / Thesis (M.S.)--Florida Atlantic University, 2018. / FAU Electronic Theses and Dissertations Collection

食物中毒感染是常見但非常重要的全球性公共健康問題。沙門氏鼠傷寒桿菌乃常被分離出來的細菌性病原體之一。隨著實驗室參考菌株LT2的基因組序列於2001年被發表之後，另外9個沙門氏鼠傷寒菌菌株的基因序列均已陸續進行測序。最近，本實驗室亦對十個本地沙門氏鼠傷寒菌臨床分離菌株的基因序列進行了測序。為了為這些基因組序列提供高品質的註釋，我們把預測的基因組提交到質量控制工具GenePRIMP以識別有潛在錯誤或異常的預測基因。本研究針對血液分離菌株78896和糞便分離菌株1047518的GenePRIMP報告進行人工檢查，並對每個菌株超過270個的基因進行了修訂。此外，本研究亦對上述的10個本地菌株進行了功能註釋。註釋項目包括沙門氏菌致病島（SPIs）、致病因子、tRNA和非編碼小分子RNA、噬菌體和CRISPRs結構等基因組及致病元素。 KEGG通路則提供了進一步的功能註釋。 / 本研究同時對本地的血液和糞便分離菌株，連同國外的臨床分離菌株，進行了廣泛的比對，用以識別全身性沙門氏菌感染的潛在遺傳因素。 本研究進行了以下基因分析：（1）多位點序列分型（MLST）；（2）在小鼠全身性感染中涉及的主調控因子和關鍵元素; 及（3）人類腸胃道感染中涉及的基因。然而，這些分析產生只能對全身性沙門氏菌感染提供有限的見解。然而，透過使用RAST註釋系統，我們於其中三個血液分離菌株中發現了一個的額外的螯鐵蛋白aerobactin鐵採集系統。儘管在體外實驗中，這些血液分離菌株並沒有明顯的生長優勢，但實驗結果表明，在缺乏鐵的培養液中，aerobactin基因的表達水平是比較高的。此外，我們亦於其中四個血液分離菌株中，發現負責細胞色素c熟成(ccm)的基因座均被中斷。這可能改變了這些血液分離菌株中細胞色素c的生物合成途徑。這些鐵採集和同化機制的觀察均為未來全身性沙門氏菌感染的研究提供了可能的發展方向。 / 本研究同時識別了用以分別本地及海外的沙門氏鼠傷寒菌菌株的分子標記，並在鮭魚和生菜的接種實驗中，展現了它們分辨本地及海外菌株的能力。然而，在投入實際應用之前，這些標記尚需要進一步的驗證和測試，以便確定快速檢測方法的有效性。 / Foodborne infection is a common but important public health issue worldwide. Salmonella enterica serovar Typhimurium is frequently isolated from outbreaks as one of the common bacterial causative agents. Following the availability of the genome sequence of the reference lab strain LT2 in 2001, nine genomes of S. Typhimurium had been sequenced since then. Recently, genomes of ten local S. Typhimurium clinical isolates have been assembled in our laboratory. In order to provide high quality annotation of these genome sequences, the predicted gene sets were submitted to the quality control tool GenePRIMP (Gene PRediction IMprovement Pipeline) to identify potentially erroneous and abnormal gene calls. The GenePRIMP reports for the local blood isolate 78896 and stool isolate 1047518 were manually inspected and more than 270 genes were amended individually for each isolate. Functional annotation had also been performed for the 10 local isolates. Genomic and virulent elements including Salmonella Pathogenicity Islands (SPIs), virulence factors, tRNAs and small non-coding RNAs, prophage elements and CRISPRs structures had been annotated. The KEGG pathways provided a further means of functional annotation. / The local blood and stool isolates, together with the sequenced foreign clinical isolates, had also been extensively compared to identify potential genetic determinants of Salmonella systemic infection. (1) Multilocus sequence typing (MLST); (2) Alignment of master regulators and key players of systemic infection in mice; and (3) Analyses of the genes responsible for human gastrointestinal tract infection had been performed. However, these analyses yielded limited insights on systemic infection. Alternatively, using subsystems annotation by RAST, an additional aerobactin siderophore iron acquisition system was shown to be prevalent among three of the blood isolates. Despite no obvious growth advantage was offered to the blood isolates in an in vitro experiment, it was demonstrated that expression of the aerobactin genes was higher in iron-depleted culturing medium. In addition, a disrupted cytochrome c maturation (ccm) locus that may alter the cytochrome c biogenesis pathway was also identified in four of the blood isolates. These observations in iron acquisition and assimilation mechanisms suggest their potential in future direction of Salmonella systemic infection studies. / Molecular markers specific to local and foreign S. Typhimurium isolates were also identified and their utility in differentiating local and foreign isolates was demonstrated in a pilot spiking experiment using raw salmon and lettuce. These markers will require further verification and testing prior to actual application in real-world settings in order to examine the validity of the rapid detection method. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Cheng, Chi Keung. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 124-146). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / Abstract of thesis entitled --- p.iii / 摘要 --- p.v / Acknowledgements --- p.vii / Table of Contents --- p.viii / List of Tables --- p.xi / List of Figures --- p.xiii / Abbreviations --- p.xiv / Chapter Chapter 1 --- Literature Review --- p.1 / Chapter 1.1 --- Introduction and Taxonomy --- p.1 / Chapter 1.2 --- Epidemiology of Salmonella Typhimurium infections --- p.2 / Chapter 1.3 --- Pathogenesis of Salmonella Typhimurium infection --- p.4 / Chapter 1.3.1 --- Infection mechanisms --- p.4 / Chapter 1.3.2 --- Salmonella Pathogenicity Islands --- p.6 / Chapter 1.3.3 --- Regulation of virulence --- p.9 / Chapter 1.4 --- Non-typhoid Salmonella (NTS) systemic infection --- p.11 / Chapter 1.4.1 --- Epidemiology of NTS systemic infection --- p.11 / Chapter 1.4.2 --- Salmonella Typhimurium multidrug resistance --- p.12 / Chapter 1.5 --- Salmonella Typhimurium genomics --- p.15 / Chapter 1.5.1 --- Salmonella Typhimurium genome sequencing --- p.15 / Chapter 1.5.2 --- Comparative studies on Salmonella genomes --- p.17 / Chapter 1.6 --- Aims of project --- p.19 / Chapter Chapter 2 --- Curation and detailed annotation of genomes of local Salmonella Typhimurium clinical isolates --- p.22 / Chapter 2.1 --- Introduction --- p.22 / Chapter 2.2 --- Materials and Methods --- p.27 / Chapter 2.2.1 --- Manual curation of GenePRIMP results --- p.27 / Chapter 2.2.2 --- Salmonella Pathogenicity Islands (SPIs) and virulence factors annotation --- p.29 / Chapter 2.2.3 --- Small RNA and t-RNA annotation --- p.29 / Chapter 2.2.4 --- Phage elements annotation --- p.30 / Chapter 2.2.5 --- CRISPRs annotation --- p.30 / Chapter 2.2.6 --- KEGG annotation --- p.30 / Chapter 2.3 --- Results --- p.32 / Chapter 2.3.1 --- Manual curation of GenePRIMP results --- p.32 / Chapter 2.3.1.1 --- Short genes --- p.35 / Chapter 2.3.1.2 --- Long genes --- p.35 / Chapter 2.3.1.3 --- Unique genes --- p.36 / Chapter 2.3.1.4 --- Overlapped genes --- p.36 / Chapter 2.3.1.5 --- Broken genes --- p.37 / Chapter 2.3.2 --- Salmonella Pathogenicity Islands (SPIs) and virulence factors annotation --- p.37 / Chapter 2.3.2.1 --- Salmonella Pathogenicity Islands (SPIs) annotation --- p.37 / Chapter 2.3.2.2 --- Virulence factors annotation --- p.44 / Chapter 2.3.3 --- Small RNA and t-RNA annotation --- p.44 / Chapter 2.3.4 --- Phage elements annotation --- p.44 / Chapter 2.3.5 --- CRISPRs annotation --- p.50 / Chapter 2.3.6 --- KEGG annotation --- p.51 / Chapter 2.4 --- Discussion --- p.53 / Chapter 2.4.1 --- Manual curation of GenePRIMP results --- p.53 / Chapter 2.4.1.1 --- Gene amendment not required --- p.54 / Chapter 2.4.1.2 --- Genes with boundaries relocated --- p.54 / Chapter 2.4.1.3 --- Genes to be discarded --- p.55 / Chapter 2.4.1.4 --- Gene pairs to be fused --- p.55 / Chapter 2.4.1.5 --- Potential pseudogenes formation --- p.56 / Chapter 2.4.2 --- Salmonella Pathogenicity Islands (SPIs) annotation --- p.57 / Chapter 2.4.3 --- Virulence factors annotation --- p.57 / Chapter 2.4.4 --- Small RNA and t-RNA annotation --- p.58 / Chapter 2.4.5 --- Phage elements annotation --- p.59 / Chapter Chapter 3 --- Identification of genetic determinants of blood invasiveness in local S. Typhimurium clinical isolates --- p.61 / Chapter 3.1 --- Introduction --- p.61 / Chapter 3.2 --- Materials and Methods --- p.66 / Chapter 3.2.1 --- Multilocus Sequence Typing (MLST) --- p.66 / Chapter 3.2.2 --- Phage elements annotation for foreign isolates --- p.67 / Chapter 3.2.3 --- Alignment of genes inferred to play important roles in NTS systemic --- p.infection67 / Chapter 3.2.4 --- Alignment of genes inferred to involved during infection in the gastrointestinal (GI) tract --- p.68 / Chapter 3.2.5 --- Subsystems assignment using Rapid Annotation using Subsystem Technology (RAST) server --- p.68 / Chapter 3.2.6 --- Growth analysis of local S. Typhimurium clinical isolates in iron-limiting environment --- p.69 / Chapter 3.2.7 --- Reverse transcription and real-time PCR --- p.70 / Chapter 3.2.7.1 --- Primer design and verification --- p.70 / Chapter 3.2.7.2 --- cDNA synthesis and real-time PCR --- p.70 / Chapter 3.3 --- Results --- p.73 / Chapter 3.3.1 --- Multilocus Sequence Typing (MLST) --- p.73 / Chapter 3.3.2 --- Phage elements annotation for foreign isolates --- p.73 / Chapter 3.3.3 --- Alignment of genes inferred to play important roles in NTS systemic infection --- p.74 / Chapter 3.3.4 --- Alignment of genes inferred to involved during infection in the gastrointestinal (GI) tract --- p.79 / Chapter 3.3.4.1 --- Acid tolerance response --- p.79 / Chapter 3.3.4.2 --- Epithelial cells attachment --- p.80 / Chapter 3.3.4.3 --- Epithelial cells invasion --- p.83 / Chapter 3.3.4.4 --- Survival within macrophages --- p.83 / Chapter 3.3.5 --- RAST subsystem analysis --- p.86 / Chapter 3.3.6 --- Growth analysis and aerobactin genes expression --- p.87 / Chapter 3.4 --- Discussion --- p.93 / Chapter Chapter 4 --- Molecular markers identification and testing on selected foodstuff for local S. Typhimurium isolates --- p.97 / Chapter 4.1 --- Introduction --- p.97 / Chapter 4.2 --- Materials and Methods --- p.101 / Chapter 4.2.1 --- Molecular markers identification --- p.101 / Chapter 4.2.2 --- Primer design and verification --- p.101 / Chapter 4.2.3 --- Spiking experiments on selected food samples --- p.103 / Chapter 4.2.4 --- Quantitative TaqMan real-time PCR --- p.103 / Chapter 4.3 --- Results --- p.105 / Chapter 4.3.1 --- Molecular markers identification --- p.105 / Chapter 4.3.2 --- Spiking experiments and TaqMan real-time PCR --- p.109 / Chapter 4.4 --- Discussion --- p.113 / Chapter 4.4.1 --- Molecular markers identification --- p.113 / Chapter 4.4.2 --- Spiking experiments and TaqMan real-time PCR --- p.114 / Chapter Chapter 5 --- General discussion --- p.116 / Chapter 5.1 --- Manual curation of GenePRIMP results --- p.116 / Chapter 5.2 --- Functional annotation of local S. Typhimurium genomes --- p.118 / Chapter 5.3 --- Systemic infection studies --- p.120 / Chapter 5.4 --- Molecular markers identification and spiking experiments --- p.121 / Chapter 5.5 --- Conclusion and future perspectives --- p.122 / References --- p.124

Diabetics often have elevated levels of serum lipids and cholesterol and increased risk of cardiovascular disease. Streptozotocin-induced diabetes was used to determine whether elevated serum cholesterol levels in diabetics are due to loss of control of hydroxymethylglutaryl coenzyme A (HMG-CoA) reductase, which catalyzes the committed step in cholesterol synthesis. Strain A/ST female mice were fed 10% corn oil diets, half with 2% cholesterol. Experimental groups were injected with 9.0 mg streptozotocin / 100g body weight. Diabetes was confirmed by weight loss, elevated blood sugars, and enlarged spleens. Reductase activity was assayed spectrophotometrically. Serum cholesterol levels were determined by gas liquid chromatography. Both diabetic and control mice fed cholesterol had elevated serum cholesterol levels and decreased reductase activities. These observations suggest that HMG CoA reductase is not the primary control point in the control of serum cholesterol levels in diabetic mice. The increase in serum cholesterol in the SI mice was not more than in the control group, suggesting that increased serum cholesterol is not a key factor in the control of coronary heart disease and related diseases in diabetics. The HMG CoA reductase activity was reduced in both SI and control mice fed 2% cholesterol, but not significantly, possibly due to a small sample size. Other substances that control serum cholesterol are all density classes of lipoproteins (high, intermediate, low, and very low) as well as the chylomicrons. / Department of Biology