Thesis (PhD)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: see item for full text / AFRIKAANSE OPSOMMING: sien item vir volteks

Mutations in BRCA1 and BRCA2, the first two breast cancer susceptibility genes identified, carry as much as an 85% lifetime risk of developing breast, ovarian or other cancers. Genetic testing for mutations in these two genes has recently become commercially available. There have been varying amounts of psychological distress noted among women with a family history of breast cancer. Distress has been observed to impact psychological functioning, activities of daily living, and the practice of breast cancer surveillance behaviors. Within the genetic screening process, psychological distress has been shown to impact the decision to undergo genetic screening, the comprehension and retention of risk assessment information, as well as affecting the subject following the receipt of the genetic test results. Little work has been done to examine predictors of distress within at risk subjects. This study examines psychological distress among 52 community women presenting for BRCA1 and BRCA2 genetic mutation testing. Predictors of distress included family cancer history, education, age, Ashkenazi ethnicity, and Internality and Powerful Others Health Locus of Control. Vulnerable sub-groups of patients include younger women, women with higher levels of education and women of Ashkenazi ethnicity.

Crohn's Disease (CD) is a debilitating condition characterised by chronic intermittent intestinal inflammation. More than 90 genetic polymorphisms are associated with CD susceptibility, including several in genes of the innate immune system. Here I present a series of experiments designed to enhance our knowledge of the roles of CD-associated polymorphisms in pathogenesis. Many therapeutic regimens are employed in CD treatment, but patients' responses to treatment and disease progression vary widely. There is great interest in studying whether analysis of patients' genotype at CD-associated polymorphisms can be used to predict their disease course, and guide clinical decision-making. To answer these questions, it is essential to be able routinely and cost- effectively to genotype patients at the full range of known CD-associated polymorphisms. The first project presented here describes the design and initial successful testing of a CD-specific genotyping microarray for use in genotype-phenotype studies. The polymorphism most strongly associated with CD susceptibility is in the pattern recognition receptor NOD2; the remaining experiments presented here study the function of NOD2 in primary human monocyte-derived Dendritic Cells (DCs). First, a microarray study is presented which characterises global transcriptional responses to NOD2 stimulation in DCs. NOD2 stimulation is shown to enhance transcriptional changes induced by Toll-Like Receptor 2 stimulation, and NOD2-mediated transcriptional regulation is shown to be lost in DCs expressing CD-associated NOD2 variants. Second, experiments are presented which describe development of a new protocol for proteomic analysis of post-translational protein modifications, and which identify a number of novel candidate targets of NOD2 signalling in DCs. Finally, a project is presented which demonstrates for the first time that NOD2 stimulation induces autophagy in DCs, in an NF-kB and RIPK2-dependent pathway. CD-associated polymorphisms in NOD2 and ATG 16Ll abolish NOD2-mediated autophagy induction, resulting in impaired bacterial handling and antigen presentation.

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A thesis submitted to the Faculty of Science under the school of Molecular and Cell Biology in fulfilment for requirements for Doctor of Philosophy Degree. February 2016 / The use of synthetic chemical pesticides has several negative implications for the Agricultural industry, which include the development of resistance to the insecticides, crop contamination and the killing of non-target insects. This has brought many scientists in the field of nematology and entomology to investigate biological control agents which can help solve identified challenges and these biocontrol agents have also included entomopathogenic nematodes. The majority of entomopathogenic nematodes species that have been isolated belong to Heterorhabditids and Steinernematids which act as vectors for insect pathogenic bacteria species belonging to the genera, Photorhabdus and Xenorhabdus, respectively. However, other species of nematodes, one of which includes a strain of Caenorhabditis briggsae, have also been shown to act as a vector for an insect pathogenic strain of Serratia marcescens. Oscheius sp. TEL-2014 EPNs have been observed to act as vectors for insect pathogenic bacteria belonging to the genus Serratia. In this study a novel insect pathogenic Serratia sp. strain TEL was isolated from the gut of infective juveniles belonging to a species of Oscheius sp. TEL-2014. Next generation sequencing of the bacteria was conducted by generating genomic DNA paired-end libraries with the Nextera DNA sample preparation kit (Illumina) and indexed using the Nextera index kit (Illumina). Paired-end (2 × 300 bp) sequencing was performed on a MiSeq Illumina using the MiSeq reagent kit v3 at the Agricultural Research Council Biotechnology Platform. Quality control and adapter trimming was performed and the genome was assembled using SPADES. 19 contigs were generated with an average length of 301767 bp and N50 of 200,110 bp. The genome of the Serratia sp. TEL was found to be 5,000,541 bp in size, with a G+C content of 59.1%, which was similar to that of other Serratia species previously identified. Furthermore, the contigs were annotated using NCBI Prokaryotic Genome Automatic Annotation Pipeline. Features of the annotated genome included protein encoding sequence or genes, rRNA encoding genes, tRNA encoding genes, ncRNA sequences and repeat regions. 4,647 genes were found and 4,495 were protein-coding sequences (CDS). The genome contains 36 pseudo genes, 2 CRISPR arrays, 13 rRNA genes with five operons (5S, 16S, 23S), 88 tRNAs genes, 15 ncRNA sequences and 9 frameshifted genes. Several genes involved in virulence, disease, defense, stress response, cell division, motility and chemotaxis were identified. This genome sequence will allow for the investigation of identified genes and that will be critical in furthering the understanding of the insect pathogenicity of Serratia sp. strain TEL. Furthermore, it will provide additional genomic insights about the insect-nematode interactions and thus help us improve their ability to be used as biological control agents in agricultural industries. Oscheius sp. TEL-2014 was tested for its entomopathogenicity and it was found that this species was able to infect and kill two model insects Galleria mellonella and Tenebrio molitor. This new nematode species brought 100% mortality within 72 h post-exposure in G. mellonella and whereas, within 96 hours in T. molitor. Following morphometrics analysis of Oscheius sp. TEL-2014 it was concluded that this nematode is described as a novel entomopathogenic nematode species based on its morphometrics and 18S rRNA gene sequence originality. Whole genome sequencing of Oscheius sp. TEL-2014 inbred lines (7 and 13) was performed using Illumina Hiseq sequencing system and paired ends library preparation protocol. Sequencing reads assembled on Velvet resulted in generation of 75965 contigs (line 7) and 53190 contigs (line 13). Gene prediction tools showed that proteins involved in gene expression and DNA replication are present in Oscheius sp. TEL-2014. The draft genome of Oscheius nematodes will support the improvement and initiation of further studies intended to help us understand the molecular and metabolic processes in this genus.

We attempted to understand the molecular regulators that impact inflammation using a rat model of human sensation-seeking/risk-taking trait for drug and stress vulnerability, based on their exploratory behavior displaying high rates (HRs) or low rates of locomotor reactivity (LRs) to environmental stress. We found that HRs have a pro-inflammatory phenotype as indicated by increased protein expression of the inflammatory cytokine TNF-(Sa(B. Furthermore, we found that HRs have a lower gene expression of the glucocorticoid receptor and histone deacetylase 2 which are known to play an immunosuppressive role. Autophagy (macroautophagy) is a homeostatic process needed for cell maintenance, growth and proliferation and known to assist in tumor survival. FYVE and coiled-coil domain containing 1 (FYCO1) is a novel protein implicated to assist in the plus-end directed trafficking and fusion of autophagosomes. In these studies, we show that FYCO1 gene expression among human breast cell lines of varying degrees of malignancy. / Lillian C. Onwuka-Ekpete. / Thesis (M.S.)--Florida Atlantic University, 2012. / Includes bibliography. / Mode of access: World Wide Web. / System requirements: Adobe Reader.

In recent years more and more researchers have begun to use data mining and machine learning tools to analyze gene microarray data. In this thesis we have collected a selection of datasets revolving around prediction of patient response in the specific area of breast cancer treatment. The datasets collected in this paper are all obtained from gene chips, which have become the industry standard in measurement of gene expression. In this thesis we will discuss the methods and procedures used in the studies to analyze the datasets and their effects on treatment prediction with a particular interest in the selection of genes for predicting patient response. We will also analyze the datasets on our own in a uniform manner to determine the validity of these datasets in terms of learning potential and provide strategies for future work which explore how to best identify gene signatures. / Includes bibliography. / Thesis (M.S.)--Florida Atlantic University, 2014. / FAU Electronic Theses and Dissertations Collection

Restrictive cardiomyopathy (RCM) is a heart muscle disease, characterized by diastolic dysfunction. The present dissertation is to understand the mechanisms underlyijng the initiation of diastolic dysfunction and the fast disease progression to early death in a RCM mouse model, the transgenic cTnI193His mouse... These data showed that myocardial ischemia occurred after diastolic dysfunction and before systolic dysfunction which proceeded congestive heart failure. The results demonstrate that myocardial ischemia causing cardiomycete death is a link between the initial diastolic dysfunction and late-stage systolic dysfunction, and accelerates the disease progression to fatal heart failure in the early age. / by Yuejin Li. / Thesis (Ph.D.)--Florida Atlantic University, 2011. / Includes bibliography. / Mode of access: World Wide Web. / System requirements: Adobe Reader.

Cytogenetics is a study on the genetic considerations associated with structural and functional aspects of the cells with reference to chromosomal inclusions. Chromosomes are structures within the cells containing body's information in the form of strings of DNA. When atypical version or structural abnormality in one or more chromosomes prevails, it is defined as chromosomal aberrations (CA) depicting certain genetic pathogeny (known as genetic disorders). The present study assumes the presence of normal and abnormal chromosomal sets in varying proportions in the cytogenetic complex ; and, stochastical mixture theory is invoked to ascertain the information redundancy as a function of fractional abnormal chromosome population. This bioinformatic measure of redundancy is indicated as a track-parameter towards the progression of genetic disorder, for example, the growth of cancer. Lastly, using the results obtained, conclusions are enumerated, inferences are outlined and directions for future studies are considered. / by Jagadeshwari Karri. / Thesis (M.S.C.S.)--Florida Atlantic University, 2012. / Includes bibliography. / Mode of access: World Wide Web. / System requirements: Adobe Reader.

目的:銀屑病是一種慢性炎症性皮膚疾病，特點是角質形成細胞過度增生。角質形成細胞過度增生在銀屑病家族中被證實與位於染色體6p2 1. 3 上的編碼人類白細胞抗原的基因組區有關。通過全基因組掃描，至少確定了10 個銀屑病易感基因位點(PSORS 卜的ORS10) ，而6p21 區的PSORS1 被認為是銀屑病最主要的易感位點，而HLA-C， CCHCRl (coiled coil alpha-helixrod homolog) 和CDSN(corneodesmosin) 是該位點上的重要的侯選基因。然而，這三個基因之間的連鎖不平衡很難將他們的個體效應分開。我們的目標是研究CCHCCRl 基因以及它的相關蛋白在皮膚上皮細胞增生過程中的作用。 / 方法:本研究應用免疫螢光技術'免疫印跡分析和即時反轉錄聚合臨鏈反應 (Real Time RT-PCR) 三種方法比較CCHCRl 基因在銀屑病人的皮損區與正常人的皮膚細胞的表達。其次比較了CCHCRl 蛋白在喜樹鹼(拓撲異構酪I 抑制劑/凋亡誘導劑)的作用下在三種細胞(永生化的人皮膚角質形成細胞-HaCaT '人直結腸癌細胞-HT29 和人子宮頸癌細胞- HeLa) 內的表達。最後，我們應用了皮膚器官樣培養物( OTC) 研究HaCaT 細胞於皮膚角質化過程中CCHCRl 蛋白的表達。 / 結果:✹HCR1 蛋白在正常皮膚和銀屑病人皮損皮膚表達模式不同，銀屑病人皮損皮膚有五種CCHCR1 蛋白染色模式，而正常人皮膚只有兩種染色模式。免疫印跡分析顯示CCHCR1 蛋白在正常皮膚含量較銀屑病人高。免役螢光顯微技術顯示角蛋白17 (K17) ，一種細胞增生的標誌性蛋白，只表達在正常皮膚基底層的角質細胞中，而在銀屑病人皮膚， K17 從基底層到顆粒層都有顯著表達，並且和CCHCR1 蛋白表達區域相同。這種現象也存在於OTC 發育過程中所有角質細胞中，而CCHCR1 蛋白表達隨OTC 的培養時間從10 天到21 天呈上升趨勢，說明CCHCR1 蛋白與細胞增殖有一定的關係。當細胞生長在培養血表面形成細胞與細胞接合全面的細胞層時， CCHCR1 的信使核糖核酸水平大幅升高，顯示CCHCR1 的信使核糖核酸表達當細胞生長速度呈負相關。CCHCR1 的信使核糖核酸水平在同步化的HaCaT 細胞G2/M 期輕度升高，而蛋白水平在G1 期達到最高。在G2/M 期，高爾基體出現崩解，而CCHCR1 蛋白和其他高爾基蛋白一樣分散到胞漿。這種現象同樣地出現在經2 州喜樹鹼刺激48小時的HeLa 和HaCaT 細胞內，免疫細胞化學染色顯示CCHCR1 和golgin-97 分散到胞漿。CCHCR1 信使核糖核酸水平在HeLa 和HaCaT 細胞都升高，而蛋白在HaCaT 細胞明顯上調。這說明CCHCR1 蛋白量的上調和細胞生長停滯有關。應用siRNA 或shRNA 抑制CCHCR1 蛋白表達後， golgin-97 分散到胞漿，但是HeLa細胞可以繼續增殖。這說明CCHCR1 蛋白可能有助於維持高爾基複合體的完整，喜樹鹼對細胞生長的抑制作用可能與CCHCR1 蛋白的上調相關。 / 結論: CCHCR1 蛋白在正常皮膚表皮細胞和銀屑病人皮損區細胞胞漿內都有表達，但是正常皮膚表達較銀屑病人高，在OTC 增生後期和細胞生長緩慢或停滯期也明顯升高，說明CCHCRl 基因的上調可能抑制細胞的增生。經喜樹鹼刺激後，細胞生長受到抑制，細胞停滯在Gl 期並誘導細胞凋亡， CCHCRl 的信使核糖核酸和蛋白都上調，表明CCHCRl 與細胞的增生抑制有關。同時，它的下調也造成高爾基複合體的崩解，說明CCHCRl 蛋白可以維持高爾基複合體的結構完整性。因此， CCHCRl 不僅可以保持高爾基複合體的完整而且和細胞的增生有關。另一方面，大量證據表明長期性的高血糖會導致胰島　細胞功能紊亂。鑒於此，揭示胰島功能調節的潛在機理并闡明胰島功能与高血糖症之間的關係變得尤為重要。 / Aim: Psoriasis is one of the common chronic inflammatory skin disorders characterized by keratinocyte hyperproliferation, T lymphocyte-mediated inflammation, and abnormal differentiation. Genome-wide scans have revealed that at least ten different susceptibility loci, PSORS1-PSORS10, were linked to psoriasis, among which PSORS 1 on chromosome 6p21.3 was unambiguously associated with families with psoriasis. Three strongly psoriasis-associated susceptibility alleles have been identified in PSORS1, namely HLA-C, CCHCR1 (coiled-coil alpha-helical rod protein 1 ), and CDSN ( comeodesmosin); their strong linkage disequilibrium, however, makes it very difficult to distinguish their individual genetic effects. Among them, we were interested in the CCHCR1 gene, and its possible roles with associated proteins in the process of skin epidermal cell proliferation were studied in this thesis. / Methods: The cellular expressiOn of CCHCR1 protein in the epidermis was compared between human skins from healthy donors and psoriasis patients by immunofluorescence microscopy and immunoblotting analysis. Its intracellular expression was investigated in cultured human immortalized skin keratinocyte cell line (HaCaT), human colorectal cancer cell line (HT29) and human cervical carcinoma HeLa cell line, as well as in the skin keratinocyte-fibroblast organotypic culture (OTC). The responses of these cells to camptothecin (CPT), a topoisomerase I inhibitor, were compared in HaCaT cells, HT29 cells and HeLa cells. In cell experiments, CCHCR1 expression was studied by immunofluorescence microscopy, immunoblotting analysis, and real-time reverse transcriptase-polymerase chain reaction (real-time RT-PCR). / Results: The immunofluorescence staining patterns for CCHCR1 protein were different between normal and psoriasis skin biopsies. There were at least five patterns (Patterns I, III-VI) in psoriatic skins, but only two (Patterns I and II) in the normal skin, implicating that the expression of CCHCR1 protein was changed in psoriasis. Besides the different cellular location of CCHCR1 protein in normal and psoriasis skins, a higher level of CCHCR1 expression was measured in normal skin than that in psoriatic skin by immunoblotting analysis, and immunofluorescence microscopy further revealed a co-localization of CCHCR1 protein with keratin 17 (K17), a proliferation marker protein and also putative autoantigen for psoriasis, to basal keratinocytes of normal skins, and extended further fro m the basal to granular keratinocytes in the epidermis of psoriasis patients, suggesting a close association of CCHCR1 protein to cell proliferation. During epidermal development in OTC, the CCHCR1 protein was expressed in all keratinocyte layers in a time-dependent manner from day 1 0 to day 21, reaching a maximal level at day 21 when cell proliferation decreased and differentiation to the corneum became active. Furthermore, immunofluorescence for K17 remained co-localized to CCHCR1-positive cells in OTC from day 10 to day 14, but the level of K17 was very weak in the keratinocytes of suprabasallayers on day 21. In HaCaT cell culture, CCHCR1 transcription level at 100% confluence was 25 folds higher than that in subconfluence. Taken all these results together, the up-regulation of CCHCR1 was closely related to cell growth inhibition and differentiation. Immunofluorescence microscopy revealed a co-localization of CCHCR1 protein with a Golgi marker protein, golgin-97, in the compact Golgi complex at the juxtanuclear region of HaCaT cells and HeLa cells. CCHCR1 gene transcribed in all phases of the cell cycle, but was slightly higher in the G2/M phase of synchronous HaCaT cells, and its translation reached its maximal level in the G 1 phase. In the G2/M phase, CCHCR1 protein was dispersed in the cytoplasm like the golgin. Such dispersal was also observed in HaCaT cells and HeLa cells treated with CPT for 24 h and 48 h, respectively. CCHCR1 expression increased in both transcriptional and translational levels as well as apoptotic changes following growth arrest in both HeLa cells and HaCaT cells. Depletion of the CCHCR1 protein by means of siRNAor shRNA-mediated knockdown induced HeLa cells proliferation, cell elongation and disassembly of the Golgi complex. / Conclusion: CCHCR1 protein was expressed in the cytoplasm of epidermal keratinocytes of both normal and psoriasis skins, with a higher level detected in the normal skins. It was also found especially abundant in the keratinocytes of skin organotypic cultures during their transition from proliferation to differentiation. CCHCR1 gene transcription was increased obviously in cultured HaCaT cells at confluence. CCHCR1 was also up-regulated at both transcriptional and translational levels in response to the antiproliferative drug, camptothecin, in HaCaT cell experiments, and was accompanied by G 1 arrest and subsequent apoptosis. When CCHCR1 was knockdowned in HeLa cells, the Golgi complex disassembled, implicating a role of CCHCRl protein in the maintenance of Golgi integrity and in the control of cell proliferation. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Wang, Lijun. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 107-133). / Abstracts also in Chinese. / Abstract --- p.i / Acknowledgements --- p.viii / List of Abbreviation --- p.ix / Chapter Chapter 1 --- General Introduction --- p.1 / Chapter 1.1 --- Psoriasis --- p.1 / Chapter 1.2 --- Skin --- p.9 / Chapter 1.3 --- An In Vitro Model for Human Skin Equivalent --- p.10 / Chapter 1.4 --- Aim of Study --- p.12 / Chapter Chapter2 --- Expression of CCHCRl Protein in Psoriasis skin --- p.13 / Chapter 2.1 --- Background of Skin and Psoriasis --- p.13 / Chapter 2.2 --- Materials and Methods --- p.18 / Chapter 2.3 --- Results --- p.21 / Chapter 2.4 --- Discussion and Conclusion --- p.25 / Chapter Chapter3 --- Expression of CCHCRl Protein in Skin Organotypic Culture --- p.37 / Chapter 3.1 --- Background about Skin Organotypic Culture --- p.37 / Chapter 3.2 --- Materials and Methods --- p.41 / Chapter 3.3 --- Results --- p.45 / Chapter 3.4 --- Discussion and Conclusion --- p.49 / Chapter Chapter4 --- Expression of CCHCRl Protein in HaCaT Cells Treated with Camptothecin --- p.60 / Chapter 4.1 --- Background --- p.60 / Chapter 4.2 --- Materials and Methods --- p.61 / Chapter 4.3 --- Results --- p.68 / Chapter 4.4 --- Discussion and Conclusion --- p.73 / Chapter Chapter 5 --- General Discussion --- p.95 / References --- p.107 / Publications --- p.172