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Helicase-like Transcription Factor (HLTF) is a DNA helicase protein which is homologous to SNF/SWI family. It has been demonstrated to be a functional homolog of yeast Rad5, required for the maintenance of genomic stability. Although the physiologic role of HLTF is largely unknown,inactivation of HLTF by promoter hypermethylation has been found in more than 40% human colon cancers. In this study, we have applied mouse transgenic approaches to determine whether loss of HLTF function could be important for colorectal carcinogenesis. HLTF knockout mice were generated by the deletion of first 5 exons of the HLTF gene. The complete loss of HLTF expression in HLTF -/- mice was confirmed by northern blot and real time RT-PCR assays. HLTF -/- mice did not show any developmental defects within a 2-year observation indicating that HLTF is dispensable for mouse development. Furthermore, HLTF -/- mice were free of intestinal or colorectal tumors or other types of tumors, suggesting that loss of HLTF function alone is not sufficient to drive oncogenic transformation in intestinal track and other tissues. To determine whether loss of HLTF function could cooperate with other tumor suppressors in the formation of colorectal cancers, we have bred HLTF knockout mice with the mutant mice for APC (adenomtous polyposis coli) and P53. In HLTF -/-APC Min/+ mice, a significantly increased formation of intestinal adenocarcinoma and colorectal cancers were observed. Although very few HLTF -/-P53 -/- mice developed colorectal cancers, these mice had increased incidence of the formation of metastatic lymphomas. Cytogenetic analysis of colorectal cancer cells derived from HLTF -/-APC Min/+ mice demonstrated a high incidence of gross chromosomal instabilities, including Robertsonian fusions, fragments and aneuploidy. All these genetic alterations were not observed in the intestinal tumor cells from APC Min/+, implicating that loss of HLTF function could induce genomic instability which contributes to intestinal carcinogenesis. To further investigate the role of HLTF in colorectal carcinogenesis, we have also applied a shRNA knockdown approach to down-regulate HLTF expression in human HCT-116 colon cancer cells. HCT-116 cells highly express HLTF and show less chromosomal instability, making these cells as a very useful model to investigate the loss of function of HLTF in human colorectal carcinogenesis. Using Western blot approach, we confirmed that HLTF knockdown HCT-116 cells had less than 5% of HLTF expression as compared to the scramble controls. By inoculating HLTF knockdown HCT-116 cells to Rag1 -/-IL2 -/- immunocompromised mice, we further demonstrated that HLTF knockdown promote tumor growth and invasion. Moreover, spectral karyotyping analysis revealed that HLTF knockdown human colon cancer cells had significantly increased chromosomal instability, including both aneuploidy and chromosomal translocation. Taken together, our work strongly indicates that loss of HLTF function can promote the malignant transformation of intestinal or colonic adenomas to carcinomas by inducing genomic instability. Given the high frequency of epigenetic inactivation by hypermethylation of HLTF in human colon cancers, our studies strongly suggest that this epigenetic alteration could be directly involved in the development of colorectal cancer rather than a consequence of this carcinogenesis.

Quantitative and qualitative analyses of the serum antibody responses of NIH, C57BL/10, BALB/c, DBA/2 and CFLP mice infected with Trichuris muris have been made using ELISA and immunoprecipitation techniques. No correlation was found between specific serum antibody titres measured using T. muris E/S products and the time of onset of expulsion in the different mouse strains examined. However, there were some differences in the antigen recognition profiles of some sera as determined by immunoprecipitation analyses. In all the strains of mice examined significant increases in detectable specific serum antibody to the parasite E/S products occurred around day 15 to 20 postinfection and continued to rise, as measured up to at least day 40 and even up to day 65. Cortisone acetate treatment during larval development, in infected CFLP mice, in order to establish heavy adult worm burdens, did not reduce specific antibody titres to T. muris E/S products. In responding and tolerant DBA/2 mice there was no marked difference in either the kinetics of specific serum antibody production during primary and secondary infections, or in the antigen specificities of secondary infection sera. The "defect" in mechanism in the tolerant DBA/2 mice, which allows primary infections of T. muris to develop to patency, was shown to be permanent as secondary infections with the parasite could also establish in these animals. An investigation was made of the phenomenon of tolerance in the DBA/2 model- system and in the cortisone treated CBA mice. The capacity of MLNC from different groups of animals to produce IL-2 in vitro upon mitogen stimulation was investigated, on the basis that IL-2 deficit during antigen presentation may result in immune tolerance. Although no differences were found in the responding and tolerant DBA/2 cell-Vpopulations, there was an apparently synergistic interaction between cortisone administration and T. muris infection which dramatically reduced the IL-2 producing capacity of the MLNC. However, IL-2 cannot yet be ruled out as a factor in the inherent tolerance of a proportion of the DBA/2 population as IL-2 receptor expression by the 2 groups of cells assayed was not examined. Basic analyses of the antigens of T. muris were performed. The major protein of adult male homogenate (AMA) was also the major protein of the excretory/secretory (E/S) products and the surface antigen preparations. In addition several common E/S and surface antigens were shown to have proteolytic enzyme activities against gelatin and/or casein. The relationship between T. muris and Trichinella spiralis was examined in greater detail, and the m. wts. of the cross-reacting antigens were determined. Evidence suggested that the stichosomes of these worms may be the source of these antigens. Both Trichuris muris adults and Trichinella spiralis infective larvae each had common major E/S and surface antigens, indeed, both were shown to have surface proteases. These studies were extended to examine the possibility of cross-reactivity between Trichuris muris and T. trichiura; mouse infection sera and human infection sera respectively were able to cross-react with heterologous antigen preparations. The demonstration that anti-Trichinella spiralis 48 kD and 50/55 kD stichocyte antigen MoAbs also reacted with Trichuris trichiura adult homogenate in ELISA supports the suggestion that common stichocyte antigens may exist amongst the trichuroid nematodes Trichuris muris, Trichuris trichiura and Trichinella spiralis. Monoclonal antibodies were produced against the E/S products of Trichuris muris, which were characterized in terms of isotype and antigen specificities. Initial experiments indicated that one of the IgA MoAbs recognizing 34,22,20 and 18 kD E/S proteins may be effective in the passive transfer of immunity.

Long ncRNAs (non-protein coding transcripts generally considered longer than 200 nucleotides to be distinguished from classes of small RNAs) are abundantly transcribed from the mammalian genome. Despite their abundance, little is known about these transcripts. Although several individual long ncRNAs have been well-characterised and ascribed important cellular functions, there remains considerable controversy as to whether long ncRNAs are, in the main, functional. Indeed, their abundance has prompted many people to argue that long ncRNAs are simply transcriptional ‘noise’ generated by spurious transcription initiation events resulting from low RNA polymerase II fidelity. This thesis demonstrates that large numbers of long ncRNAs are specifically expressed along both temporal and spatial axes of mouse development in a manner consistent with a biological function. Custom-designed microarrays were employed to analyse the expression profiles of large numbers of long ncRNAs, along with protein-coding genes, in two models of cellular differentiation; the differentiation of mouse embryonic stem (ES) cells from pluripotency to differentiation along a hemopoietic lineage; and the commitment and differentiation of neural stem cells to oligodendrocytes. The core networks that include gene expression, transcription factor binding sites and chromatin domains that regulate ES cell pluripotency and lineage specification have been the subject of considerable attention and provide a detailed context in which to analyse ncRNA expression. Of those ncRNAs examined, 945 (26% of total) ncRNAs were expressed during the differentiation of ES to embryoid body (EB), of which 174 were significantly differentially expressed. Many of these ncRNAs were transcribed from genomic locations that overlapped modified chromatin domains, and in two further studied cases directly engaged with epigenetic machinery. Similarly, 332 long ncRNAs (9% of those examined) were expressed during processes of neuronal-glial fate switching, neurogenesis and oligodendrocyte progressive differentiation and termination, of which around half were also significantly differentially expressed. Furthermore, many of these ncRNAs exhibited expression profiles that coincided with pivotal events during the commitment and differentiation of neural stem cells (NSC) to mature myelinating oligodendrocytes. Consideration of the genomic context revealed many long ncRNAs were expressed from diverse places including intergenic, intronic, and imprinted loci and may overlap with, or are transcribed antisense to, protein-coding genes with previously described roles in either ES or NSC pluripotency and differentiation. This association also extended to expression profiles, where a comparative analysis often showed complex relationships of expression between ncRNAs and associated protein coding genes, suggesting a potential role for ncRNAs in regulating the expression of associated gene loci. The complexity and specificity of the long ncRNAs expression was illustrated by analysis of the in situ hybridisation (ISH) data conducted in collaboration with the Allen Brain Atlas. Of 1328 long ncRNAs, 849 (64%) were expressed in the mouse brain, 623 (47%) of which exhibited specific expression profiles associated with distinct neuroanatomical regions, cell types, or subcellular compartments. Again, examination of their genomic context revealed long ncRNAs were often associated with protein-coding genes of neurological importance and this association often extended to include linked expression profiles in the mouse brain. The comparative analysis of protein-coding gene expression relative to associated noncoding transcription also revealed an additional level of complexity in gene structure and genomic architecture. Analysis of both microarray and ISH data show 3’UTRs can exhibit discordant expression profiles relative to their associated protein coding genes, often in a tissue- and developmentally-specific manner. Indeed, a genome-wide analysis showed that the independent expression of 3’UTR transcripts is prevalent throughout the mouse genome where they may function intrinsically as long ncRNAs during development. Together, these genome-wide analyses indicate a large proportion of long ncRNAs exhibit specific expression profiles that are inconsistent with the notion they are meaningless transcriptional noise. Taken together with numerous studies published in recent years, this thesis provides evidence to support the emergence of long ncRNAs as a major functional component of the regulatory network that underpins differentiation and development in mammals and other complex organisms.

Title page, table of contents and abstract only. The complete thesis in print form is available from the University of Adelaide Library. / The enzyme 25-hydroxyvitamin D la-hydroxylase or CYP27Bl is the key enzyme in the two-step activation process by which vitamin D is converted to its biologically active form 1,25-dihydroxyvitamin D (1,25D). The actions of a number of regulators on the renal CYP27B1 enzyme activity have been recognized for some years, although the underlying molecular mechanisms remain largely unknown and the DNA regions involved in the in vivo regulation of gene expression by these factors have not been delineated as yet. In order to identify the regulatory regions through which these factors control CYP27B1 expression in the kidney in vivo and to study the spatial and temporal expression of the CYP27B1 gene during development, a transgenic mouse model was established. This model was developed using pro-nuclear injection of a DNA construct containing the firefly luciferase reporter gene under the control of the 1541 bp region of the human CYP27B1 promoter. Following pro-nuclear injection, three transgenic founders were obtained and bred to establish three independent transgenic lines. In all three lines, a very similar expression pattern of the luciferase reporter gene was detected. High levels of luciferase activity were detected in the kidney, brain, testis, skin and bone. Lower levels of luciferase activity were detected in heart, lung, liver, distal small intestine, skeletal muscle and spleen extracts. No reporter gene expression could be detected in the proximal small intestine. This animal model was used to identify the ability of the 1541 bp promoter region of the CYP27B1 gene to respond in the kidney to a number of physiological challenges including dietary calcium, vitamin D and the immunomodulator LPS. In addition, the temporal expression of the reporter gene was studied by sacrificing animals at 6 different time points (2, 4, 6, 8, 12 and 64 weeks of age). The functionality of the CYP27B1 promoter was verified by comparing the regulation of the expression of the reporter gene with that of the endogenous CYP27B1 gene. The expression of endogenous CYP27B1 mRNA levels was therefore determined using Real-Time RT-PCR. The expression of the reporter gene and the endogenous CYP27B1 mRNA levels in the kidney were increased during early development (2 week old animals) and fell with increasing age. Reporter gene expression and CYP27BI mRNA levels were down-regulated in response to increasing amounts of dietary calcium in a dosedependent manner. Vitamin D-deficiency resulted in an increase in both the reporter gene and CYP27B1 expression. However, the increase in CYP27B1 mRNA levels was substantially higher than the increase in reporter gene expression, suggesting that other regulatory elements are required to maximize the effect of vitamin D-deficiency. LPS administration did not affect the expression of either luciferase or the endogenous CYP27B1 gene in the kidney. Immunohistochemistry was used to identify the cell-specific location of the luciferase and the endogenous CYP27B1 protein in the kidney in kidney sections of vitamin D-deficient animals. Both luciferase protein and the endogenous CYP27B1 protein were identified in the proximal tubular cells of the kidney. The regulation of the expression of the reporter gene was also studied in the transgenic mouse model in a number of extra-renal tissues that have been shown to express CYP27Bl and to be responsive to 1,25D. These tissues include heart, liver, lung, femora, bone marrow, skeletal muscle, testis, skin, brain, spleen and proximal and distal small intestine. Although in most tissues, the expression of luciferase was highest in the 2 week old animals and fell with increasing age, in the testis, the expression levels were low in the developing animals and increased with increasing age. No physiological significant effects were detected in any of the extra-renal tissues examined in response to dietary calcium and vitamin D, suggesting that these factors control CYP27Bl expression in a kidney-specific manner. In addition, no physiologically significant effect of the LPS administration could be detected in these tissues. Future studies employing transgenic animals which express transgenic constructs containing both the CYP27Bl promoter and upstream and/or intronic sequences are required to identify the factors that regulate CYP27Bl expression in the different tissues and to delineate the DNA regulatory regions through which these factors exert their effects in vivo. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1140412 / Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 2004

Thesis (M.S.)--School of Dentistry. University of Missouri--Kansas City, 2008. / "A thesis in oral biology." Advisor: Mark L. Johnson. Typescript. Vita. Title from "catalog record" of the print edition Description based on contents viewed Aug. 07, 2008. Includes bibliographical references (leaves 57-65). Online version of the print edition.

Thesis (Ph. D.)--University of Texas at Austin, 1998. / Vita. Includes bibliographical references (leaves 193-229). Available also in a digital version from Dissertation Abstracts.

Thesis--Wisconsin. / Vita. Includes bibliographical references.

Thesis (Ph. D.)--University of Wisconsin--Madison, 1982. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 193-228).

Thesis (Ph.D.)--Boston University / The role of specific streptococcal products in the production of infection by these organisms has been the subject of many studies by previous investigators. To a great extent these workers have attempted to correlate the formation of these substances by certain streptococcal strains with their virulence and disease manifestations in man as well as with the lethal effects produced by these organisms in mice. The purpose of this study was to investigate further the role of individual streptococcal products in the pathogenicity of group A streptococci, mainly by observing the effects of these products on local and systemic infections in the hamster and the mouse, respectively. Specifically, the M protein, streptokinase, streptodornase, streptolysin 0, the erythrogenic toxin, the hyaluronic acid capsule, and hyaluronidase were studied. [TRUNCATED]