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.

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.

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

No description available.

Friedreich ataxia (FRDA) is an autosomal recessive, neurodegenerative disorder with severely debilitating effects and no current cure. FRDA is mainly caused by the hyper-expansion of a GAA repeat present in intron 1 of the FXN gene, which results in decreased gene expression and consequently a deficiency of the mitochondrial protein frataxin. In the first instance, frataxin deficiency renders an impaired protection from oxidative stress. Antioxidant therapy with cannabinoids (CBD and THC) and CTMIO was investigated in GAA repeat FXN YAC transgenic mouse models of FRDA, but no significant improvements were detected on functional measurements such as rotarod performance and locomotor activity. Additionally such compounds failed to protect the brain of treated mice from oxidative insults. Therefore, the use of such antioxidant compounds cannot be advocated for FRDA therapy. Recent findings indicate that FXN silencing in FRDA may be mediated by repressive heterochromatin, suggesting the use of histone deacetylase inhibitors (HDACi) as FXN up-regulators. Therefore, therapy with a benzamide-type HDACi (106) was similarly investigated on the FXN YAC GAA mouse model. No significant improvements were detected by functional and histochemical analysis. However, significant changes were produced in global acetylation levels of H3 and H4 in the brain of treated mice, suggesting that the drug is capable of crossing the blood-brain barrier and producing an effect. Additionally, significant increases in frataxin expression were detected in the brain of treated mice. To identify further FRDA disease mechanisms, characterization of the FXN gene for the presence of the CCCTC-binding factor (CTCF) was also performed on FRDA patient cerebellum samples. Overall, lower levels of CTCF were detected in FRDA-associated FXN alleles, suggesting the potential involvement of CTCF in the regulation of FXN transcription.

Clinical cases of bovine mastitis are most severe in the early stages of lactation. The causes of this increased propensity for severe mastitis during early lactation, compared to mid and late lactation are unclear. In order to better understand the early lactation immune response to mastitis, a murine model of mastitis was employed. Intramammary inoculation of a mastitis causing Escherichia coli strain was performed in lactating mice at various stages of lactation to model the immune response seen in cows during lactation. In our experiments, mice in the early stages of lactation exhibited altered mRNA expression of cytokines IL-1β, IL-6, IL-10, and TNFα over the course of infection when compared to mice at mid lactation. Additionally, increased bacterial growth was observed in the mammary gland of mice infected during early lactation compared to late lactation. These results are consistent with the immune response observed in cows at early lactation. These results suggest that the mouse may provide a useful model to study differences in the immune response seen during different times in lactation.

The New Zealand Black (NZB) mouse chromosome 13 (c13) is linked to development of autoimmunity. B6 mice containing a portion of NZBc13 (B6.NZBc13 (c13)) develop a lupus phenotype that includes: autoantibody production, increased B and T cell activation, and marginal zone B cell and myeloid dendritic cell expansions. c13 mice have a B cell intrinsic dsRNA-sensing defect, leading to increased TLR3 expression and survival. The role of the aberrant dsRNA sensing in the generation of the c13 autoimmune phenotype was assessed by generating c13 mice with TLR3 knocked out (c13.TLR3KO). Marginal zone B cell expansion and B cell activation were attenuated in c13.TLR3KO mice, but other cellular phenotypes were not affected. Autoantibody production was partially reduced. These results indicate that altered dsRNA-sensing contribute to a portion of the altered cellular phenotypes in c13 mice, but that other susceptibility loci in the c13 interval are required for full development of autoimmunity.

The New Zealand Black (NZB) mouse chromosome 13 (c13) is linked to development of autoimmunity. B6 mice containing a portion of NZBc13 (B6.NZBc13 (c13)) develop a lupus phenotype that includes: autoantibody production, increased B and T cell activation, and marginal zone B cell and myeloid dendritic cell expansions. c13 mice have a B cell intrinsic dsRNA-sensing defect, leading to increased TLR3 expression and survival. The role of the aberrant dsRNA sensing in the generation of the c13 autoimmune phenotype was assessed by generating c13 mice with TLR3 knocked out (c13.TLR3KO). Marginal zone B cell expansion and B cell activation were attenuated in c13.TLR3KO mice, but other cellular phenotypes were not affected. Autoantibody production was partially reduced. These results indicate that altered dsRNA-sensing contribute to a portion of the altered cellular phenotypes in c13 mice, but that other susceptibility loci in the c13 interval are required for full development of autoimmunity.

Thesis(M Tech(Biomedical Technology))--Cape Peninsula University of Technology, 2009 / This thesis provides the first scientific evidence of the photoprotective properties of rooibos and honeybush herbal tea extracts and to some extent, two major honeybush polyphenols, hesperidin and mangiferin. These properties were demonstrated using in vivo models by: Providing evidence for the inhibition of tumour promotion by ultraviolet B (UVB) radiation in a two-stage skin carcinogenesis mouse model. Topical application of polyphenol-rich extracts of rooibos and honeybush prior to UVB tumour promotion of 7,12-dimethylbenz[a]anthracene (DMBA)-initiated mouse skin, inhibited the formation of tumours. The rooibos and honeybush extracts decreased the incidence and volume of the tumours. Topical application of hesperidin and mangiferin were less effective than the honeybush extracts as only the tumour volume was decreased, but not the incidence. Providing evidence for the inhibition of photodamage of the skin by UVB exposure in a mouse model. Topical application of polyphenolic rich extracts of honeybush prior to UVB irradiation of mouse skin reduced erythema, peeling, oedema and hyperplasia. The depletion of antioxidant enzymes catalase and superoxide dismutase (SOD) was prevented. The extracts protected the skin from oxidative and direct DNA damage, and reduced lipid peroxidation. The induction of cyclooxygenase-2 (COX-2) and ornithine decarboxylase (ODC) was also reduced. Topical application of the polyphenols hesperidin and mangiferin showed reduced protective effects compared to the extracts. Suggesting the possible mechanisms by which honeybush and the polyphenols protect against photocarcinogenesis such as reducing tumour promotion, inflammation and oxidative stress. Suggesting the benefits of including honeybush and rooibos as cosmeceuticals in skin care products and sunscreens as part of the strategy for preventing skin cancer. Discussing the recommendations for further study such as investigating more specific chemopreventive activities of these two South African herbal teas and their polyphenols, dose response studies and clinical evaluations.

archives@tulane.edu / BRCA1 faithfully repairs damaged DNA by promoting homology-directed repair (HDR). Loss of Brca1 and other HDR genes are incompatible with embryonic viability and cause severe genomic instability. Cells lacking BRCA1 are sensitive to cellular stresses such as DNA damage caused by ionizing radiation (IR). Homozygous loss of Brca1 is embryonic lethal in mice, and the few tissue-specific knockouts generated develop abnormally. Therefore, we created an inducible Cre mouse model to study Brca1 loss in all adult mouse tissues allowing for examination of viability, longevity, and stress response in the absence of HDR and the importance of HDR in different tissues of an adult mouse. After validating the inducible Cre system using a reporter allele in mice, we generated mice with alleles of the inducible Cre system and floxed Brca1 alleles. Cre was induced in adult mice at ten weeks of age, resulting in extensive, widespread deletion of Brca1. Contrary to the embryonic lethality observed in all previously tested germline Brca1 knockout mouse models, adult mice with Brca1 deletion displayed no overt phenotypes. Brca1Δ/Δ mice showed extensive, widespread deletion of Brca1 and survived up to 1 year after Brca1 recombination. Targeted, high-depth sequencing of recombined tissues indicated mutations accumulated in both the mammary gland and the intestine. However, only the mammary gland had an HDR deficiency signature. Next, we examined Brca1Δ/Δ mice survival after exposure to ionizing radiation and mitomycin C (MMC). Surprisingly, Brca1Δ/Δ mice responses are DNA damage specific. Brca1Δ/Δ mice deficient for HDR showed no increased sensitivity to IR but died four to eight days following MMC exposure. Our results show that BRCA1 is not required for long-term viability or DNA double-strand break repair, but BRCA1 is essential for DNA crosslink repair to maintain viability in an adult mouse. / 1 / JoyOlayiwola