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No description available.

A non-replicating recombinant adenovirus, Ad5MCMVlacZ, which expresses the 13-galactosidase (l3-gal) reporter gene, was used to examine both constitutive and inducible repair of UVC-damaged DNA in Chinese hamster ovary (CHO) cells. Host cell reactivation (HCR) of 13-gal activity for UVC-irradiated Ad5MCMVlacZ was examined in non-irradiated and UVC-irradiated nucleotide excision repair (NER) proficient parental CHO-AA8 and m mutant CHO-UV61 cells which are deficient in the transcription-coupled repair (TCR) pathway of NER. Cells were infected with either UVC-irradiated or non-irradiated Ad5MCMVlacZ and scored for 13-gal activity 24 h later. HCR of 13-gal activity for UVC-irradiated Ad5MCMVlacZ was significantly reduced in non-irradiated CHO-.UV61 cells compared to that in non-irradiated CHO-AA8 cells suggesting that repair in the transcribed strand of the UVC-damaged reporter gene in untreated CHO-AA8 cells utilizes TCR. Prior UVC-irradiation of cells with low UV fluences resulted in a transient enhancement of HCR for expression of the UVC-damaged reporter gene in CHO-AA8 cells but not m TCR deficient CHO-UV61 cells. Pre-UVC-treatment of cells resulted also in an enhanced expression of 13 -gal for unirradiated Ad5MCMVlacZ in both CHO-AA8 and CHO-UV61 cells. However, compared to CHO-AA8 cells, the CHO-UV61 cells exhibited comparable levels of enhanced 13-gal activity following significantly lower UVC exposures to cells suggesting that persistent damage in active genes plays a direct role in enhancing 13-gal activity driven by the MCMV promoter in CHO cells. These results suggest that prior UVC treatment results in a transient enhancement in repair of UVC-damage DNA in the transcribed strand of the active reporter gene in CHO-AA8 cells through an enhancement of TCR or a mechanism that involves the TCR pathway and that the upregulation of reporter gene expression alone is not sufficient for enhanced repair of the reporter gene in CHO-UV61 cells. The HCR assay was used also to examine both constitutive and inducible repair of UVC-damaged DNA in mouse embryonic fibroblast (MEF) cells. HCR of B-gal activity for UVC-irradiated Ad5MCMVlacZ was examined in non-irradiated and UVC-irradiated NER proficient parental wild type MEF cells and in MEF cells with specific knockouts in the p53 (p53-/-), pRb (pRb-/-), and p107 (p107-/-) genes. Cells were infected with either UVC-irradiated or non-irradiated Ad5MCMVlacZ and scored for ~-gal activity 24 h later. HCR of ~-gal activity for UVC-irradiated Ad5MCMVlacZ did not show a significant difference in non-irradiated cells for any of the MEF knockouts cells compared to the parental strain suggesting that p53, pRb and p107 does not play a role in repair of the UV -damaged reporter gene in untreated MEF cells. Prior UVC-irradiation of cells with low UVC fluences resulted in an enhancement of HCR for expression of the UV C-damaged reporter gene in MEF wild type cells, low passage pRb-/-and p 1 07 -I-MEF cells but not in p53-/-MEF cells or in high passage pRb-/-and p107-/-MEF cells. These results suggest that prior UVC treatment MEF cells results in an induced repair of UVC-damaged DNA that is dependent on p53. The presence of an enhancement of HCR for the UVC-damaged reporter gene in pre-UVC treated cells in low passage, but not in high passage, pRb-/-and p 1 07-I-cells suggests that the lack of pRb or pI 07 expression per-se does not result in a deficiency in inducible DNA repair. However, these results suggest that the lack of pRb or p 1 07 expression results in alterations in MEF cells at high passage number that abrogate inducible repair of UVC-damaged DNA. UVA produces predominantly single base damage that is repaired through base excision repair (BER), whereas UVC and UVB produce predominantly cyclobutane pyrimidine dimers (CPDs) and 6-4 photoproducts (6-4PP) that are repaired through NER. The colony survival following exposure to various UV sources was examined in cells proficient and deficient in (NER). The UV sources were a UVC source from a germicidal lamp emitting predominantly at 254 nm. and a UVA source from a lKW Hg-Xe arc lamp using either a Band pass filter (BPF) or a 335 Cut-off-filter (335COF). NER deficient CHO-UV5 and CHO-UV61 cells were more sensitive to UVC exposure compared to NER proficient CHO-AA8 cells, consistent with the production of UVC-induced DNA damage predominantly in the form of CPDs and 6-4PPs which are repaired through the NER pathway. NER deficient xeroderma pigmentosum cells from complementation group D (XPD) were more sensitive compared to NER proficient normal human cells following exposure to the UVA-BPF source. In addition XPDdenV cells, which express the denY gene from bacteriophage T4, were more resistant than XPD cells following exposure to the UVA-BPF source. Since the denY protein is specific for excision ofCPDs these results indicate a substantial proportion of the induced DNA damage resulting from the UV A-BPF is in the form of CPDs, presumably due to a significant UVB component in the beam. In contrast, the NER deficient CHO-UV5 and CHO-UV61 cells showed a similar sensitivity compared to the NER proficient CHO-AA8 cell line following UVA-335COF exposures up to 60 KJ/m2• However, for UVA-335COF exposures greater than 60 KJ/m2 the NER deficient cells were more sensitive compared to the NER proficient CHO-AA8 cells, although the difference in sensitivity between NER deficient and NER proficient cells was less than that detected following UV A-BPF exposure. These results suggest that the UVA-335COF exposure produces predominantly DNA damage of the single base type for exposures less 60 KJ/m2. This is consistent with the calculated spectral distribution, which showed a 5.62% UVB component for the UVA-BPF, but only 0.14% UVB component for the UVA-335COF. / Thesis / Master of Science (MS)

Photodynamic therapy (PDT) for cancer utilizes the localised delivery of light to activate a photosensitizing drug (such as Photofrin) which is selectively retained by tumour tissues. Although this treatment modality is undergoing clinical trials, the mechanism of PDT cytotoxicity is not fully understood. One approach to understanding the mechanism of action of PDT is to study cell mutants showing alterations in their response to PDT. The capacity of mammalian cells to support virus replication has been used as an assay to compare cellular response to ultraviolet (UV) light and other cytotoxic agents. Cellular capacity has been previously employed to measure cellular recovery following UV irradiation and to predict the sensitivity of cells to several anticancer drugs. In this work, I have examined the use of the capacity assay to examine the sensitivity of cells to PDT treatment. RIF-1 mouse fibrosarcoma cells and a PDT resistant derivative, RIF-8A; as well as Chinese hamster ovary (CHO) cells and CHOMDR (multi-drug resistant) mutant cells were studied. Consistent with the clonogenic survival of these cells after PDT, the viral capacities of RIF-8A and CHO-MDR cells· were greater than those of RIF-1 and CHO cells respectively following PDT treatment. The capacity assay was also used to examine the relative ability of RIF cells to recover from PDT damage. These capacity experiments show that recovery from PDT damage is greater in RIF-SA cells compared to RIF-1 cells, suggesting that RIF-SA cells have an enhanced repair capacity for PDT damage compared to RIF-1 cells. The response of CHO cells and the RIF cells to ultraviolet (UV) light irradiation was also examined. No difference in the UV sensitivity of CHO-N and CHO-MDR cells was found. However, the RIF-SA cell line showed a cross-resistance to UV. The survival of viral DNA synthesis for UV-irradiated virus was also greater in RIF-SA cells compared to RIF-1 cells, suggesting that RIF-SA cells have an increased capacity for the repair of UV-induced DNA damage. It is possible that the increased resistance to PDT of RIF-SA may also result from an increased repair of POT-induced DNA damage in RIF-SA cells. The identification and characterization of a POT-sensitive cell mutant was also investigated. The CHO-AUXB1 cell mutant was found to show an increased sensitivity to PDT and UV compared to the CHO-PRO- parent line. This sensitivity of the CHO-AUXB1 mutant may also result from a deficiency in the repair mechanism of PDT-induced and UV-induced damage. / Thesis / Master of Science (MS)

The purpose of this research is, first, to determine by laboratory methods, that species specificity does not exist in closely allied taxonomic animals; second, to determine a unit of activity for the gonadotropic hormone. For this purpose a quantitative method for determining potency is necessary to ascertain the seasonal production of the gonadotropic factor in fish. A further aim in this investigation is to demonstrate that the diversity of the gonadotropic factor, in relation to phylogenetic variations, is not as ineffectual as previously reported.

<p dir="ltr">Diabetes has become an international health crisis with type 2 diabetes composing the majority of cases. Along with a variety of other systemic effects, type 2 diabetes increases fracture risk. This aspect of type 2 diabetes has become a topic of interest in preclinical research and has been investigated using rodent models of type 2 diabetes. Of these models, the Yellow Kuo Kondo (KK-A^y) mouse model has shown promise as an obese model of type 2 diabetes. In the KK-A^y model, mice heterozygous for a mutation in the agouti gene (A^y) are treated as an obese model of type 2 diabetes. Those that are homozygous (no mutation) are treated as non-diabetic, obese controls. While this model has been indicated to be non-diet dependent, recent data has revealed the efficacy of this model may be reliant on diet. Following approval from the Indiana University-Purdue University at Indianapolis School of Science Institutional Animal Care and Use Committee, mice of each sex and genotype were placed on separate diets. Half on a standard chow diet and the other half on a diet recommended by Jackson Laboratory for this strain. Animals were aged to 16 weeks of age with blood glucose and body weight monitored every other week. Animals were then sacrificed to collect whole blood, blood serum, the pancreas, bilateral tibiae, and bilateral femora. End-point metabolic impacts were assessed through hemoglobin A1c and serum insulin measures while skeletal measures were quantified using microcomputed tomography scanning and analysis. Through this research, it was determined diet did have a significant impact on the skeletal and metabolic phenotype associated with type 2 diabetes in the KK-A^ymodel. </p>

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MENVSC / Department of Ecology and Resource Management / Rodents can quickly respond to land use changes whether the change positively or negatively influences their life. In the case of positive influence, rodents exploit the additional food resources and increase their numbers to potential pest level, especially in the absence of predators. Such a population increase can potentially be harmful to humans due to the diseases that rodents carry and the costs due to damage to crops, stored foods and personal possessions that they may cause to small holder farmers. Small holder farmers live in a mixed landscape that is constantly changing. Such changes are changes in land use and they do not only affect rodent population dynamics and species composition, but also their ecosystem services and integrity. Understanding how rodents respond to these land use changes (crop, grazing and settlement) will not only improve the implementation of Ecologically Based Rodent Management (EBRM), but might also enable the monitoring of ecosystem integrity. Rodent trapping was conducted in two different study sites which experience different rainfalls during wet and dry season. A 70 m x 70 m grid was set in three different land uses (crops, grazing and settlement) per study site. A mark-recapture technique was applied and all captures were processed on a temporal station on site. In all grids at a distance of 30 m a line of 20 snap traps were set. With the tapping effort of 1470 trap nights per season for both seasons in this study we captured 839 rodents and 2 shrews, which represented 469 individual rodents and 1 individual shrew. At Vyeboom, cropping land use had the highest number capture (210) and the highest in species richness (9) rodent species and 1 shrew. The settlement land use was second at 144 captures for 8 rodent species and lastly the grazing land use at 80 captures with 7 rodent species. On the other hand, at Ka-Ndengeza also cropping land use had the highest capture (186) with highest richness at 7 rodent species. When it comes to settlement and grazing, settlement was second (129) to cropping in terms of the number of capture but last in terms of richness (5) whilst grazing was last in terms of number of captures 92 and second in terms of richness (6). Despite the high diversity of rodents, only Mastomys natalensis, Gerbilliscus leucogaster, Steatomys pratensis and Rattus rattus were captured in meaningful sample sizes to allow for robust density estimation. Similarly there were strong seasonal effects on rodent captures, with almost no captures during the wet season.

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