Ataxia telangiectasia-mutated (ATM) is a serine/threonine protein kinase that has critical functions in the cellular responses to DNA damage, including cell cycle checkpoint activation and DNA repair. Since ataxia telangiectasia individuals, who have homozygous mutations in ATM, are exquisitely radiosensitive there is considerable interest in inhibiting the kinase activity of ATM to increase the efficacy of targeted radiotherapy. In this dissertation work, I sought to understand the cellular responses to radiation when ATM kinase activity is transiently inhibited using the small molecule ATM kinase inhibitor KU55933. During my PhD, our laboratory has shown that transient ATM kinase inhibition one hour post-irradiation results in radiosensitization, increased chromosome aberrations and abrogation of sister chromatid exchange. I contributed to these findings by showing that the cellular radiosensitization seen in H460 cells with kinase-inhibited ATM was identical to that seen when ATM protein was disrupted using siRNA prior to the insult. In addition, I demonstrated that 15 minutes of ATM kinase activity post-irradiation is sufficient to trigger the G2/M cell cycle checkpoint, and that subsequent transient inhibition of ATM with KU55933 does not affect recovery from this checkpoint. To gain a more global view of the functional consequences of kinase-inhibited ATM following irradiation, I utilized a SILAC-based tandem mass spectrometry approach, combined with a subcellular fractionation protocol, to determine ATM kinase-dependent spatial proteome dynamics in response to radiation-induced DNA damage. Analysis of the chromatin-associated proteome revealed that the retention of 53BP1 at chromatin is decreased when the kinase activity of ATM is inhibited following ionizing radiation (IR). Using fluorescence recovery after photobleaching in live cells, I determined that the stability of IR-induced GFP-53BP1 foci is decreased when the kinase activity of ATM is inhibited following IR. These results provide a roadmap for understanding ATM kinase-dependent spatial protein dynamics in response to DNA damage.

Thesis (M.S.)--Villanova University, 2008. / Biology Dept. Includes bibliographical references.

Ph.D.

Thesis (Ph. D.)--University of Chicago, Committee on Neurobiology, June 1999. / Includes bibliographical references. Also available on the Internet.

Made available in DSpace on 2014-06-11T19:32:08Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-03-17Bitstream added on 2014-06-13T20:23:03Z : No. of bitstreams: 1 satrapa_ra_dr_botib.pdf: 444664 bytes, checksum: ea4f185d0abddba64ab9a9ce073f5407 (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / A fim de melhor compreender as diferenças entre zebuínos e taurinos em relação à resistência ao estresse térmico (ET), objetivou-se com o presente trabalho: (1) verificar se a adição de IGF-I ao meio de cultivo, na ausência de Soro Fetal Bovino (SFB), seria capaz de manter as taxas de produção embrionária semelhantes àquelas obtidas em meio de cultivo com SFB; (2) verificar se a adição de IGF-I ao meio de cultivo é mais eficiente em diminuir os efeitos deletérios do ET em embriões da raça Holandesa preto e branco (HPB) quando comparados aos da raça Nelore; (3) verificar se o efeito deletério do ET no desenvolvimento embrionário e na taxa de apoptose é mais acentuado em embriões da raça HPB, quando comparado aos da raça Nelore. No experimento 1, oócitos de vacas aneloradas oriundas de matadouro foram maturados, fertilizados com sêmen de touros da raça Nelore e, 10 horas pós inseminação (hpi), os embriões foram distribuídos ao acaso em quatro grupos, de acordo com a composição do meio de cultivo: SFB (5% de SFB + 0 ng/mL de IGF-I; n=165); IGF (0% de SFB + 51 100 ng/mL de IGF-I; n=163); SFB+IGF (5% de SFB + 100 ng/mL de IGF; n=169) e controle (0% de SFB + 0 ng/mL de IGF-I; n=168). Foram avaliadas as taxas de clivagem, mórula, blastocisto e blastocisto eclodido. Nos experimentos 2 e 3, oócitos de vacas Nelore e HPB oriundas de matadouro foram maturados em meio TCM 199, fertilizados com sêmen de touros das raças Nelore (n=6) e HPB (n=6), respectivamente, e cultivados em meio SOF (synthetic oviduct fluid, na ausência de SFB) até o estágio de blastocisto. Dadas 10 hpi, os embriões foram distribuídos ao acaso em quatro grupos: controle (cultivados na ausência de IGF a 39 oC); ET (cultivados na ausência de IGF e expostos a 41 oC, 96 hpi, por 9 horas, retornando a seguir para 39 oC); IGF (cultivados na presença de IGF a 39 oC) e ET/IGF (IGF e ET). No... / Not available

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Title page, table of contents and abstract only. The complete thesis in print form is available from the University of Adelaide Library. / The aim of the work described in this thesis was initially to screen the ethanol extracts of eleven Indonesian ginger species (Zingiberaceae family) for anticancer activity. MCF-7 breast and HT-29 colon cancer cells were used for the investigations. Extracts of Zingiber aromaticum and Boesenbergia pandurata were found to be the most active species, similar to that of Curcuma langa which has been shown to possess anticancer activity in vitro and in vivo (Aruna and Sivaramakrishnan, 1992; Azuine and Bhide, 1992). These two active species were then further investigated. Bioactive compounds from the species were isolated and identified using various chromatography procedures and nuclear magnetic resonance (NMR) and their anticancer activities were further tested on MCF-7 breast and HT-29 colon cancer cells including cell cycle analysis and measurements of apoptosis. The ethanol extracts of these two active species were also investigated using the AOM-induced colon cancer model in rats. The antiinflammatory activity of the ethanol extract of Z. aromaticum was also investigated using dextran sulfate sodium (DSS) induced ulcerative colitis (UC) in rats. The inhibitory activity of ethanol extracts of rhizomes of 11 ginger species was initially tested against MCF-7 breast and HT-29 colon cancer cells using colorimetric tetrazolium salt (MTT) assay. Ethanol extracts of eight species (Amommum cardamomum, C. longa, C. mangga, C. xanthorrhiza, Boesenbergia pandurata, Zingiber aromaticum, Z. officinale, Z. cassumunar) showed a strong inhibitory effect on the growth of the cancer cells with the IC50 concentrations between 100-100 g/ml. The ethanol extract of Curcuma aeruginosa was less active (IC5o between 100-120 g/ml) and extracts of Kaempferia galangal and K. rotunda had no effect on the growth of either cell lines at concentrations up to 250 g/ml. Ethanol extract of C. longa was used as a comparison since curcumin, an active compound isolated from this species, has had demonstrated its anticancer activity in vitro, in vivo and is currently undergoing clinical trial against colon cancer (Greenwald, et al., 2001; Sharma et al., 2001). Extracts of Z. aromaticum and B. pandurata had very strong inhibitory activity similar to the extract of C. longa. Curcumin was not detectable in either Z. aromaticum or B. pandurata. The ethanol extracts of the active species were not toxic on human skin fibroblast cells (SF 3169). The ethanol extracts of Z. aromaticum and B. pandurata were further fractionated using two different solvents by reversed phase preparative HPLC. Fraction A was eluted with a mobile phase containing 5% vlv aqueous methanol containing 0.025% v/v trifluoroacetic acid (TFA) and fraction B was eluted with 100% methanol. The inhibitory activity of fractions was then investigated against HT-29 colon cancer cells and assayed using the MTT assay. Zerumbone, a sesquiterpenoid compound was isolated from fraction B of the extract of Z. aromaticum and a chalcone derivative, panduratin A was isolated from fraction B of the extract of B. pandurata. Curcumin was in fraction A of extract of C. longa. The anticancer activity of zerumbone and panduratin A was investigated using MCF-7 breast. HT-29 and CaCo-2 colon cancer cells. The inhibitory activity of the active compounds was assessed using the MTT assay. The ICso of zerumbone in each of the cell lines was about 10 uM and of curcumin on HTU29 cells was 25 uM. The IC50 of panduratin A in HT-29 cells was 16 uM and in MCF-7 cells was 9 uM. Zerumbone and panduratin A showed antiproliferative effects by alteration of the DNA distribution in the cell cycle and induction of apoptosis. HT-29 cells treated with zerumbone at concentrations of 10 -25 uM or panduratin A at concentrations of 9 -65 uM for 24 h were stained with propidium iodide (PI) to determine cell cycle distribution and analysed using FACScan flow cytometry. The proportion of cells in the S phase was reduced from 18.7% in untreated cells to 10.2% in HT-29 cells after treatment with zerumbone at 10 uM to 3.1% at 25 uM. Cells in the G2 phase increased from 18.5% at 10 uM to 40% at a concentration of 25 uM. Panduratin A increased the proportion of cells in the GO/G1 phase from 33% of untreated cells to 71% after treatment with 65 uM for 24 h. Panduratin A slightly reduced the proportion of cells in S phase and cells in G2/M phase also decreased from 36,8% in untreated cells to 15.4% at 65 M. Apoptosis was determined using double labelled (Annexin-V-Fluos and PI) and then evaluated using FACScan Flow Cytometry. Morphological features of apoptosis were also examined using DiffQuick stain and fluorescent Hoechst 3355 and 4,6-diamino-2-phenylindole (DAPI). Zerumbone induced apoptosis in HT-29 cells in a dose dependent rnanner, At 48 h, 2% of cells treated with 10 M of zerumbone underwent apoptosis, which increased to 8% when treated with 50 M, Panduratin A at 28 M increased the number of cells undergoing apoptosis from 2,2% to 16.7% when treated with a concentration of 65 M. The ethanolic extracts of Z. aromaticum and B. pandurata were also investigated using the azoxymethane (AOM) induced aberrant crypt foci (ACF) model of colon cancer in rats in a short and long term study. Ethanolic extracts of C. tonga and curcumin were used as comparison. The basal diet used throughout all animal studies in this thesis was a semi-purified AIN-93 G diet (Reeves et aI., 1993). ACF were induced by two doses (15 mg/kg BW) subcutaneously of AOM one week apart and ACF were visualised in the formalin fixed colon using methylene blue stain. The ACF study was run over a short (5 weeks) and long (13 weeks) experiments. Diets containing ethanol extracts prepared from the equivalent of 2% (w/w) dried rhizome of Z. aromaticum, B. pandurate or C. tonga in a short term study did not affect the formation of ACF in rats compared to those in the control diet group. The ACF formation in a short term study was dominated by small numbers of aberrant crypts (1 or 2) per focus. It is suggested that large ACF (4 or more ACs/focus) are better predictors of colon cancer (Uchida et aI., 1997; Jenab et aI., 2001). Diets containing ethanol extracts of the equivalent of 4% by weight of dried rhizomes of Z. aromaticum, B. pandurata, C. longa were investigated over 13 week study, Total ACF were significantly reduced by Z. aromaticum extract (0.34%) in the diet (down 21%, p<0.05) relative to rats fed the control diet. A similar reduction was observed with C, longa extract (0.86%) in the diet (down 24%, p<0.01) and with 2000 ppm curcumin. There was no significant different in small ACFs (1-2 ACs/ focus) between dietary treatments. The number of foci containing 3-4 ACs/focus was significantly reduced (35%, p<0,001) in animals fed the Z. aromaticum extract and 34% (p<0.001) of animals fed the C. tonga extract. The total number of ACF containing 5 or more ACs per focus of animals fed 0.34% Z. aromaticum extract was 41 % lower than control (p<0.05) and for 0.86 % C. tonga extract was 22% (not significant). A diet containing extract (0.56%) of B. pandurata did not significantly affect the formation of ACF compared to the control AIN group. The concentration of zerumbone in the Z.aromaticum extract diet was assayed at 300 ppm, and of curcumin in the C. tonga extract diet was also 300 ppm. The concentration of panduratin A was not assayed in the diet due to late identification of the active compound. The antiinflammatory activity of ethanol extract of Z. aromaticum was investigated using dextran sulfate sodium (DSS) induced ulcerative colitis in rats. Sulfasalazine, a widely used compound to treat inflammatory bowel disease (IBD) in humans was used as the positive control. Diets containing ethanol extracts (0.34% and 0.68%) prepared from the equivalent of 4% and 8% by weight of dried rhizomes of Z. aromaticum were given to the animals throughout the experiment. On day three, rats were given 2% DSS in drinking water for 5 d and then just water for 3 d and then were killed. During the DSS treatment rats were maintained in metabolic cages, body weight, food and fluid intake and clinical symptoms such as consistency of stools and blood in faeces were recorded daily. There was slight but not significant reduction in the body weight of rats fed 0.68% extract of Z. aromaticum in the diet due to reduced food consumption. The extract of Z. aromaticum (0.34%) and sulfasalazine suppressed clinical signs of ulcerative colitis. Eleven percent of the controls were hemoccult positive on day 2 after DSS administration, which progressed further by day three with 67% being hemoccult positive and 100 % on day five. By comparison, blood appeared on day 3 of rats treated with diet containing 0.34% and 0.68% extract of Z. aromaticum and 0.05% sulfasalazine, and only 33%, 67% and 22%, of rats being hemoccult positive on day 5 respectively. The disease activity index (DAI) of rats fed diet containing 0.34% extract of Z. aromaticum was about 0.4 and similar to those which were fed with diet containing sulfasalazine. The DAI of untreated rats was 1.4. The crypt score of rats fed the extract of Z. aromaticum was slightly reduced but it was not significantly different from those of untreated rats. Other histological scores were not significantly different between dietary treatments. Extract of Z. aromaticum significantly decreased the content of PGE-2 in colon tissue compared to that of untreated animals. There was a reduction of TX8-2 content in colonic tissue of rats fed with extracts of Z. aromaticum but this was not significant. The activity of myeloperoxidase (MPO) activity in the colonic tissue of rats fed with sulfasalazine was significantly lower than that of the untreated controls and those which fed with extracts of Z. aromaticum. The results from the studies performed in this thesis showed that extract of Z. aromaticum which contains an active sesquiterpenoid zerumbone have anticancer and antiinflammatory activity suggesting that the extract may have benefits as a chernopreventative agent. However further studies are needed to elucidate their other pharmacological actions. Panduratin A showed potential anticancer activity in cell culture in vitro. However an extract of B. pandurata did not have effect on the AOM-induced colon cancer model. Different cancer models such as breast and prostate cancer could be used to further investigate the anticancer activity of extract of B. pandurata and panduratin A and to elucidate their mechanism. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1097849 / Thesis (Ph.D.) -- University of Adelaide, Dept of Medicine, 2003

Orientador: Ciro Moraes Barros / Banca: Fabíola Freitas de Paula Lopes / Banca: Fernanda Alvarenga / Banca: Gisele Zoccal Mingoti / Banca: Marcelo Fábio Gouveia Nogueira / Resumo: A fim de melhor compreender as diferenças entre zebuínos e taurinos em relação à resistência ao estresse térmico (ET), objetivou-se com o presente trabalho: (1) verificar se a adição de IGF-I ao meio de cultivo, na ausência de Soro Fetal Bovino (SFB), seria capaz de manter as taxas de produção embrionária semelhantes àquelas obtidas em meio de cultivo com SFB; (2) verificar se a adição de IGF-I ao meio de cultivo é mais eficiente em diminuir os efeitos deletérios do ET em embriões da raça Holandesa preto e branco (HPB) quando comparados aos da raça Nelore; (3) verificar se o efeito deletério do ET no desenvolvimento embrionário e na taxa de apoptose é mais acentuado em embriões da raça HPB, quando comparado aos da raça Nelore. No experimento 1, oócitos de vacas aneloradas oriundas de matadouro foram maturados, fertilizados com sêmen de touros da raça Nelore e, 10 horas pós inseminação (hpi), os embriões foram distribuídos ao acaso em quatro grupos, de acordo com a composição do meio de cultivo: SFB (5% de SFB + 0 ng/mL de IGF-I; n=165); IGF (0% de SFB + 51 100 ng/mL de IGF-I; n=163); SFB+IGF (5% de SFB + 100 ng/mL de IGF; n=169) e controle (0% de SFB + 0 ng/mL de IGF-I; n=168). Foram avaliadas as taxas de clivagem, mórula, blastocisto e blastocisto eclodido. Nos experimentos 2 e 3, oócitos de vacas Nelore e HPB oriundas de matadouro foram maturados em meio TCM 199, fertilizados com sêmen de touros das raças Nelore (n=6) e HPB (n=6), respectivamente, e cultivados em meio SOF (synthetic oviduct fluid, na ausência de SFB) até o estágio de blastocisto. Dadas 10 hpi, os embriões foram distribuídos ao acaso em quatro grupos: controle (cultivados na ausência de IGF a 39 oC); ET (cultivados na ausência de IGF e expostos a 41 oC, 96 hpi, por 9 horas, retornando a seguir para 39 oC); IGF (cultivados na presença de IGF a 39 oC) e ET/IGF (IGF e ET). No ... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Not available / Doutor