Four experiments were conducted to investigate methods of producing transgenic bovine embryos entirely in vitro. Experiment 1 examined the effect of DNA microinjection at 11, 15 and 19 h after fertilization (haf) on survival rate and DNA detection frequency by polymerase chain reaction (PCR). There was no difference in transgene detection frequency between treatments (53% at 11; 50% at 15; 48% at 19 haf). Of all injected embryos developing to the morula or blastocyst stage after 7 d in culture, 89% tested positive for the presence of the transgene by PCR. Greater developmental efficiencies can be obtained when injection is performed early in pronuclear formation (7% (11/161) at 11; 4% (61159) at 15; 1 % (1/165) at 19 haf; p<0.05). Experiment 2 examined the effect of microinjection of DNA into the germinal vesicle (gv) of bovine oocytes on subsequent development and detection of the transgene. Injection of the transgene into the gv reduced developmental rates compared to controls (control=23% (89/384); non-injected=9% (23/250); GV injected=5% (12/259); p<O.05). Transgene detection frequency was 64% (37/58). Injection of bovine oocytes before fertilization results in viable embryos containing the transgene, although at low frequencies. Experiment 3 was designed to examine whether the frequency of microinjected DNA detection by peR In whole bovine embryos would decline over a 21 d culture period. At d 0, the transgene was detected in 100% (46/46) of embryos analyzed. At d 7, detection frequency was 84% (51/62) in viable embryos, at d 14 49% (18/37), and at d 21 38% (3/8). DNA detection frequency in microinjected bovine embryos by PCR analysis does not give a reliable indication of live transgenic birth rates until after 14 d in culture. Experiment 4 examined microinjected bovine embryos for their potential use as donor embryos in nuclear transfer, or cloning. There was no difference in development between embryos cloned from microinjected donor embryos and those from control donor embryos (injected=11 % (37/377); control=9% (7/81); p>0.05). Of the embryos developing from microinjected donors, 32% (12/37) were PCR positive. Microinjected embryos can be successfully used in a nuclear transfer program to produce more viable embryos, and the resulting embryos may be more reliably screened by PCR. The efficiency of producing viable bovine embryos positive for the injected gene may be increased by performing microinjection early in pronuclear formation, and entering the resulting embryos into a nuclear transfer program. / Ph. D.

DNA is known to be a mechanically and thermally stable structure. In its double stranded form it is densely packed within the cell nucleus and is thermo-resistant up to 70 °C. In contrast, we found a sudden loss of cell nuclei integrity at relatively moderate temperatures ranging from 45 to 55 °C. In our study, suspended cells held in an optical double beam trap were heated under controlled conditions while monitoring the nuclear shape. At specific critical temperatures, an irreversible sudden shape transition of the nuclei was observed. These temperature induced transitions differ in abundance and intensity for various normal and cancerous epithelial breast cells, which clearly characterizes different cell types. Our results show that temperatures slightly higher than physiological conditions are able to induce instabilities of nuclear structures, eventually leading to cell death. This is a surprising finding since recent thermorheological cell studies have shown that cells have a lower viscosity and are thus more deformable upon temperature increase. Since the nucleus is tightly coupled to the outer cell shape via the cytoskeleton, the force propagation of nuclear reshaping to the cell membrane was investigated in combination with the application of cytoskeletal drugs.

Made available in DSpace on 2014-10-09T12:48:22Z (GMT). No. of bitstreams: 0 / Made available in DSpace on 2014-10-09T13:57:22Z (GMT). No. of bitstreams: 0 / Dissertacao (Mestrado) / IPEN/D / Instituto de Pesquisas Energeticas e Nucleares - IPEN/CNEN-SP

Made available in DSpace on 2014-10-09T12:48:22Z (GMT). No. of bitstreams: 0 / Made available in DSpace on 2014-10-09T13:57:22Z (GMT). No. of bitstreams: 0 / Neste estudo, foi determinado o dano celular em linfócitos periféricos após exposição ao 153Sm-EDTMP (Samário-153 etilenodiaminotetrametilenofosfonato) por meio da técnica de análise de micronúcleos e coloração diferencial. O 153Sm-EDTMP é um radiofármaco utilizado para alívio da dor em pacientes com metástase óssea. A análise da freqüência de micronúcleos em amostras sangüíneas de pacientes obtidas uma hora após a administração endovenosa do radiofármaco (41 MBq/kg) mostrou que não houve diferença estatística em relação aos valores basais em células binucleadas. Porém, a análise da distribuição do dano em células mononucleadas mostrou que os pacientes sem tratamento radioterápico prévio apresentaram um aumento significativo na freqüência de células com um micronúcleo e aqueles com tratamento radioterápico prévio, nas células com dois ou mais micronúcleos. Os experimentos in vitro realizados com exposição de sangue total a três concentrações radioativas de 153Sm-EDTMP (0,370; 0,555 e 1,110 MBq/mL) por uma hora mostraram um aumento na freqüência de micronúcleos e de células necróticas e apoptóticas com o aumento da dose de radiação. Foram construídas curvas dose-resposta para os indivíduos sadios e para os pacientes com metástases óssea sem prévio tratamento radioterápico. A comparação das curvas mostrou que os pacientes apresentaram uma radiossensibilidade mais alta que os indivíduos sadios tanto quanto a porcentagem de células com micronúcleos como de células mortas (necróticas e apoptóticas). / Dissertacao (Mestrado) / IPEN/D / Instituto de Pesquisas Energeticas e Nucleares - IPEN/CNEN-SP

Neste estudo, foi determinado o dano celular em linfócitos periféricos após exposição ao 153Sm-EDTMP (Samário-153 etilenodiaminotetrametilenofosfonato) por meio da técnica de análise de micronúcleos e coloração diferencial. O 153Sm-EDTMP é um radiofármaco utilizado para alívio da dor em pacientes com metástase óssea. A análise da freqüência de micronúcleos em amostras sangüíneas de pacientes obtidas uma hora após a administração endovenosa do radiofármaco (41 MBq/kg) mostrou que não houve diferença estatística em relação aos valores basais em células binucleadas. Porém, a análise da distribuição do dano em células mononucleadas mostrou que os pacientes sem tratamento radioterápico prévio apresentaram um aumento significativo na freqüência de células com um micronúcleo e aqueles com tratamento radioterápico prévio, nas células com dois ou mais micronúcleos. Os experimentos in vitro realizados com exposição de sangue total a três concentrações radioativas de 153Sm-EDTMP (0,370; 0,555 e 1,110 MBq/mL) por uma hora mostraram um aumento na freqüência de micronúcleos e de células necróticas e apoptóticas com o aumento da dose de radiação. Foram construídas curvas dose-resposta para os indivíduos sadios e para os pacientes com metástases óssea sem prévio tratamento radioterápico. A comparação das curvas mostrou que os pacientes apresentaram uma radiossensibilidade mais alta que os indivíduos sadios tanto quanto a porcentagem de células com micronúcleos como de células mortas (necróticas e apoptóticas). / In this study the cellular damage in peripheral lymphocytes after exposure to 153Sm-EDTMP (Samarium-153 ethylenediaminetetrametylenephosphonate) was determined using the technique of micronuclei analysis and differential coloration. 153Sm- EDTMP is a radiopharmaceutical used for pain relief in patients with bone metastases. The analysis of the frequency of micronuclei in patient blood samples obtained one hour after endovenous administration of radiopharmaceutical (41 MBq/kg) showed no statistical difference in relation to basal values in binucleated cells. However the analysis of damage distribution in mononucleated cells, showed that the patients without previous radiotherapic treatment presented a significant increase in the frequency of cells with one micronucleus and in those who had taken previous radiotherapic treatment, in cells with two or more micronuclei. The in vitro experiments conducted with the exposition of total blood to three radiation concentrations of 153Sm-EDTMP (0.370, 0.555 and 1.110 MBq/mL) during one hour showed an increase in the frequency of micronuclei and necrotic and apoptotic cells with increasing radiation dose. Dose-response curves for healthy donors and patients with bone metastasis without previous radiotherapic treatment were constructed. The comparison of the curves showed that patients presented higher radiosensitivity, either micronuclei or dead cell (necrotic or apoptotic) percentages, than healthy donors.

Neste estudo, foi determinado o dano celular em linfócitos periféricos após exposição ao 153Sm-EDTMP (Samário-153 etilenodiaminotetrametilenofosfonato) por meio da técnica de análise de micronúcleos e coloração diferencial. O 153Sm-EDTMP é um radiofármaco utilizado para alívio da dor em pacientes com metástase óssea. A análise da freqüência de micronúcleos em amostras sangüíneas de pacientes obtidas uma hora após a administração endovenosa do radiofármaco (41 MBq/kg) mostrou que não houve diferença estatística em relação aos valores basais em células binucleadas. Porém, a análise da distribuição do dano em células mononucleadas mostrou que os pacientes sem tratamento radioterápico prévio apresentaram um aumento significativo na freqüência de células com um micronúcleo e aqueles com tratamento radioterápico prévio, nas células com dois ou mais micronúcleos. Os experimentos in vitro realizados com exposição de sangue total a três concentrações radioativas de 153Sm-EDTMP (0,370; 0,555 e 1,110 MBq/mL) por uma hora mostraram um aumento na freqüência de micronúcleos e de células necróticas e apoptóticas com o aumento da dose de radiação. Foram construídas curvas dose-resposta para os indivíduos sadios e para os pacientes com metástases óssea sem prévio tratamento radioterápico. A comparação das curvas mostrou que os pacientes apresentaram uma radiossensibilidade mais alta que os indivíduos sadios tanto quanto a porcentagem de células com micronúcleos como de células mortas (necróticas e apoptóticas). / In this study the cellular damage in peripheral lymphocytes after exposure to 153Sm-EDTMP (Samarium-153 ethylenediaminetetrametylenephosphonate) was determined using the technique of micronuclei analysis and differential coloration. 153Sm- EDTMP is a radiopharmaceutical used for pain relief in patients with bone metastases. The analysis of the frequency of micronuclei in patient blood samples obtained one hour after endovenous administration of radiopharmaceutical (41 MBq/kg) showed no statistical difference in relation to basal values in binucleated cells. However the analysis of damage distribution in mononucleated cells, showed that the patients without previous radiotherapic treatment presented a significant increase in the frequency of cells with one micronucleus and in those who had taken previous radiotherapic treatment, in cells with two or more micronuclei. The in vitro experiments conducted with the exposition of total blood to three radiation concentrations of 153Sm-EDTMP (0.370, 0.555 and 1.110 MBq/mL) during one hour showed an increase in the frequency of micronuclei and necrotic and apoptotic cells with increasing radiation dose. Dose-response curves for healthy donors and patients with bone metastasis without previous radiotherapic treatment were constructed. The comparison of the curves showed that patients presented higher radiosensitivity, either micronuclei or dead cell (necrotic or apoptotic) percentages, than healthy donors.

Thesis (Ph. D.)--University of Missouri--Columbia, 1998. / Typescript. Vita. Includes bibliographical references (leaves : 325-355). Also available on the Internet.