Würzburg, Universiẗat, Diss., 2005.

München, Techn. Univ., Diss., 2000.

University, Diss., 2002--Kiel.

University, Diss., 2005--Düsseldorf.

Thesis (Ph. D.)--Ohio State University, 2007. / Full text release at OhioLINK's ETD Center delayed at author's request

Thesis (Ph. D.)--Case Western Reserve University, 2007. / [School of Medicine] Department of Environmental Health Science. Includes bibliographical references. Available online via OhioLINK's ETD Center.

The aim of this project was to study ATP binding cassette (ABC) transporters in the human placenta, in particular their regulation and role in trophoblast differentiation and survival. The presence and localisation of four major placental drug transporters, multidrug resistance gene product 1 and 3 (MDR1 and 3)/ABC subfamily B members 1 and 4 (ABCB1 and 4), multidrug resistance associated proteins 1 and 2 (MRP1 and 2)/ABCC1 and 2 and breast cancer resistance protein (BCRP)/ABCG2 was initially studied in term human placenta, cultured primary trophoblast and BeWo and Jar trophoblast-like cell lines. Jar cells were found to be more similar to nondifferentiated cytotrophoblast with respect to their ABC protein expression profile, whereas BeWo cells more closely reflected differentiated syncytiotrophoblast. Treatment of primary term trophoblasts in vitro with cytokines (TNF- or IL-1) decreased expression and activity of apical transporters ABCB1/MDR1 and ABCG2/BCRP. Growth factors, on the other hand, increased BCRP expression and activity, while estradiol stimulated BCRP, MDR1 and MDR3 expression MDR1/3 functional activity. The ability of BCRP/ABCG2 to abrogate the apoptotic effects of TNF- and ceramides was studied in primary trophoblast and BeWo cells using pharmacological and molecular (siRNA) approaches. The results suggest that BCRP/ABCG2 contributes to the resistance of trophoblast cells to cytokine-induced (extrinsic) apoptosis, whereas its effects on apoptosis activated via the intrinsic mitochondrial pathway is minimal. This altered resistance was associated with increased intracellular accumulation of ceramides and reduced ability to maintain phosphatidylserine in the inner leaflet of the plasma membrane. A role for BCRP/ABCG2 in cell protection from differentiation-induced stressors was also demonstrated during the process of cell fusion associated with transient loss of plasma membrane lipid asymmetry. Finally, expression of BCRP/ABCG2 (and 9 other genes) was studied in 50 placentas from normal pregnancy and pregnancies complicated with fetal growth restriction (FGR). A marked reduction of BCRP/ABCG2 and MDR1/ABCB1 expression was observed in FGR placentas, while other transporter genes were unaffected. Collectively these data suggest that BCRP/ABCG2 and probably other ABC transporters may play a hitherto unrecognised survival role in the placenta, conferring a “stress resistance” to trophoblast cells.

Thesis (Ph. D.)--University of Texas at Austin, 2008. / Vita. Includes bibliographical references.

Orientador: Licio Augusto Velloso / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas. / Made available in DSpace on 2018-08-17T07:03:20Z (GMT). No. of bitstreams: 1 Mansur_Eli_D.pdf: 1693041 bytes, checksum: a5b3d9f350100a50b16d835341ff4bc9 (MD5) Previous issue date: 2007 / Resumo: A leptina, hormônio com semelhança funcional e estrutural às citocinas, é conhecida por exercer, além das ações clássicas de controle da ingestão alimentar e termogênese, importantes funções na modulação das respostas do sistema imune. Alguns destes efeitos são dependentes da propriedade da leptina em modular a apoptose das células tímicas. Neste trabalho, utilizamos ratos Wistar para investigar os mecanismos moleculares envolvidos no controle, dependente da leptina, da apoptose no timo. A apoptose foi avaliada por citometria de fluxo e ELISA para determinação de nucleossomos, enquanto que a transdução do sinal foi avaliada por imunoprecipitação, imunoblot e microscopia confocal. O ObR estava expresso na maioria das células tímicas e a sua quantidade relativa reduziu-se progressivamente durante a maturação dos timócitos. A expressão do ObR estava co-localizada com JAK-2 e STAT-3, e a injeção aguda, in vivo , de leptina promoveu a fosforilação em tirosina de JAK-2 e o engajamento de STAT-3. O tratamento com leptina, também, levou à fosforilação em tirosina de IRS1 e fosforilação em serina de Akt. O tratamento crônico com leptina reduziu a apoptose tímica, e este efeito não foi inibido pelo AG490, um inibidor de JAK, mas foi significativamente inibido por LY294002, um inibidor de PI3-Quinase, e por um oligonucleotídeo antisense para IRS1. Portanto, a leptina inibe a apoptose em células tímicas via um mecanismo independente da ativação de JAK-2 mas dependente do engajamento da via IRS1/PI3-Quinase / Abstract: The cytokine-like hormone leptin is known to exert important functions on the modulation of immune responses. Some of these effects are dependent on the property of leptin to modulate the apoptosis of thymic cells. In the present study, we employed Wistar rats to investigate the molecular mechanisms involved in leptin-dependent control of apoptosis in thymus. Apoptosis was evaluated by flow cytometry and ELISA for nucleosome determination, while signal transduction was evaluated by immunoprecipitation, immunoblot and confocal microscopy. The ObR was expressed in most thymic cells and its relative amount reduced progressively during thymocyte maturation. ObR expression was co-localized with JAK-2 and STAT-3, and an acute, in vivo , injection of leptin promoted the tyrosine phosphorylation of JAK-2 and the engagement of STAT-3. The treatment with leptin also led to the tyrosine phosphorylation of IRS1 and serine phosphorylation of Akt. Chronic treatment with leptin reduced thymic apoptosis, an effect that was not inhibited by the JAK inhibitor AG490 but was significantly inhibited by the PI3-kinase inhibitor LY294002 and by an antisense oligonucleotide to IRS1. Thus, leptin inhibits the apoptosis of thymic cells through a mechanism that is independent of the activation of JAK-2 but depends on the engagement of the IRS1/PI3-kinase pathway / Doutorado / Medicina Experimental

As vitafisalinas sÃo lactonas esteroidais (C28), estruturalmente baseadas no esqueleto do ergostano, comumente encontradas em plantas da famÃlia Solanaceae. A fim de avaliar as propriedades anticÃncer desses compostos, cinco vitafisalinas [O, F, M, N e (17S, 20R, 22R) -5 β, 6β :18,20-2-diepÃxi β-4, 18 - diidrÃxi-1 - oxovita-3-24-enolido] isoladas da Acnistus arborescens, planta tÃpica do nordeste brasileiro, foram analisadas utilizando diversos modelos biolÃgicos. Todas as cinco vitafisalinas mostraram efeitos citotÃxicos em linhagens de cÃlulas tumorais, sendo a vitafisalina O a mais potente e a vitafisalina (17S, 20R, 22R) -5 β, 6β :18,20-2-diepÃxi β-4, 18 - diidrÃxi-1-oxovita-3 - 24-enolido) a menos potente. Ao compararmos as estruturas quÃmicas das vitafisalinas e suas atividades, foi observado que a ligaÃÃo dupla entre os carbonos 2 e 3 à essencial para os efeitos citotÃxicos desses compostos. No entanto, as vitafisalinas (O, F e N) nÃo mostraram qualquer especificidade para linhagens tumorais, jà que tambÃm apresentaram efeitos citotÃxicos e genotÃxicos, semelhantes, em cÃlulas leucÃmicas (HL-60) e em cÃlulas normais (PBMC). A viabilidade celular e curvas de crescimento foram determinadas, para as linhagens de HL-60 e K-562, utilizando o ensaio de exclusÃo de azul de tripan. As vitafisalinas O, F, M e N reduziram o nÃmero de cÃlulas viÃveis de modo dose e tempo dependente, apresentando valores de CI50 variando de 0,7 a 3,5 μM apÃs 72 horas de incubaÃÃo. Nas mesmas linhagens leucÃmicas, as vitafisalinas tambÃm inibiram a sÃntese de DNA, causaram alteraÃÃes morfolÃgicas tÃpicas de apoptose, e apenas na linhagem HL-60 induziram a ativaÃÃo da caspase-3. AlÃm disso, foi realizado, em cÃlulas de HL-60, a anÃlise da integridade da membrana celular, distribuiÃÃo do ciclo celular, fragmentaÃÃo de DNA e o potencial transmembrÃnico de mitocÃndria, utilizando citometria de fluxo. Nestes experimentos, as vitafisalinas O e F, somente na concentraÃÃo de 10 μM, reduziram o nÃmero de cÃlulas viÃveis para 60 e 40%, respectivamente. Na anÃlise do ciclo celular, ambas vitafisalinas, na concentraÃÃo de 5 μM, causaram um acÃmulo de cÃlulas na fase G2/M do ciclo celular. Ambas vitafisalinas tambÃm causaram um aumento significativo do nÃmero de cÃlulas apresentando fragmentaÃÃo de DNA. Os resultados da anÃlise do potencial transmembrÃnico de mitocÃndria mostraram um aumento na despolarizaÃÃo de 4,7, 17,5 e 9,1% causado pela vitafisalina O e de 7,6, 16,6 e 5,6% pela vitafisalina F. O efeito antitumoral (in vivo) da vitafisalina F foi analisado em camundongos transplantados com o tumor Sarcoma 180, nas doses de 5, 10 e 20 mg/Kg/dia por via intraperitoneal e na dose de 20 mg/Kg/dia por via oral. O crescimento do tumor foi inibido em mais de 76% na maior doses testada (20mg/Kg/dia), tanto por via intraperitoneal quanto por via oral. A anÃlise histopatolÃgica dos ÃrgÃos dos animais mostraram que a vitafisalina F provoca efeitos tÃxicos moderados, principalmente no fÃgado e nos rins, mas esses podem ser considerados como reversÃveis. Tendo em vista todos estes dados, pode concluir-se que as vitafisalinas podem ser consideradas como uma classe emergente de novos compostos anticÃncer. / Withaphysalins are C28-steroidal lactones structurally based on the ergostane skeleton commonly found in Solanaceae species. In order to evaluate the anticancer properties of these compounds, five withaphysalins [O, F, M, N and (17S,20R,22R)-5β,6β: 18,20-diepoxy-4β,18-dihydroxy-1-oxowitha-24-enolide] isolated from Acnistus arborescens, a plant from the northeastern Brazilian flora, were analyzed in several biological models. All five withaphysalins showed cytotoxic effects against tumor cell lines, being withaphysalin O the most potent and withaphysalin (17S,20R,22R)-5β,6β: 18,20-diepoxy-4β,18-dihydroxy-1-oxowitha-24-enolide, the less potent. Based on these results, its shown that a double-bond between carbons 2 and 3 is essential for the cytotoxic activity of withaphysalins. Withaphysalins (O, F and N) did not show any specificity to tumor cell lines, showing similar cytotoxic and genotoxic effects against leukemic cells (HL-60) and normal cells (PBMC). Cell viability and growth curves of HL-60 and K-562 treated cells were determined using trypan blue exclusion assay, where all withaphysalins reduced the number of viable cells in a dose-and time-dependent fashion, with IC50 values ranging from 0.7 to 3.5 μM after 72 h of incubation. In HL-60 and K-562 cells, the withaphysalins inhibited DNA synthesis, induced morphological alterations, typical of apoptosis, and only in the HL-60 cell line, and they induced activation of caspase-3. Moreover, it was performed the analyzes of cell membrane integrity, cell cycle distribution, DNA fragmentation and the mitochondrial membrane potential using flow citometry. In these experiments, withaphysalins O and F, only at concentration of 10ÂM, reduced the number of viable cells to 60 and 40% respectively. In the cell cycle analysis, both withaphysalins led to a cell cycle arrest at G2/M, at the concentration of 5μM. Cells treated with both withaphysalins also showed a significant increase in DNA fragmentation when compared to the negative control. Results of the mitochondrial transmembrane potential showed depolarization changes in accordance to the tested concentration (2.5, 5 and 10μg/mL) with 4.7, 17.5 and 9.1% for withaphysalin O and 7.6, 16.6 and 5.6% for withaphysalin F, respectively. The in vivo antitumor effects of withaphysalin F was performed in animals bearing the sarcoma 180 tumor, and at the highest dose tested (20mg/Kg/day), growth tumor was inhibited in 77%. Histopatological analysis of mice organs showed that withaphysalin F causes moderate toxic effects mostly in liver and kidney, but they may be considered reversible effects. Taking in account all these data, it can be concluded that withaphysalins could be considered as an emerging class of new anticancer compounds.