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Call number: LD2668 .T4 1957 H3 / Master of Science

A dissertation submitted to the Faculty of Medicine, University of the Witwatersrand, Johannesburg in fulfilment of the Degree of Master of Science in the branch of Medicine / The guinea pig clears circulating tissue ferritin in a manner different from other mammals. Previous work in man and in rats has shown that injected tissue ferritin is removed from the plasma predominantly by the liver; however, in the guinea pig it is cleared predominantly by red cell precursors and furthermore, the ferritin iron is utilised directly for haem synthesis. During pregnancy, the foeto-placental complex also has a high requirement for iron. / IT2018

The placenta is responsible for mediating fetal growth and development, thereby influencing health across the lifespan. Physical activity (PA) confers benefits to mother and baby during pregnancy, but little is known about its impact on the placenta. There were two purposes of this study: i) to determine if maternal PA during pregnancy influences placenta mitochondrial protein content and function, and ii) to determine if there were differences in placenta mitochondrial protein content and function in different regions of the placenta, namely proximal or distal to the centre of the placenta. Healthy women between 12-28 weeks gestation were recruited, and free-living PA was objectively assessed at multiple time points during pregnancy using an accelerometer. Participants were grouped by minutes of moderate-to-vigorous PA (MVPA) per day. Placenta tissue samples were collected from central and distal placental regions immediately post-birth and were used for two separate analyses. Half of the samples were flash frozen in liquid nitrogen and used for western blot analysis of mitochondrial complex I-V proteins. Fresh mitochondria were isolated from the other half of the samples, and high-resolution respirometry was used to measure placental mitochondrial respiration. There were significant positive correlations between maternal PA and mitochondrial protein content in peripheral tissue samples, but protein content was significantly higher in central tissue compared to peripheral tissue samples. In addition, state 3 respiration was higher in central tissue samples of placentas from participants with high MVPA compared to participants with low MVPA. Finally, complex I protein was higher in central tissue samples of placentas from female offspring compared to placentas of male offspring. However, many of these results are underpowered and further study is warranted. This study provides new avenues to explore the relationship between PA and placenta mitochondria in healthy populations.

The placenta provides a protective barrier for the developing foetus during gestation. Physiological barriers including the placenta, liver, kidney, intestine and blood-brain barrier are known to express ATP-Binding cassette transporters (ABC transporters) and metabolising enzymes. These specialised proteins have the ability to transport or metabolise xenobiotics. There is evidence to suggest that ABC transporters and metabolising enzymes are located at the interface between the maternal and foetal blood supplies (a cell layer referred to as the syncytiotrophoblast) and therefore may help protect the foetus from harmful xenobiotics. During new compound development prenatal developmental toxicity testing forms an important part of safety assessment. In order to predict potential toxicity of a new chemical entity to humans, rodent and non-rodent species are currently used. This thesis investigates the rat and human placental barrier properties in order to help facilitate our knowledge of species differences and contribute to our understanding of the limitations of these surrogate models. The approaches taken include: genomic analyses using microarray data to compare the overall expression of ABC transporters and metabolising enzymes throughout gestation in both species, immunohistochemical techniques to localise transporters and metabolising enzymes in the rat placenta, and in vitro functionality assays of selected transporters performed in rat and human placental cell line models. The main findings have shown a similar mRNA expression level of ABCG2/BCRP (breast cancer resistance protein) throughout gestation in the rat and human, however different mRNA expression levels of other transporters (slco4a1/oatp4a1 in particular) and metabolising enzymes were also highlighted. Immunohistochemistry localised selected transporters to the syncytiotrophoblast region of the rat placenta (the interface of maternal and foetal circulations). Functional in vitro assays were successfully utilised in rat and human placental cell lines which showed functional ABCB1/P-gp in both species. Overall, these findings provide a genomic characterisation of the rat and human protective placental barrier properties and show transporter functionality in in vitro cell-based assays which will prove useful in prenatal and developmental toxicity tests. Alternatives to using animals have been explored by using functional in vitro assays which could potentially be implored during the new compound discovery phase. This could help to make animal testing more selective for given compounds and ensures the new chemical entity is being tested in the model closest resembling the human.

Orientador: Maria Cristina Cintra Gomes Marcondes / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-11-01T14:27:16Z (GMT). No. of bitstreams: 1 Toledo_MerciaTancredo_D.pdf: 3837085 bytes, checksum: 8ec6cd217ab21fa4523eaf5e631553fd (MD5) Previous issue date: 2003 / Resumo: A placenta provê todas as necessidades para o crescimento e desenvolvimento fetal. Na presença de neoplasia é difícil predizer qual será a duração, evolução e termo da gravidez. O crescimento tumoral é prejudicial à mãe e á unidade materno fetal. O substrato energético fundamental usado pelo feto é a glicose proveniente da placenta, já que o fígado fetal é incapaz de processar a gliconeogênese. A placenta armazena glicogênio para sua própria atividade metabólica e provê lactato para o metabolismo fetal. Em estudos prévios, observamos danos à unidade feto-placentária, através de alterações bioquímicas e morfológicas na placenta, associadas ao crescimento do tumor de Walker ou à inoculação do líquido ascítico. Apoptose placentária pode afetar as funções da placenta e pode estar associada ao retardo de crescimento fetal. Por isto, este trabalho teve por objetivos investigar: 1) Reserva de nutrientes placentários através da análise de armazenamento de glicogênio; 2) Precursores de apoptose placentária possivelmente produzidos em função do crescimento do Carcinossarcoma de Walker 256 ou pela inoculação de líquido ascítico; 3) Envolvimento do sistema ubiqu.itina-proteossomo no processo seletivo de proteínas placentárias. Ratas Wistar grávidas adultas foram distribuídas em três grupos: controle (C); tumor de Walker (W); inoculadas com líquido ascítíco (A); sacrificadas no 16°, 19° e 21 ° dia após a cruza. Os tecidos placentários foram homogenizados e analisados através de: I) ensaios bioquímicos para glicogênio, conteúdo protéico, fosfatase alcalina e atividade glutationa-S-transferase; II) ensaios imunohistoquímicos, contando-se células positivas para P ARP, caspase-3 e citocromo c, e IV) quantificação de precursores apoptóticos (PARP, anti-caspase 3 e citocromo c) e do sistema ubiquitina-proteossomo subunidade 205, P42 e ubiquitina E2 por Westem blotting. Os pesos placentário e fetal foram significativamente reduzidos em ratos implantados com tumor e inoculados com líquido ascítico nos três dias de análise da gravidez. O efeito do crescimento tumoral aumentou os índices de reabsorções fetais que foram semelhantes ao dos grupos inoculados com líquido ascítico. O labirinto trofoblasto apresentou-se reduzido no 210 dia em ambos os grupos W e A. Os estoques de glicogênio e a atividade da fosfatase alcalina foram reduzidos no grupo implantado com tumor. Os efeitos produzidos pelo crescimento tumoral foram semelhantes aos observados nos grupos inoculados com líquido ascítico. Diminuição do conteúdo protéico placentário foi observado nos grupos W e A. Placenta, de ratos implantados com tumor e inoculados com líquido ascítico, apresentaram diminuição na atividade de GST no 190 e 210 dia; paralelamente houve aumento significativo de PARP, caspase-3 e citocromo c placentário tanto na análise imunohistoquímica quanto na análise por Westem blotting. O crescimento do tumor de Walker causou prejuízo ao tecido placentário e, especialmente, promoveu modificações nos precursores da apoptose. Considerando-se que o espongio trofoblasto é um dos responsáveis por manter a função placentária normal, a apoptose descontrolada destas células pode resultar em impacto negativo ao desenvolvimento fetal através de fatores produzido pelas células do tumor, ou, indiretamente, pelos efeitos causados pela inoculação do líquido ascítico aumentando a apoptose. Em animais implantados com tumor e inoculados com líquido ascítico os tecidos placentários não apresentaram dif¿!rença significativa de ubiquitina E2, embora as subunidades proteossômicas 208 e P42 fossem significativamente mais expressivas nos grupos W e A, com relação aos respectivos controles. 0 sistema ubiquitina-proteossomo está envolvido na reorganização e seleção de proteínas nucleares e citoplasmáticas; sendo assim, a presença de tumor ou a inoculação do líquido ascítico promoveu alterações neste sistema comprometendo, provavelmente, a viabilidade dos tecidos placentários e, conseqüentemente, a evolução da gravidez. Esses resultados podem ajudar na compreensão de eventos e identificação de métodos que venham garantir o bem-estar fetal e desenvolvimento da placenta. Assim o crescimento do tumor de Walker, juntamente com a inoculação de líquido ascítico, promovem danos irreversíveisá placenta alterando sua homeostasia assim prejudicando o desenvolvimento e a sobrevida fetal no ambiente uterino / Abstract: The placenta provides ali needs for healthy fetus development. The cancer development difficulties to predict the period, evolution and term of pregnancy. The tumour growth can be prejudicial to mother, foetus and, especially to maternal-fetal unit. The fundamental energy substrate used by fetus and placenta is glucose, although fetalliver is incapable to process gluconeogenesis. The placenta cells mobilise glycogen storage for its own energy activity and provides lactate to earlier fetal metabolism. In previous studies, we observed damage in the maternal-foetal unity, showed by biochemical and morphological alterations in placenta associated to tumour growth or ascitic fluid inoculation. Placental apoptosis may affect a range of placental functions and can be associated foetal growth retardation. For this reason the aim of this work was to analyse the effects of Walker 256 carcinosarcoma growth in pregnant rats studing 1) the nutrient reserves as glycogen storage 2) the effects on placental apoptotic precursors and 3) the involvement of the ubiquitin-proteasome system on the selective process of placental proteins. Adult pregnant Wistar rats were distributed into three groups: control, C; tumour bearing, W; injected ascitic fluid, A, and sacrificed on 16th, 19th or 21 st day after breeding. The placental tissue aliquots were analysed after homogenising and measuring I) placental glycogen, and protein content and alkaline phosphatase and glutathione-8-transferase activity; II) histochemically the glycogenceHs presence; III) Immunohistochemically assays counting [positive cells for P ARP I caspase-3 and cytochrome c, and IV) The apoptotic precursors (PARP, anti-caspase-3 and cytochrome c) and ubiquitin-proteasome proteins (ubiquitin E2, P42 and subunit 208) expression. The placenta and fetal weight were significantly reduced in both tumour bearing and ascitic fluid injected rats since 16th, 19th and 21st day of pregnancy. The effects of tumour growth, inducing high fetal reabsorption sites were similar in ascitic fluid injected group. Labyrinth layer was significantly reduced on 21 st day in both tumour bearing and ascitic fluid injected. The placental glycogen cells andalkaline phosphatase activity were reduced in tumour groups. The effect produced by tumour growth were similar that observed in ascitic ftuid injected group. Decrease in placenta I protein content was observe in both W and A Placental P ARP, caspase-3 and cytochrome c positive cells, analysed by immunohistochemistry and cells and Westem blot, were increased in tumour- bearing and ascitic fluid injection groups, showing that tumour growth clearly produced damage in placental tissue, especially inducing changes in apoptotic precursors. Since the trophoblastic cells are the responsible ones for maintaining normal placental function, the uncontrolled death of this cells could result in a negative impact to foetal development, as many factors either directed produced by tumour cells or indirectly effect produced by ascitic fluid substances, increasing the apoptosis rate. In tumour-bearing and ascitic fluid groups the placenta tissues showed no difference in ubiquitin E2 quantity, although the proteasome subunits 208 and P42 contents were significantly higher in the W and A rats. This results suggest that the ubiquitin proteasome system was involved in placental tissues probably being responsible for organizer and selection of citoplasmatic and nuclear proteins. The presence of tumour or ascitic fluid injected probably promoted alterations in the placenta tissues that committed the pregnancy evolution. The knowledge of this events resulted by tumour effects may help to identify the better way to guarantee the welfare of fetal and placenta development. In conclusion the tumour growth and, especially, ascitic fluid injected promoted irreversible damage in a placenta tissue altering the homeostasis and compromising the foetal development / Doutorado / Fisiologia / Doutor em Biologia Funcional e Molecular