The Hippo pathway is a master regulator of cell proliferation and organ size, namely through regulation of transcriptional co-activators YAP and TAZ which bind TEAD1-4 transcription factors. The Hippo effector YAP is dysregulated in many human solid tumours including rhabdomyosarcoma and oesophageal cancer. Additionally, persistent hyperactivity of YAP in activated but not quiescent satellite cells can give rise to embryonal rhabdomyosarcoma. However, the question of exactly how YAP acts as an oncogene and actively gives rise to tumour progression in these cancers remains unknown. In this thesis I characterised the mechanisms which determine the functional role of YAP in driving instability in the genome. Secondly, lentiviral mediated knockdown of YAP is performed to determine and investigate its effect on tumorigenesis. Thirdly, gene sets from constitutive YAP S127A induced mouse ERMS tumours subjected to array-CGH were further analysed. Finally, I cloned chicken Yap1, Tead1 and Fstl5 to identify its role during chick embryonic development, by the retroviral mediated loss of function approach. The results demonstrated that constitutive YAP S127A expression in-vitro as well as in-vivo induces chromosomal instability by increasing the rate of mitotic chromosome segregation errors and copy number alterations of oncogenes and other cancer related genes. Recurrent copy number gains of the p53 inhibitor Mdm2 were observed in YAP S127A-driven ERMS tumours. Moreover, lentiviral mediated YAP knockdown showed significant reduction in proliferation, migration and invasion as well as transformation potential in human cultured cancer cells. Moreover, retroviral YAP S127A expression during early stages of chick embryo development did not lead to an overt phenotype and showed poor survival. Additionally, I have cloned RCAS-RNAi vectors to study the loss of function effect on Hippo targets and Fstl5 during chicken embryo development. Collectively, my data provides insight into the mechanisms with which YAP could drive tumorigenesis and that YAP knockdown can be considered a potential therapeutic target to reduce cancer progression.

O assoalho pélvico é anatomicamente dividido em fáscia endopélvica, diafragma pélvico e diafragma urogenital e o músculo levantador do ânus é considerada a estrutura mais importante na manutenção da estática dos órgãos pélvicos. Durante a gravidez, altos níveis de progesterona e relaxina associadas às alterações anatômicas promovidas pelo crescimento uterino são fatores que contribuem para o aumento da prevalência de incontinência urinária durante a gravidez e o puerpério. O treinamento dos músculos do assoalho pélvico (TMAP) durante a gravidez tem sido tema de vários estudos que tentam comprovar a sua eficácia na prevenção e tratamento da incontinência urinária e nos resultados do trabalho de parto e do parto. Na literatura, encontram-se trabalhos que avaliaram as repercussões fetais associadas à realização de exercício físico aeróbico, porém não existem estudos que realizaram essa investigação com exercícios específicos do assoalho pélvico. O objetivo deste estudo foi, portanto, avaliar os efeitos do TMAP sobre os parâmetros doplervelocimétricos da hemodinâmica uteroplacentária e fetal. Realizou-se um estudo do tipo experimental, prospectivo, randomizado e controlado com gestantes de baixo risco gestacional. Um grupo foi submetido aos exercícios de TMAP através de sessões semanais durante 16 semanas (grupo exercício) e outro grupo não foi submetido aos exercícios (grupo controle). Os exames ultrassonográficos foram realizados com 28, 32 e 36 semanas sendo avaliados os índices de resistência (IR) e pulsatilidade (IP) das artérias uterinas maternas e artérias umbilical e cerebral média fetal antes e após os exercícios. Na análise estatística foi utilizado o modelo de regressão linear com efeitos mistos (efeitos aleatórios e fixos). Este procedimento foi realizado através do software SAS® 9.0, utilizando a PROC MIXED. Foram incluídas 26 gestantes no grupo exercício e 11 no grupo controle. A análise da artéria uterina mostrou diferença na comparação do estudo Doppler com redução significativa de IP e IR após o exercício na avaliação com 36 semanas. Com este resultado, é possível concluir que a execução de exercícios desse tipo de TMAP, durante a gestação, não altera IP e IR na artéria umbilical e cerebral média com 28, 32 e 36 semanas de gravidez e nas artérias uterinas com 28 e 32 semanas, porém ocorre redução com 36 semanas de gravidez. / The pelvic floor is anatomically divided into endopelvic fascia, pelvic diaphragm and urogenital diaphragm, and the levator ani muscle is considered to be the most important structure for the static maintenance of the pelvic organs. During pregnancy, the high levels of progesterone and relaxin, together with the anatomical changes promoted by uterine growth, are factors that contribute to the increased prevalence of urinary incontinence during pregnancy and the puerperium. Pelvic floor muscle training (PFMT) during pregnancy has been investigated in several studies that have attempted to prove its efficacy in the prevention and treatment of urinary incontinence and in the results of labor and delivery. Studies evaluating the fetal repercussions associated with aerobic physical exercise are available in the literature, but there are no studies that perform this investigation with specific exercises for the pelvic floor. Thus, the objective of the present study was to assess the effects of PFMT on the Doppler velocimetry and uteroplacental and fetal hemodynamic parameters. An experimental, prospective, randomized and controlled study was conducted on pregnant women of low gestational risk. One group (N = 26) performed PFMT exercises once a week for 16 weeks (exercise group) and the other (N = 11) did not perform exercises (control group). Ultrasound exams were performed at 20, 32 and 36 weeks for the evaluation of resistance index (RI) and pulsatility index (PI) of the maternal uterine arteries and of the fetal umbilical and middle cerebral arteries before and after exercise. A mixed effects linear regression model (random and fixed effects) was employed for statistical analysis using the PROC MIXED method of the SAS® 9.0 software. Analysis of the uterine artery showed a difference in the comparison of the Doppler study, with a significant reduction of PI and RI after exercise at the 36 week evaluation. With this result, it is possible to conclude that the execution of this type of PFMT exercises during pregnancy does not alter the PI or RI of the umbilical and middle cerebral arteries at 28, 32 and 36 weeks of pregnancy or of the uterine arteries at 28 and 32 weeks, although there was a reduction at 36 weeks of pregnancy.

INTRODUÇÃO: Não há, até o momento, método ideal de avaliação da função renal em fetos com dilatação de vias urinárias. A ultrassonografia é utilizada como um método não invasivo e alguns parâmetros, como o índice de líquido amniótico, foram descritos na predição de insuficiência renal. Entretanto, a sensibilidade é baixa e a detecção de alterações, muitas vezes, ocorre tardiamente na gestação. A avaliação bioquímica da urina ou sangue fetais acrescenta risco à gestação e, apesar de melhorar a detecção de insuficiência renal, tem sensibilidade e especificidade baixas. O Power Doppler tridimensional é um método capaz de quantificar fluxo em órgãos parenquimatosos e tem sido utilizado na quantificação de fluxo sanguíneo de órgãos fetais e placenta. Como fetos com obstrução de vias urinárias e insuficiência renal apresentam diminuição no número de glomérulos, a quantificação do fluxo renal ao Power Doppler tridimensional poderia aprimorar a avaliação da função renal desses fetos. OBJETIVOS: quantificar o fluxo renal ao Power Doppler tridimensional em fetos com suspeita de obstrução de vias urinárias e naqueles com morfologia renal normal, avaliar a influência da profundidade nos índices vasculares e comparar os índices nos fetos que evoluíram com e sem insuficiência renal no período pós-natal. MÉTODOS: fetos com hidronefrose bilateral e/ou dilatação vesical foram prospectivamente comparados com fetos sem malformações em relação à quantificação do fluxo renal ao Power Doppler tridimensional. Os parâmetros avaliados foram IV, IVF, IF e a profundidade. Após o nascimento, as crianças foram seguidas por uma equipe de nefrologia e urologia e, de acordo com a função renal, foram classificadas em 2 grupos: insuficiência renal e função renal normal. A vascularização renal foi avaliada em cada grupo e comparada ao grupo controle. RESULTADOS: vinte e três fetos com dilatação de vias urinárias e setenta e três com morfologia renal normal foram considerados para a análise estatística. Cinco crianças (21,7%) apresentaram insuficiência renal após o nascimento. IV e IVF foram significativamente mais baixos nos casos que apresentaram insuficiência renal do que naqueles com função renal normal (p=0,009 e 0,036, respectivamente). Os três índices corrigidos pela profundidade (IVCP, IFCP e IVFCP) variaram com a idade gestacional e a variação inter-observador melhorou quando eles foram utilizados. A porcentagem do IVCP e do IVFCP em relação à controles de mesma idade gestacional foi menor nos casos que desenvolveram insuficiência renal do que naqueles que evoluíram com função renal normal. CONCLUSÕES: IV e IVF foram significativamente mais baixos em fetos que evoluíram com insuficiência renal pósnatal, porém a profundidade foi um fator interferente importante. Desta forma, IVCP e IVFCP são potencialmente melhores na avaliação de fetos com suspeita de obstrução de vias urinárias. Como os índices corrigidos pela profundidade variam de acordo com a idade gestacional, é necessária a construção de curvas de normalidade por idade gestacional para que os referidos índices possam ser avaliados na prática clínica / INTRODUCTION: There is no ideal method for prenatal evaluation of renal function whether there is a urinary tract dilatation in the fetus. Although ultrasound is a noninvasive method and some parameters have been described to evaluate fetal renal function, as amniotic fluid index, there is a lack of sensitivity to renal failure when ultrasound is used alone. Furthermore, ultrasound changes may appear late in pregnancy. Biochemical evaluation of fetal urine or blood may expose the fetus to some risk, and still lack sensitivity and specificity for renal failure. Threedimensional Power Doppler evaluation has been used to quantify blood flow in fetal organs and placenta. As urinary tract obstruction lead to decrease in renal glomeurli and consequently to a decrease in parenchymal renal flow, three-dimensional quantification of renal flow may improve the evaluation of fetal renal function in fetuses with renal dilatation. OBJECTIVES: To evaluate the ability of threedimensional evaluation of renal vascularization to predict postnatal renal prognosis in fetuses with suspicion of urinary obstruction and to analyze depth influence in vascular indexes. METHODS: Fetuses with bilateral hydronephrosis and/or bladder dilatation had renal vascularization evaluated by three-dimensional ultrasound and VOCAL and were prospectively compared to healthy fetuses. Parameters evaluated were VI, VFI, FI and the distance between the probe and the renal cortex. Follow up by urologists and nephrologists allowed us to allocate these fetuses in two groups: renal impairment and normal renal function. Renal vascularization was evaluated in each group and compared to controls. RESULTS: Twenty-three fetuses with urinary dilatation and seventy-three fetuses with normal renal morphology where considered for statistical analysis. Five fetuses (21,7%) developed renal impairment. VI and VFI where significantly lower in fetuses that developed renal impairment than in those with normal renal function (p=0.009 and 0.036 respectively). Depth-corrected indexes (VIDC, FIDC and VFIDC) varied with gestational age and inter-observer variability was improved when depth was taken into account. The percentage of VIDC and VFIDC of cases in relation to gestational aged matched controls were lower in fetuses that developed post-natal renal impairment than in fetuses with normal renal function. CONCLUSION: Although VI and VFI were significantly lower in fetuses that developed post-natal renal impairment, depth seemed to be an important confounding variable. Thus, VIDC and VFIDC were potentially useful in this context. However, since depth-corrected indexes are related to gestational age, nomograms are needed to further evaluate the role of these parameters in predicting renal impairment

Includes list of papers arising from this thesis. "July 2004" Includes bibliographical references (leaves 253-297)

Posterior urethral valves cause bladder outflow obstruction and damage to the developing fetal kidney. Posterior urethral valves affect 1 in 8000 new-born males. A third of these children develop end stage renal failure by adolescence, despite valve ablation in the early post-natal period, implying that majority of the damage to the kidneys occurs in utero. How does this damage occur, and should we intervene in utero? The answers to these questions require further research, and are the basis to this thesis. This thesis focused on the effect bladder outflow obstruction has on morphology and apoptosis in the fetal kidney in a fetal lamb model. It also looked at the effect of bladder outflow obstruction on fetal stress hormone levels. Bladder outflow obstruction was created surgically in fetal lambs at day 70 of gestation, and fetal kidneys were analysed at day 2, 5, 10, 20 and 30 after creation of obstruction. Controls undergoing sham surgery were used for comparison. Four aspects were investigated: - effects of bladder outflow obstruction on renal histology effects of bladder outflow obstruction on expression of pro-apoptosis gene Bax and anti-apoptosis gene Bcl-X - effects of bladder outflow obstruction on renal regional apoptosis effects of bladder outflow obstruction on serum fetal ACTH and cortisol levels. Bladder outflow obstruction resulted in sequential morphological change in the fetal kidney over time. By 2 days post-obstruction, cystic change was noted. In addition, patchy attenuation of the nephrogenic blastema was evident by 5 days post-obstruction, with more confluent blastemal attenuation as well as generalized renal architectural disorganization by 10 days post-obstruction. By 20 and 30 days post-obstruction, cystic renal dysplasia had developed. Bladder outflow obstruction resulted in an increase in the ratio of renal expression of pro-apoptosis gene Bax to anti-apoptosis gene Bcl-X. Regional apoptosis counts showed increased tubular apoptosis compared to controls at 2 days post-obstruction, and increased blastemal apoptosis compared to controls at 5 days post-obstruction. By 10 days post-obstruction, blastemal apoptosis counts were reduced compared to controls. There were no significant differences in fetal serum ACTH and cortisol levels between fetal lambs with bladder outflow obstruction and controls. In conclusion, the results of this thesis outline the spectrum of morphological change in the fetal kidney over 30 days of bladder outflow obstruction. They show that detectable changes in morphology occur within two days of bladder outflow obstruction. Likewise, detectable changes in gene expression occur within 2 days of bladder outflow obstruction. The increased ratio of expression of Bax to Bcl-X suggests a swing towards increased apoptosis in response to bladder outflow obstruction. Further research is required to ascertain if these changes are reversible. However, the early onset of these changes as shown in this thesis suggests that any fetal intervention to protect the fetal kidney from the effects of bladder outflow obstruction may need to be instituted very early in gestation

In this work, I argue that the Alexandiran position on feticide found in Hellenistic Judaism and appropriated by patristic, medieval and reformed theologians is defensible in the 21st Century. I formulate an argument from the Alexandrian position as it appears in several representative Christian traditions. This argument contends that that: [1] killing a human being without justification violates the law of God, [2] a formed conceptus (i.e. a fetus) is a human being and [3] that in the case of feticide (at least in the majority of cases) no justification is forthcoming. In developing my case, I argue that the objections raised against the premises of this argument by contemporary philosphers are unsound. I defend the intellectual acceptability of belief in and appeals to the existence of a divine law, the notion that a formed fetus is a human being and the claim that feticide lacks any justification in the vast majority of cases. In addition, I examine and critique theologians who claim the Alexandrian position is motivated by misogyny and those who claim it appropriates a translation error found in the Septuagint. I conclude that the traditional position is defensible and that contemporary dismissals of it are unconvincing.

Fetal alcohol spectrum disorders (FASD), caused by maternal alcohol consumption during pregnancy, has been extensively studied in the human. Animal studies show that alcohol exposure during very early development may result in severe brain damage, often incompatible with a postnatal life. However, for surviving offspring it is unknown whether they suffer long term brain damage. The final assembly of the mature brain results from a controlled balance between proliferation of glial and neuronal precursors and programmed cell death. The overall aim of the current study was to use a physiologically relevant mouse model to assess the acute and long-term effects of binge alcohol exposure on the early embryo, to simulate human pregnancy at the third week of gestation when pregnancy may be undetected. A number of paradigms were used to assess the acute dose-response effect, the blood alcohol concentration (BAC) profile and the extent of cell death following alcohol exposure on gestational day (G) 7.5. The exposure paradigms were single binge IG6.5, IG4.5, IP4.5, or an extended binge IG4.5+, IG3.0+. Two control groups were Con6.5 and Con4.5+. Acute cell death was determined using terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), activated caspase-3 staining, and transmission electron microscopy. Cell proliferation was investigated using S-phase immuno-labeling, bromodeoxyuridine (BrdU) birthdating and immuno-detection (BrdU/anti-BrdU). The long-term effects were investigated at G18.5 and postnatal day (PN) 60. Unbiased stereological methods were used to assess the effect of ethanol exposure at G7.5 on neocortical volume, cell number and density of neurons, glial cells, and capillary cells at PN60. The first principal finding of the present study was that binge ethanol exposure during gastrulation resulted in acute apoptotic cell death in the ectoderm of the mouse embryo. Cell death was dependent on both peak BAC and the duration of elevated BAC. Significant increased cell death (TUNEL labeling) was observed in groups IG6.5 (9.43 � 2.08%) and IG4.5+ (8.97 � 2.12%) compared with control groups Con6.5 (2.14 � 0.09%) and Con4.5+ (2.81 � 0.36%). There was no significant increased cell death in ethanol exposed groups IG4.5 (3.43 � 0.45%), IP4.5 (3.68 � 0.67%), or IG3.0+ (1.72 � 0.24%). TEM analysis revealed that cell death exhibited characteristics of the apoptotic pathway. The second principal finding of the present study was that binge ethanol exposure during gastrulation resulted in acute arrested proliferation in the ectoderm of the mouse embryo. The S-phase proliferation was significantly decreased within the whole ectoderm in the ethanol exposed group IG6.5 (45.58 � 2.34%) compared with control group Con6.5 (62.08 � 3.11%). The third principal finding of the present study was that binge ethanol exposure during gastrulation induced the long term effect of laminate disorganization in the neocortex. The incidence of abnormal lamination was 87.5% in IG6.5 compared with 16.7% in IG3.0+ and 14.3% in Con6.5. Although ethanol exposure increased embryonic reabsorption, decreased litter size, and increased abnormal offspring, neocortical volume, and the total number of neurons, glial cells, and capillary cells was not affected. The total number (10⁶) of neurons, glial cells, and endothelial cells respectively was 12.221 � 0.436, 4.865 � 0.167, and 2.874 � 0.234 in IG6.5; 11.987 � 0.416, 4.942 � 0.133, and 2.922 � 0.130 in IG3.0+; and 11.806 � 0.368, 5.166 � 0.267, and 3.284 � 0.217 in controls, at PN60. These results provide important information pertinent to fetal outcome for those women who drink heavily in early pregnancy. The results also demonstrate the importance of the pattern of ethanol exposure and blood alcohol concentration in determining the magnitude of ethanol�s teratogenic impact. Ethanol exposure on G7.5 that resulted in a high transient BAC, induced disorganized neocortical lamination, indicative of a permanent structural change. This disruption may result in altered neocortical function and requires further investigation.

"July, 2001" / Addendum inserted in back. / Includes bibliographical references (leaves 250-284) / xxiii, 284 leaves : ill. (some col.), plates (some col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Surgery, 2001

This research was conducted to test the hypothesis that parenteralarginine supplementation is effective in enhancing birth weights of intrauterinegrowth restricted (IUGR) fetuses. Underfed and prolific ewes were used asexperimental models. The first study characterized the pharmacokinetics ofarginine and citrulline and assessed the potential of citrulline to serve as aprecursor for enhancing arginine availability in fetal and maternal plasma. Sixlate pregnant ewes and their fetuses were instrumented to access arterial andvenous circulations. Intravenous boluses of 155 mol of L-arginine-HCl or Lcitrullineper kg body weight were administered to each ewe. Administration ofcitrulline was more effective than arginine in achieving a sustained increase inconcentrations of arginine in maternal and fetal blood. Accordingly, theclearance rate of citrulline was lower and its biological half-life in maternal bloodgreater, when compared with arginine. The second experiment determined ifadministration of arginine to underfed ewes is effective in ameliorating orpreventing IUGR. Ewes were fed either 100% or 50% of the National ResearchCouncil recommended nutrient requirements for pregnant sheep. Between Day60 of pregnancy and parturition control-fed ewes received saline solution and underfed ewes received either saline solution or L-arginine-HCl solution (155mol of arginine/kg body weight) intravenously three times daily (n=5 / treatmentgroup). Birth weights of lambs were lower in saline-infused underfed ewes.There was no difference in birth weights of lambs from control-fed and argininetreatedunderfed ewes. The third experiment determined whether administrationof arginine could improve survival rates of lambs and enhance fetal growth inewes carrying multiple fetuses. Between Days 100 and 121 of pregnancy, ewesreceived an intravenous infusion of either saline solution (n= 14) or L-arginine-HCl solution (345 mol of arginine/kg body weight, n=20) three times daily.Parenteral administration of arginine increased the percentage of lambs bornalive and enhanced the birth weights of quadruplets. Collectively, these resultsindicate that 1) parenteral administration of arginine improves pregnancyoutcomes in underfed and prolific ewes; and 2) the use of arginine or citrullinemay have important implications for the design of an effective treatment forpreventing or ameliorating IUGR in mammals.

This research was conducted to test the hypothesis that parenteralarginine supplementation is effective in enhancing birth weights of intrauterinegrowth restricted (IUGR) fetuses. Underfed and prolific ewes were used asexperimental models. The first study characterized the pharmacokinetics ofarginine and citrulline and assessed the potential of citrulline to serve as aprecursor for enhancing arginine availability in fetal and maternal plasma. Sixlate pregnant ewes and their fetuses were instrumented to access arterial andvenous circulations. Intravenous boluses of 155 mol of L-arginine-HCl or Lcitrullineper kg body weight were administered to each ewe. Administration ofcitrulline was more effective than arginine in achieving a sustained increase inconcentrations of arginine in maternal and fetal blood. Accordingly, theclearance rate of citrulline was lower and its biological half-life in maternal bloodgreater, when compared with arginine. The second experiment determined ifadministration of arginine to underfed ewes is effective in ameliorating orpreventing IUGR. Ewes were fed either 100% or 50% of the National ResearchCouncil recommended nutrient requirements for pregnant sheep. Between Day60 of pregnancy and parturition control-fed ewes received saline solution and underfed ewes received either saline solution or L-arginine-HCl solution (155mol of arginine/kg body weight) intravenously three times daily (n=5 / treatmentgroup). Birth weights of lambs were lower in saline-infused underfed ewes.There was no difference in birth weights of lambs from control-fed and argininetreatedunderfed ewes. The third experiment determined whether administrationof arginine could improve survival rates of lambs and enhance fetal growth inewes carrying multiple fetuses. Between Days 100 and 121 of pregnancy, ewesreceived an intravenous infusion of either saline solution (n= 14) or L-arginine-HCl solution (345 mol of arginine/kg body weight, n=20) three times daily.Parenteral administration of arginine increased the percentage of lambs bornalive and enhanced the birth weights of quadruplets. Collectively, these resultsindicate that 1) parenteral administration of arginine improves pregnancyoutcomes in underfed and prolific ewes; and 2) the use of arginine or citrullinemay have important implications for the design of an effective treatment forpreventing or ameliorating IUGR in mammals.