published_or_final_version / Paediatrics and Adolescent Medicine / Master / Master of Philosophy

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Doctor of Philosophy / Department of Animal Sciences and Industry / John M. Gonzalez / This series of studies focuses on the impact of intra-litter variation on fetal myogenesis, and the ability of porcine progenitor cells to respond to growth promoting stimuli. In study 1, the smallest (SM), median (ME), and largest (LG) male fetuses from each litter were selected for muscle morphometric analysis from gilts at d-60 ± 2 and 95 ± 2 of gestation. On d-60 and 95 of gestation LG fetuses had greater whole muscle cross-sectional area (CSA) than ME and SM fetuses, and ME fetuses had greater whole muscle CSA than SM fetuses. Indicating that SM and ME fetuses are on a delayed trajectory for myogenesis compared to LG fetuses. At d-60 the advanced trajectory of LG compared to ME fetuses was due to increased development of secondary muscle fibers; whereas, the advanced myogenic development of LG and ME fetuses compared to SM fetuses was due to the presence of fewer primary and secondary muscle fibers. At d-95 of gestation the advanced myogenic development of LG and ME was due to increased hypertrophy of secondary muscle fibers. For study 2, porcine fetal myoblasts (PFM) were isolated from SM, ME, and LG fetuses from d-60 ± 2 of gestation fetuses and for study 3, porcine satellite cells (PSC) were isolated from the piglet nearest the average body weight of the litter. Both myogenic cell types were utilized to evaluate effects of porcine plasma on proliferation, differentiation, and indications of protein synthesis. For the proliferation assay, cells were exposed to one of three treatments: high serum which consisted high-glucose Dulbecco's Modified Eagle Medium supplemented with 10% (vol/vol) fetal bovine serum, 2% (vol/vol) porcine serum, 100 U penicillan/mL, 100 µg of strepmycin/mL, and 20 µg of gentamicin/mL (HS), low serum which consisted of HS without 10% FBS (LS), and LS supplemented with 10% (wt/vol) porcine plasma (PP). Treatments for the differentiation and protein synthesis assays consisted of either HS or LS media that either contained porcine plasma at 10% (wt/vol; PPP) or 0% (wt/vol; PPN). The HS-PFM had a greater proliferation rate compared to the LS and PP-PFM, and PP-PFM had a greater proliferation rate compared to LS-PFM. The LG fetuses’ PFM had a reduced proliferation rate compared to SM and ME fetuses’ PFM, which were similar. The PPP-PFM had a decreased myotube diameter compared to PPN-PFM. Small fetuses’ PFM had a greater myotube diameter compared to ME and LG fetuses’ PFM, and ME fetuses’ PFM had a greater myotube diameter compared to LG fetuses’ PFM. The proliferation rate of PP-PSC was decreased compared to the HS- and LS-PSC, and HS-PSC had a greater proliferation rate compared to LS-PSC. The PPP-PSC had greater differentiation capacity and myotube diameter than PPN-PSC. In conjunction these results indicate divergent myogenic development among different fetal sizes within a litter and suggest that porcine plasma supplementation stimulates myogenic progenitor cell activity in an age specific manner.

In five separate experiments, weight-adjusted doses of TEM, EMS, and ENU were injected intraperitoneally into twelve week-old female mice six hours after mating. On day seventeen of gestation, the females were sacrificed and their uterine contents were examined. The effect of each agent was determined by its ability to cause malformations and death to the developing embryos. All treatment groups showed statistically significant elevated levels of malformations in comparison to their corresponding control groups. The reproductive damage induced in these experiments cannot be singularly attributed to teratogenesis or mutagenesis but a combination of the two.

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

A Distrofia Muscular de Duchenne (DMD) é uma doença genética neuromuscular hereditária, ligada ao cromossomo X, sendo encontrada em humanos do sexo masculino. Esta doença muscular é descrita em outras espécies. O modelo de estudo pré-clínico GRMD (Golden Retrievier Muscular Dystrophy) apresenta sintomas clínicos fenotipicamente característicos da DMD em humanos e, por esta razão, tem sido amplamente utilizado como modelo de estudos pré-clínicos. O objetivo da presente pesquisa foi avaliar o tecido muscular, no modelo canino distrófico, ao longo da gestação. Quatro fêmeas, portadoras do gene distrófico, foram inseminadas com sêmen fresco de cães distróficos. No 25º dia, pós-inseminação, as fêmeas foram submetidas a exames de ultrassonografia para confirmar a gestação. As fêmeas gestantes passaram por uma ovariosalpingohisterectomia (OSH) para a retirada dos embriões e fetos nos seguintes períodos gestacionais: 28º , 33º , 38º e 42º dias. Em seguida fragmentos de tecido muscular foram analisados macroscopicamente e microscopicamente. Para verificar expressões proteicas, amostras de tecido foram submetidas a técnicas imunológicas, e PCR para distrofina, miostatina, e utrofina. Aos, 33º e 38º dias de gestação, no grupo distrófico, foram observadas características teciduais que corroboram com desenvolvimento tardio do tecido muscular. Os resultados para detecção proteica sugerem que, a distrofina, miostatina e utrofina foram expressas igualmente nos grupos controle e distrófico, durante todos os períodos do desenvolvimento gestacional analisado. Por fim, os dados sugerem que animais distróficos apresentam músculo sadio durante a fase gestacional, o que pode ser benéfico para testes farmacológicos em idade precoce. / Duchenne Muscular Dystrophy (DMD) is a hereditary neuromuscular genetic disease linked to the X chromosome, being found in male humans. This muscle disease is described in other species. The pre-clinical GRMD (Golden Retrievier Muscular Dystrophy) study model presents phenotypically characteristic clinical symptoms of DMD in humans and,for this reason, has been widely used as a model for preclinical studies. The aim of the present study was to evaluate the muscular tissue, in the dystrophic canine model, throughout the gestation. Four females, carriers of the dystrophic gene, were inseminated with fresh semen from dystrophic dogs. On the 25th day, post-insemination, the females were submitted to ultrasonography to confirm the pregnancy. The pregnant females underwent an ovariosalpingohisterectomy (OSH) for the removal of the embryos and fetuses in the following gestational periods: 28º, 33º, 38º and 42º days. Then fragments of muscle tissue were analyzed macroscopically and microscopically. To verify protein expression, tissue samples were submitted to immunological techniques, and PCR for dystrophin, myostatin, and utrophin. At the 33 and 38th days of gestation, tissue characteristics were observed in the dystrophic group, which corroborate the late development of muscle tissue. The results for protein detection suggest that dystrophin, myostatin and utrophin were also expressed in the control and affected groups, during all periods of the gestational development analyzed. Lastly, the data suggest that dystrophic animals present healthy muscle during the gestational phase, which may be beneficial for pharmacological tests at an early age.

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.

Introduction: Many medications have an unproven safety profile for use during pregnancy, leading to issues when chronic diseases, such as hypertension and depression, present during pregnancy. The focus of this research programme is to determine whether in utero exposure to antihypertensive and antidepressant medication is associated with increased risk of adverse events at birth, and up to 27 months of age in the child. Methods: Two systematic reviews were performed to identify current published literature and knowledge gaps. Following this, using Scottish healthcare data, a cohort of 268,711 children born 2010-2014 were identified. Following cleaning of the data, multiple imputation was used to account for missing values. Poisson, linear and multinomial regressions were performed to identify the relationship between in utero medication exposure and child outcomes. Results: In utero antihypertensive exposure was associated with preterm birth, low birth weight, small for gestational age, but not developmental issues. However, untreated hypertension was associated with low birth weight, preterm birth, and small for gestational age. In utero antidepressant exposure was associated with preterm birth, low birth weight, small for gestational age, preeclampsia, having a special needs indicator at 10 days and 6-8 weeks post-birth, developmental issues at 27 months Conclusions: This research programme identified several adverse outcomes following in utero exposure to antihypertensive and antidepressant medication.

The aim of this thesis was to examine fetal behavioural responses to auditory and tactile stimulation. Responses were examined for the second- and third-trimester fetuses (second-trimester ≤ 27 gestational weeks (wGA), third- trimester >27 wGA), in both experiments, respectively. Experiment 1 of this thesis examined fetal behavioural responses to the mother’s recorded and live voice, contrasting findings to an environmental sound and silent control conditions. Behavioural responses of 30 fetuses trimester (20-33 wGA, N = 13 in the 2nd and N = 17 in the 3rd trimester) Were examined in the following conditions were explored: (1) mother’s live, (2) and recorded voice, (3) an environmental sound, and (4) a silent control condition. Findings showed the strongest responses to maternal sounds as well as differential responses between gestational age groups. Younger fetuses displayed an arousal response to maternal voice, whereas third-trimester fetuses displayed an orientating response. The aim of Experiment 2 was to examine whether fetuses can differentiate between different human sources of tactile stimulation of the maternal abdomen. Behavioural responses of 28 fetuses (20-33 wGA, N = 15 in the 2nd and N = 13 in the 3rd trimester) were examined across four conditions: (1) mother’s, (2) father’s, and (3) stranger’s touch, as well as a (4) silent control condition. Differential responses to the tactile stimulation were found, especially in reaching of the uterine wall, and self- touch across the four conditions. Third- trimester fetuses touched the uterus wall for significantly longer than fetuses in the second-trimester. The strongest responses were found to the mother’s touch. Further differential responses were found between age groups, with third-trimester fetuses clearly differentiating between different sources of tactile stimulation, while second-trimester fetuses hardly showed differentiated responses. It is suggested that maturational differences in both experiments are due to the fetal development of the central nervous system, and might indicate the emergence of a proprioceptive self-awareness by the 3rd trimester.

Kwan Pui-Chun. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (leaves 140-155). / Abstracts in English and Chinese. / Chapter Chapter 1 --- Introduction / Chapter 1.1 --- Overview --- p.1 / Chapter 1.2 --- Iron homeostasis --- p.5 / Chapter 1.3 --- Natural Resistance Associated Marcophage Protein (Nramp) Family --- p.15 / Chapter 1.4 --- Divalent Metal Transporter 1 (DMT1) --- p.18 / Chapter 1.5 --- Iron Responsive Element (IRE) and Iron Regulatory Protein (IRP) --- p.23 / Chapter 1.6 --- Expression and localization of DMT-1 in human --- p.27 / Chapter 1.7 --- Iron and the developing feus --- p.31 / Chapter 1.8 --- Objectives of the study --- p.36 / Chapter Chapter 2 --- Materials and Method / Chapter 2.1 --- Study population --- p.37 / Chapter 2.2 --- Procedure of surgical termination of pregnancy --- p.38 / Chapter 2.3 --- Tissues collection and preparation --- p.39 / Chapter 2.4 --- Semi-quantitative Reverse Transcription-Polymerase Chain Reaction --- p.44 / Chapter 2.5 --- Immunohistochemistry --- p.49 / Chapter 2.6 --- Statistical analysis --- p.55 / Chapter Chapter 3 --- Results / Chapter 3.1 --- Description of subjects --- p.56 / Chapter 3.2 --- Existence of human DMT-1 isoforms at early pregnancy --- p.58 / Chapter 3.3 --- Relative expression of DMT-1 isoforms to β -actin mRNA expression at different week gestation --- p.67 / Chapter 3.4 --- Cellular localization of DMT-1 isoforms at early pregnancy --- p.91 / Chapter 3.5 --- Relative expression of DMT-1 proteins at early pregnancy --- p.101 / Chapter Chapter 4 --- Discussion / Chapter 4.1 --- Existence of DMT-1 at early pregnancy --- p.116 / Chapter 4.2 --- Expression of DMT-1 isoforms at early pregnancy at gene level --- p.118 / Chapter 4.3 --- Expression of DMT-1 isoforms at early pregnancy at protein level --- p.120 / Chapter 4.4 --- "Comparison expression of DMT-1 between human fetus, human adult and animal studies" --- p.121 / Chapter 4.5 --- Functional importance of DMT-1 at developing fetus at early pregnancy --- p.130 / Chapter 4.6 --- Conclusion --- p.138 / Chapter 4.7 --- Further study --- p.139 / Chapter Chapter 5 --- Reference --- p.140 / Appendix I: Calculation of EM --- p.156