Orientador: Cláudia Maria Bertan Membrive / Banca: Guilherme Pugliesi / Banca: Flávia Lombardi Lopes / Resumo: Mortalidade embrionária em bovinos, entre 15 e 19 dias de prenhez, pode ser promovida pela liberação de PGF2 endometrial. A síntese de PGF2 é inibida pela suplementação com compostos ricos em ácido linoléico, como a semente de girassol. Assim, objetivou-se avaliar o efeito e os mecanismos pelos quais a suplementação com semente de girassol atua na performance reprodutiva de fêmeas bovinas de corte. Hipotetizou-se que tal suplementação promove um incremento na taxa de concepção de receptoras de embriões produzidos in vitro submetidas à TETF e que tal efeito decorra de modificações na composição lipídica plasmática e endometrial, alterações na expressão de transcritos envolvidos na biossíntese de eicosanoides e/ou modificações no número e/ou morfometria das glândulas endometriais. No Experimento 1, novilhas mestiças zebuínas submetidas ao protocolo de TETF suplementadas com semente de girassol, por 22 dias a partir da remoção do dispositivo de progesterona, apresentaram maior taxa de concepção em relação ao grupo controle (55,66% vs. 36,94%; P < 0,01). No Experimento 2, vacas Nelore suplementadas com semente de girassol apresentaram maiores concentrações plasmáticas de colesterol total, LDL e HDL; modificações na composição lipídica endometrial e alterações na expressão de transcritos envolvidos na biossíntese de eicosanoides. Conclui-se que a suplementação com semente de girassol aumenta a taxa de concepção por modificar a composição lipídica plasmática e endometrial, a expressão de transcritos envolvidos na biossíntese de eicosanoides e número... ((Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Embryonic mortality in cattle, between 15 and 19 days of pregnancy, can be promoted by the release of endometrial PGF2. Synthesis of PGF2 is inhibited by supplementation with compounds rich in linoleic acid such as sunflower seed. This study aimed to evaluate the effects and the mechanisms by which supplementation with sunflower seed acts on reproductive performance of beef cows. It was hypothesized that such supplementation promotes an increase on conception rate of in vitro-produced embryos subjected to FTET; and that this effect arises from changes in plasma lipid composition and endometrial changes in the expression of transcripts involved in the biosynthesis of eicosanoids and/or changes in the number and/or endometrial gland morphology. In Experiment 1, zebu crossbred heifers submitted to the FTET protocol supplemented with sunflower seed, for 22 days from the removal of the progesterone device, showed higher conception rate in the control group (55.66% vs. 36.94% ; P < 0.01). In Experiment 2, Nelore cows supplemented with sunflower seed showed higher plasma concentrations of total cholesterol, LDL and HDL, changes in endometrial lipid composition and changes in the expression of transcripts involved in the biosynthesis of eicosanoids. It was concluded that supplementation with sunflower seeds increases the conception rate by modifying the lipid composition of the plasma and the endometrium, the expression of transcripts involved in the biosynthesis of eicosanoids and number and morphometry of the endometrial glands / Mestre

Orientador: Raquel Fantin Domeniconi / Resumo: O ambiente desempenha papel crucial durante o desenvolvimento fetal, podendo influenciar diretamente a saúde da prole. Assim, o estado nutricional materno é essencial para a saúde e bem-estar do feto. Há relatos de alterações em parâmetros relacionados à função epididimária, em animais adultos, cujas mães sofreram restrição de proteína durante as fases de gestação e lactação. A origem das alterações funcionais no epidídimo pode estar relacionada à fatores hormonais, bem como na expressão de seus receptores, e por meio da modulação de moléculas de sinalização como FGFs (Fator de crescimento de Fibroblastos), vias da ERK e Wnt, e à funcionalidade da Src, ou seja, proteínas relacionadas aos processos do desenvolvimento epididimário. No entanto, não há informações que esclareçam as causas dessas alterações ou como a restrição proteica atua ao longo do desenvolvimento pré e pós-natal do epididímo. Desta forma, o objetivo deste trabalho foi avaliar o impacto da restrição proteica materna, no ducto mesonéfrico e no epidídimo nas fases iniciais do desenvolvimento. Assim, ratas Wistar prenhes foram divididas em dois grupos experimentais que receberam uma dieta de normoproteica (NP; 17% de proteína; n=19) ou hipoproteica (HP; 6% de proteína; n=19) durante a gestação e a lactação. Cinco ratas prenhes de cada grupo foram eutanasiadas para coleta dos ductos mesonéfricos dos embriões no dia gestacional (DG)17,5 e as demais permaneceram até os dias pós-natais (DPN) 7 ou 14. Os filhotes mach... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The environment plays a crucial role during fetal development and can directly influence the health of the offspring. Thus, maternal nutritional status is essential for fetus' health and well-being. There have been reports of alterations in parameters related to epididymal function in adult animals whose mothers suffered protein restriction during the gestation and lactation. The origin of the functional alterations in the epididymis can be related to the hormonal factors, as well as in the expression of its receptors, and on signaling molecules modulation like FGFs (Fibroblast Growth Factor), ERK and Wnt pathways, and functionality of Src, or proteins related to epididymal development. However, there is no information to clarify the causes of these changes or how protein restriction acts throughout pre and postnatal development. The aim of this study was to evaluate the impact of maternal protein restriction on the mesonephric duct and in the early stages of epididymal development. Pregnant female Wistar rats were divided into two groups that received either normoprotein (17%; NP) or low-protein (6%; LP) diet ad libitum during gestation and lactation. Five pregnant rats from each group were euthanized to collect the mesonephric ducts on gestational day (DG) 17.5 and the others rats remained until the postnatal days (PND) 7 or PND14. The male offspring had their biometric parameters verified and were euthanized. Blood was collected for sex hormones analysis and genital organs... (Complete abstract click electronic access below) / Doutor

During the last quarter of the twentieth century, there has been a significant expansion in the means by which parents in the United States might use technologies to watch their children. Watching and worrying about children are not new to the job of parenthood, but the ways of watching now available to parents represents a change of degree so great as to represent a change in kind. The parental gaze has become technologized. This dissertation investigates what happens when man-made devices insert themselves into this most basic of human endeavors. Parenting desires, social expectations, and technological capacities have co-evolved in the United States to a point where the norms of parental watching are increasingly technology-based. This is a "mixed methods," cross-case study. It delves into the particulars of three distinct media while looking for patterns of use and effects across the different technologies. The core of this investigation is three case studies of particular surveillance technologies that all came to prominence, in terms of their popularity or frequency of use, in the United States in the last thirty years. The three subjects of these case studies--fetal ultrasound, Eisenberg, Murkoff, and Hathaway's 1984 pregnancy advice and guide book What to Expect When You're Expecting, and baby monitors--are all media that offer parents the opportunity to be better and less anxious parents by enhancing their powers of parenting observation. They form an optical--textual--acoustic triad that demonstrates the breadth of media that are enlisted into surveillance practices. These new anxiety technologies change thinking, perceptions, and attitudes. They serve both to introduce new human capacities and to direct and to mold existing capacities. They have also helped to change our ideas of what is possible. A few overarching characteristics of American parental thinking have helped to pushed surveillance to prominence. Middle class American parents of the last quarter of the twentieth century have come to feel that the world is a more dangerous place for their children. They perceive their offspring as more vulnerable to dangers and as less capable of avoiding these dangers on their own. Parents also feel an increased sense of personal responsibility for the safety of their children. It is not that that contemporary parents have warmer or deeper feelings toward their children, but rather that contemporary parents believe that they both can and should control a much broader range of dangers to their children than parents in the past believed they could control. The "anxiety technologies" of this study serve in part to bring home to their users the riskiness of parenting and the vulnerability of the fetus/infant. These technologies have come to promote responsibility expansion, efficiency orientation, and risk focus for parents. While these technologies do provide parents with a great deal more focused information, many of the perceived enhancements in powers to effect outcomes are presumptive, illusory effects of actual increases in information. Information without influence is as likely to contribute to anxiety as to power.

Fetal testosterone (FT) exposure influences sexual differentiation and may promote well-established sex differences in externalizing (EXT) behavior. Although puberty may be a critical period for these effects, it is unknown how FT exposure influences EXT as a function of pubertal development. We used a longitudinal, multi-sample design to test the relationships between two proxy indices of FT exposure and EXT as a function of age and pubertal development (approximately ages 6, 9, 11, 14, and 16). Twin data were used to approximate FT exposure (TT-FT) because testosterone is thought to cross the intrauterine membrane and cause variability in co-twin gonadal hormone exposure, with increasing exposure for males and participants with male co-twins. Increasing number of older siblings may also approximate increasing FT exposure (SI-FT), although existing research has yet to disentangle possible postnatal socialization effects from potential FT exposure using this variable. Given that biologically related siblings share a fetal and social environment while non-biologically related siblings simply share a social environment, we tested the independent effect of SI-FT on EXT using a sibling adoption design. Across four independent samples, SI-FT and TT-FT predicted externalizing for males alone. SI-FT predicted EXT over-and-above socialization influences and interacted with pubertal development in two independent samples, with elevated EXT for those in mid-late puberty that were exposed to increased FT. TT-FT predicted EXT differentially as a function of developmental period. Our data are consistent with the notion that exposure to FT promotes sexually differentiated, sexually selected behavior during reproductively relevant periods.

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

The fetal lung undergoes extensive physiological and biochemical maturation prior to birth in preparation for its postnatal function as an organ for gas exchange. Pulmonary surfactant, a substance that reduces surface tension and prevents alveolar collapse, is produced by type II pneumocytes within the lung. Reduced ability to produce surfactant leads to neonatal respiratory distress syndrome. Synthesis of the phospholipid component of surfactant, phosphatidylcholine (PC), is stimulated by fibroblast-pneumocyte factor (FPF), a protein expressed by fibroblast cells within the fetal lung. Although its function is well known, the identity of this important protein has remained a mystery. Recent research has suggested that FPF may be neuregulin-1, a growth factor found in many tissues during development. Enhanced synthesis of PC (and therefore detection of FPF) is measured using a tissue culture-based method. Primary cultures of lung fibroblasts and type II pneumocytes are prepared, and fibroblast-conditioned medium (FCM) is exposed to the type II cells. Resultant PC synthesis is measured using radioisotope-labeled PC-precursor and a chloroform-based lipid extraction method. Initial results using this method were very inconsistent, so a study was undertaken to determine which parts of the method could be contributing to this inconsistency. Cell density of type II cultures (measured in μg DNA.plate-1) was shown to have a significant effect on results. Treatment of fibroblasts with 100 nM dexamethasone and exposure of type II cultures to the resultant FCM caused a mean 9.17% increase in PC synthesis, but when only type II cultures with a cell density below 25 μg DNA.plate-1 were analyzed, this value increased to 17.56%. Type II cultures with cell density above this threshold value showed a mean increase in synthesis of only 3.39%. The consistent application of [3H]-choline chloride also had a significant effect on results. Experiments utilizing phorbol 12-myristate 13-acetate to stimulate fibroblasts were very inconsistent. The mean activity of the initial [3H]-choline chloride solution prepared for these experiments was found to be 2.04 μCi.mL-1, compared to a mean of 4.79 μCi.mL-1 for all other experiments. Observations from this section of the study led to considerable revision of the method used to measure PC synthesis. Quadrupolar ion trap mass spectrometry (MS) was used to analyze FCM and determine if neuregulin-1 (NRG1) could be FPF. A mass spectrum was obtained for recombinant NRG1, with predominant ions of 1068, 1142 and 1246 m/z. All three of these ions were also detected in both control and dexamethasone-treated FCM. Partial fragmentation of 1068 m/z of NRG1 was achieved using MS2, and generated a base peak of 1047 m/z. This fragmentation was also observed in 1068 m/z from FCM. LC/MS was utilized to quantify NRG1 in FCM, using a standard curve generated using recombinant NRG1. Control FCM had a NRG1 concentration of 19.85 μg.mL-1, while the concentration in dexamethasone treated FCM was 41.59 μg.mL-1. FCM which had given no positive response to dexamethasone when tested using the indirect cultured cell system had a control NRG1 concentration of 20.85 μg.mL-1, and a dexamethasone treated concentration of 22.84 μg.mL-1. These values were not significantly different from the control value for FCM in those fibroblast cultures that had generated a positive response to dexamethasone. Results of this section of the study have provided strong evidence that NRG1 is a major component of FPF, and a review of the NRG1 signaling pathway further supports this conclusion. Insulin-like growth factors (IGFs) are functionally related to neuregulins and are known to be important in fetal development. The effect of IGF-II on synthesis of surfactant PC and its subsequent secretion from type II pneumocytes was studied. In terms of PC synthesis, IGF-II was tested at concentrations of 0.4, 0.6 and 0.8 μM. The mean increase in synthesis was found to be 6.00, 6.15 and 6.91%, respectively. These values were not significantly different from control values. Secretion of PC was tested over the concentration range of 0.1 to 1.6 μM, with no significant effect observed. Possible inhibition by IGF-II was also studied, using the known stimulants of secretion, neuromedin C and isoproterenol. No significant effect on the enhanced level of secretion was observed when IGF-II was added with either secretagogue. Lack of an appropriate receptor and/or the possibility that cultured cells may not exactly mimic the situation in vivo are probably the reasons IGF-II has no effect on either synthesis or secretion.

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

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.

Gestational diabetes (GDM) is a potentially serious disorder requiring timely diagnosis and management to prevent adverse maternal and fetal outcomes. Of increasing concern today, when treating the woman with GDM, is the need to provide every woman with an intensive management plan to optimise the likelihood of favourable pregnancy outcomes. Early identification of those women with GDM who require insulin therapy in addition to diet therapy would be beneficial in the planning and standardisation of clinical management protocols, to enhance pregnancy outcomes and increase cost benefits with improved allocation of resources. The aim of this study was to evaluate the ability of the fasting plasma glucose level (FPG) at diagnosis to predict an increased risk to the fetus and the need for insulin therapy in a pregnancy complicated by GDM. A prospective longitudinal study design and recruitment by convenience sample was used. Data were obtained from 327 women and their babies. Diagnosis of GDM was made by a 75 gram oral glucose tolerance test (OGTT) using Australasian Diabetes in Pregnancy Society (ADIPS) criteria with the exception of seven women diagnosed on a blood glucose level >11.1mmol/l. Following consent of the women data were collected by a self report questionnaire and the medical record system at three points; at first intervention, following delivery and at the postpartum OGTT. Demographic, social, medical, maternal and neonatal outcome data were collected. The management protocol was similar for all of the women. Following nutritional intervention any woman who could not meet the glycemic targets of <= 5mmol/l fasting and/or <= 6.5mmol/l two hours postprandial was commenced on insulin therapy. The women had a mean age of 32 years, body mass index (BMI) of 25.7 and parity of 2 (range 1-12). Diagnosis was made at an average of 30 weeks and 70 women required insulin therapy with a mean dose of 34 IU per day, commencing at a mean of 31 weeks gestation. Mean birthweight was 3400G. Of the babies 12% were >4000G. Congenital abnormalities occurred in 3%, neonatal morbidities in 2% and there was 1 death in utero. Logistic regression analysis found the following significant associations: Increasing maternal BMI was related to increasing FPG levels at diagnosis and the requirement of higher insulin doses. There was a negative linear relationship to weight gain. Ethnicity was associated with maternal BMI and ethnicity with BMI was associated with birthweight in the specific ethnic group. BMI with insulin therapy as a covariate and the FPG value at OGTT were predictive of persistent glucose intolerance in 14% of women postpartum. Each value of the OGTT was a significant predictor of the need for insulin therapy as a function of the week of gestation. The FPG level was the statistical model of best fit. A 50% probability for requiring insulin was reached with a FPG at diagnosis of 4.0 mmol/l if tested at 10 weeks gestation, 5.1mmol/l at 20 weeks and 6.1 mmol/l at 30 weeks (p<.001). These results support the substantive research aim of the study. The model has the power to predict the probability (risk) of requiring insulin therapy based on the maternal FPG level at the OGTT according to the week of gestation. The study results demonstrate that glucose intolerance is linked to a number of adverse maternal and fetal outcomes in a continuous and graded fashion. The degree of reversibility of maternal and fetal risk through therapeutic interventions such as nutrition therapy, blood glucose monitoring, exercise and active patient participation aimed at improving glucose tolerance is unknown. Therefore, the rationale for, and feasibility of, new treatment strategies such as the application of this statistical model as a management approach require large scale randomised intervention studies, oriented toward measuring maternal and fetal outcomes amongst different populations.

<p>Environmental stress during embryonic development could lead to growth restriction of the embryo, and act as a risk factor for the development of cardiovascular disease in adult life. A common environmental stressor that causes growth restriction is prenatal hypoxia, which has been shown to adversely affect adult health in mammalian models. Prenatal hypoxia causes an increase in catecholamines which results in over stimulation of the cardiac β-adrenergic receptors. Previous work on chickens has shown that prenatal hypoxia causes an increase in the sensitivity of β-adrenergic receptors to epinephrine in the embryonic heart. The sensitivity of these receptors was found to be decreased in prenatal hypoxic juvenile. Prenatal hypoxia has no significant effect on the density of these receptors in neither the embryo nor the juvenile. The lack of change in receptor density implies that the effects of hypoxia are further down stream in the signalling cascade. The β2 adrenergic receptor can couple to both the stimulatory Gα subunit (Gsα) and the inhibitory Gα subunit (Giα). We hypothesized that prenatal hypoxia would cause an increase in the Gsα in the sensitized embryos, while increasing Giα in the desensitized juveniles. This study evaluated the relative levels of Gsα and Giα in the hypoxic chicken embryo, and in the prenatally hypoxic juvenile, Using western blotting. Hypoxia considerably increased Giα in the chicken embryo while having no effect on Gsα. In the prenatally hypoxic juvenile Gsα was significantly increased while no changes were found in Giα. This dissociation between the levels of Gα subunit and receptor sensitivity implies that that hypoxia affects the signaling cascade downstream of the Gα subunit.</p>