Neste trabalho foi proposto o estudo prospectivo de gestações após a 40a semana, objetivando: a) verificar os índices de morbidade e mortalidade perinatais; b) identificar os testes de avaliação da vitalidade fetal mais adequados para a vigilância destas gestações; c) comparar os resultados dos testes de avaliação da vitalidade fetal com os resultados perinatais na 1a e 2a semana, após a 40a semana de gestação, para testar o protocolo do Serviço. Para a realização do estudo, selecionaram-se 52, gestantes divididas em 2 grupos: GI (1a semana) com 32 gestantes e GIl (2a semana) com 20 gestantes. Acompanhou-se a vitalidade fetal com a Cardiotocografia de Repouso e Intraparto, Teste da Estimulação Sônica, Avaliação do Volume do Líquido Amniótico através da Técnica dos Quatro Quadrantes, Perfil Biofisico Fetal e Dopplerfluxometria Uterina e Umbilical, realizados 2 vezes na semana. Os resultados neonatais e os índices de morbidade foram: Índices de Apgar no 10 e 50 minutos (alterados < 7), pH da artéria umbilical (alterado < 7,20), peso dos recém-nascidos, tempo de internação dos recém-nascidos, oligoidrâmnia, líquido amniótico meconial, alterações na cardiotocografia com presença de desacelerações e índices de cesárea. O estudo permitiu como resultados e conclusões: a) a incidência de oligoidrâmnio foi de 44,23%, líquido meconial de 28,85%, cardiotocografia alterada, 50,00% e partos cesáreos, 57,70%, não havendo óbito fetal ou neonatal. Não houve alterações significativas nos índices de Apgar, pH da artéria umbilical e tempo de internação dos recém-nascidos. b) A cardiotocografia e, principalmente, a avaliação do volume do líquido amniótico, pelo índice de líquido amniótico, foram os métodos mais adequados na detecção de alterações verificadas neste grupo de gestantes. Do parâmetro ultra-sonográfico do Perfil Biofisico Fetal, apenas o Volume do Líquido Amniótico demonstrou ser importante. A Dopplerfluxometria (uterina e umbilical) não revelou nenhuma utilidade na vigilância destas gestações. c) A distribuição dos casos com oligoidramnia, líquido meconial, cardiotocografia alterada, índices de cesárea e pH da artéria umbilical < 7,20, semelhante na 41a semana (GI) e 42a semana (GIl), valida o protocolo do Serviço. d) Adicionalmente, este estudo permite ainda as seguintes observações: 1) importância da oligoidrâmnia e líquido meconial na determinação dos elevados índices de cesáreas; 2) incidência elevada (50,00) de nulíparas nesta casuística. / This study proposed pros.pectively the evaluation of the gestations after 40 weeks with these objectives: a) Analysis of the perinatal outcome. b) Identification of the proper test for fetal well-being assessment for this gestation. c) Comparation of these tests results with perinatal outcome at the first and second weeks after 40 weeks, therefore, testing the protocol of this Service. It recruited 52 patients divided in two groups: GI (1st week) with 32 patients and Gil (2nd week) with 20 patients. The fetal surveillance was assessed by antepartum and intrapartum cardiotocography, acoustic stimulation test, amniotic fluid volume assessment by the ultrasonographic four quadrant technique (amniotic fluid index), fetal biophysical profile and umbilical and uterine doppler velocimetry, ali tests were performed twice weekly. The neonatal outcome results and morbidity parameters were: Apgar index in 1st and s\" minutes (alterated < 7), umbilical artery pH (alterated < 7,20), the new born weight, oligohydramnios, meconium stained, deceleration (DIP 11 or umbilical deceleration) and cesarean section rates. The study permitted these results and conclusions: a) The oligohydramnios, meconium stained, cardiotocography alterations and cesarean section incidences were 44,23%, 28,85%, 50,00% and 57,70%, respectively. There was no fetal death. b) The cardiotocography and amniotic fluid assessment by the amniotic fluid index, were the best tests to detect the alterations verified. The amniotic fluid volume was the most important parameter in the fetal biophysical profile. Doppler (uterine and umbilical) revealed no utility. c) The equal distribution of the oligohydramnios, meconium stained, altereted cardiotocography, cesarean section and umbilical artery pH < 7,20 cases in the group studied reassure the Service protocol. d) In addition this study also permitted observation of: 1) The importance of the meconium stained in the cesarean section rate. 2) The nuliparus elevated incidence (50,00%) in this group.

Made available in DSpace on 2017-07-10T14:17:09Z (GMT). No. of bitstreams: 1 Heloisa Confortim.pdf: 5400864 bytes, checksum: 6b96ff3a1a320e827bc7c09adf7eed57 (MD5) Previous issue date: 2014-02-14 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Inadequate maternal nutritional may predispose to neuromuscular disorders in the offspring, a phenomenon known as fetal programming. In this context, the present study aimed to evaluate the effects of maternal protein restriction (during pregnancy and lactation) on muscle fibers and neuromuscular junctions (NMJs) of the soleus muscle from pups at 21 and 365 days old. Male Wistar rats were divided in two experimental groups: Control (CG) - offspring of mothers fed a normal protein diet (17%) and restricted (RG) - offspring of mothers fed a low protein diet (6%). All pups were maintained with their mothers during the lactation period (21 days) and after weaning, one part of males from each group were euthanized to collect samples of the soleus muscle. The remaining rats received standard food ad libitum until 365 days, when they were also euthanatized. The samples of the soleus muscle from animals with 21 days old were collected for analysis of muscle fibers (by HE staining and ultrastructure) and NMJs (by Nonspecific Esterase technique). In animals with 365 days of age, soleus muscle samples were obtained for verification of muscle fibers (by HE staining, NADH-TR reaction and ultrastructure), quantification of intramuscular collagen (picrosirius red staining) and also for analysis of NMJs (Nonspecific Esterase technique). Regarding the results, at 21 days muscle fibers was immature and the presence of myotubes, central nuclei, fetal fibres and fibroblasts were observed in both experimental groups. An increase in the number of muscle fiber and nuclei in the RG compared to controls was observed. Muscle fibers with rarefied or loosely arranged myofibrils, Z-line disorganized and nuclei in central position were observed in CG and RG. Regarding the NMJs, the RG showed a decreased in area, larger and smaller diameter compared to the CG. At 365 days, the RG showed a decrease in the cross sectional area in type I and IIa fibers, associated with an increase in type IIb fibers. The percentage of intramuscular collagen was lower in RG. Myofibrils and Z line disorganization was also observed at ultrastructural level, with the presence of mitochondria clusters in both groups studied. A reduction in the area and smaller diameter of NMJs was observed in the GR, along with an increase in the larger diameter of these structures compared to CG. These results indicate that maternal protein restriction affects the morphology and morphometry of the neuromuscular junctions and muscle fibers. Such changes can be detected early and persist throughout adulthood and senescence, seeming irreversible / Condições nutricionais maternas inadequadas podem predispor ao aparecimento de alterações neuromusculares na prole, um fenômeno conhecido como programação fetal. Nesse contexto, o presente estudo teve como objetivo avaliar os efeitos da restrição proteica materna, durante os períodos gestacional e de lactação, sobre as fibras musculares e junções neuromusculares (JNMs) do músculo sóleo dos filhotes aos 21 e 365 dias de idade. Ratos Wistar machos foram separados em dois grupos experimentais: Controle (GC) - prole de mães alimentadas com ração normoproteica (17%) e restrito (GR) - prole de mães alimentadas com ração hipoproteica (6%). Os filhotes foram mantidos com a mãe durante o período de lactação (21 dias) e, após o desmame, parte dos machos de cada grupo foi eutanasiado para a coleta de amostras do músculo sóleo. Os demais filhotes receberam ração padrão ad libitum até os 365 dias, quando também foram eutanasiados. Amostras do músculo sóleo dos animais com 21 dias de idade foram coletadas para análise das fibras musculares (através da coloração em HE e ultraestrutura) e das JNMs (técnica de Esterase-Inespecífica). Nos animais com 365 dias de idade, amostras do músculo sóleo foram obtidas para análise das fibras musculares (através da coloração em HE, reação de NADH-TR e ultraestrutura), quantificação de colágeno intramuscular (coloração Picrosirius-red) e também para a análise das JNMs (técnica de Esterase-Inespecífica). Quanto aos resultados, aos 21 dias de idade as fibras musculares apresentaram-se imaturas, com a presença de miotubos, núcleos centrais, fibras fetais e fibroblastos nos dois grupos experimentais. Foi observado um aumento na quantidade de fibras musculares e de núcleos no GR em comparação ao GC. Fibras musculares com miofibrilas rarefeitas ou frouxamente arranjadas, linha Z desorganizada e núcleos em posição central também foram observadas nos GC e GR. Em relação as JNMs, o GR apresentou redução na área, diâmetro maior e menor quando comparadas ao GC. Aos 365 dias, o GR apresentou redução na área da secção transversal das fibras tipo I e IIa, além de um aumento nas fibras tipo IIb. A porcentagem de colágeno intramuscular foi menor no GR. Também foi observada uma desorganização das miofibrilas e da linha Z em nível ultraestrutural, com a presença de aglomerados de mitocôndrias nos dois grupos estudados. Quanto às JNMs, foi observada uma redução na área e diâmetro menor, além de um aumento no diâmetro maior no GR comparado ao GC. Estes resultados indicam que a restrição proteica materna afeta a morfologia e morfometria das fibras musculares e JNMs. Tais alterações são detectáveis precocemente e perduram ao longo da vida adulta e senescência, parecendo ser irreversíveis

Fong, Oi Ning. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 170-195). / Abstracts in English and Chinese. / Abstract --- p.i / Acknowledgements --- p.vi / Contents --- p.viii / List of Abbreviations --- p.xii / Chapter CHAPTER ONE --- Introduction --- p.1 / Chapter 1.1 --- Bacterial Infection in Neonates --- p.1 / Chapter 1.2 --- Gram-positive and Gram-negative Bacterial Cell Wall --- p.3 / Chapter 1.2.1 --- Gram-negative Bacterial Cell Wall Component - Lipopolysaccharide --- p.3 / Chapter 1.2.2 --- Gram-positive Bacterial Cell Wall Component - Peptidoglycan --- p.4 / Chapter 1.3 --- Differential Host Response against Gram-specific Bacterial Infection --- p.6 / Chapter 1.4 --- Role of Neutrophils in Host Defense against Bacterial Infection --- p.8 / Chapter 1.4.1 --- Recognition of Bacterial Components --- p.9 / Chapter 1.4.2 --- Neutrophil Functions --- p.10 / Chapter 1.5 --- Expression Profiling of Activated Neonatal Neutrophils --- p.15 / Chapter CHAPTER TWO --- Objectives --- p.26 / Chapter CHAPTER THREE --- Materials and Methodology --- p.27 / Chapter 3.1 --- Overview of the Experimental Procedure --- p.27 / Chapter 3.2 --- Cord Blood Sample Collection --- p.28 / Chapter 3.3 --- Cord Blood Neutrophil Isolation --- p.30 / Chapter 3.3.1 --- Isolation of Neutrophils --- p.30 / Chapter 3.3.2 --- Analysis of Neutrophil Purity by Flow Cytometry --- p.31 / Chapter 3.3.3 --- Cell Viability Test by Trypan Blue Exclusion Assay --- p.31 / Chapter 3.4 --- In Vitro Stimulation of Neutrophils by LPS or PGN --- p.33 / Chapter 3.5 --- Total RNA and Protein Isolation --- p.34 / Chapter 3.5.1 --- Total RNA Isolation --- p.34 / Chapter 3.5.2 --- Protein Isolation --- p.35 / Chapter 3.6 --- Preparation of Total RNA Samples for Expression Profiling and Quantitative Real Time Polymerase Chain Reaction (qPCR) --- p.37 / Chapter 3.6.1 --- DNase Treatment --- p.37 / Chapter 3.6.2 --- Total RNA Cleanup --- p.37 / Chapter 3.6.3 --- Purity Assessment of the Purified Total RNA Sample --- p.38 / Chapter 3.6.4 --- Integrity Assessment of the Purified Total RNA Sample --- p.39 / Chapter 3.6.5 --- Assessment of Tumor Necrosis Factor Alpha (TNF-α) mRNA Expression Level in Neutrophils --- p.42 / Chapter 3.7 --- Determination of the PGN Concentration for Neutrophil Activation --- p.43 / Chapter 3.8 --- "Expression Profiling of the LPS, PGN Stimulated or Unstimulated CB Neutrophils" --- p.44 / Chapter 3.8.1 --- cRNA Preparation and Array Hybridization --- p.44 / Chapter 3.8.2 --- Expression Profiling Data Analysis --- p.46 / Chapter 3.9 --- Validation of Candidate Genes Using qPCR --- p.48 / Chapter 3.10 --- Gram-Negative Bacterial Endotoxin Assay --- p.50 / Chapter CHAPTER FOUR --- LPS Stimulation Induced Transcriptional Changes in Cord Blood Neutrophils --- p.61 / Chapter 4.1 --- Result --- p.61 / Chapter 4.1.1 --- Gene Expression Profile of CB Neutrophils in Response to LPS Stimulation --- p.61 / Chapter 4.1.1.1 --- Up-regulated Genes in LPS-stimulated CB Neutrophils --- p.61 / Chapter 4.1.1.2 --- Down-regulated Genes in LPS-stimulated CB Neutrophils --- p.62 / Chapter 4.1.1.3 --- Network Analysis of Genes Induced by LPS Stimulation --- p.63 / Chapter 4.2 --- Discussion --- p.64 / Chapter 4.2.1 --- Robust Transcriptional Response in CB Neutrophils --- p.64 / Chapter 4.2.2 --- LPS Modulated Transcriptional Responses --- p.64 / Chapter 4.2.2.1 --- LPS-induced NF-kB Pathway --- p.64 / Chapter 4.2.2.2 --- LPS-induced Expression of Various Transcription Factors --- p.66 / Chapter 4.2.2.3 --- LPS-induced Regulation of Apoptosis --- p.67 / Chapter CHAPTER FIVE --- PGN Stimulation Induced Transcriptional Changes in Cord Blood Neutrophils --- p.83 / Chapter 5.1 --- Result --- p.83 / Chapter 5.1.1 --- Gene Expression Profile of PGN-stimulated CB Neutrophils --- p.83 / Chapter 5.1.2 --- Up-regulated Genes in PGN-stimulated CB Neutrophils --- p.83 / Chapter 5.1.3 --- Down-regulated Genes in PGN-stimulated CB Neutrophils --- p.84 / Chapter 5.1.4 --- Network Analysis of Genes Induced by PGN Stimulation --- p.84 / Chapter 5.2 --- Discussion --- p.86 / Chapter 5.2.1 --- Robust Transcriptional Response in CB Neutrophils --- p.86 / Chapter 5.2.2 --- PGN Modulated Transcriptional Responses --- p.86 / Chapter 5.2.2.1 --- PGN-induced NF-kB Pathway --- p.86 / Chapter 5.2.2.2 --- Possible Role of STAT3 in PGN-stimulated CB Neutrophil --- p.89 / Chapter 5.2.2.3 --- Possible Role of c-Jun in PGN-stimulated CB Neutrophil --- p.90 / Chapter CHAPTER SIX --- Comparison and Validation of LPS- and PGN-activated Transcriptomes in Cord Blood Neutrophils --- p.106 / Chapter 6.1 --- Result --- p.106 / Chapter 6.1.1 --- Comparison of the Transcriptional Changes of LPS- and PGN- stimulated CB Neutrophils --- p.106 / Chapter 6.1.2 --- Common Transcriptional Changes of LPS- and PGN-Stimulated CB Neutrophils --- p.106 / Chapter 6.1.2.1 --- Commonly Up-regulated Genes in LPS- and PGN- Stimulated Neutrophils --- p.107 / Chapter 6.1.2.2 --- Commonly Down-regulated Genes in LPS- and PGN- Stimulated Neutrophils --- p.107 / Chapter 6.1.2.3 --- Network Analysis of Genes Commonly Regulated by LPS and PGN --- p.108 / Chapter 6.1.3 --- Differential Transcriptional Changes of LPS- and PGN- Stimulated CB Neutrophils --- p.108 / Chapter 6.1.4 --- Real Time qPCR Validation of the Expression Levels of Selected Genes --- p.109 / Chapter 6.1.5 --- Expression Changes of the Confirmed Target Genes in Response to High-dose LPS Stimulation --- p.110 / Chapter 6.2 --- Discussion --- p.111 / Chapter 6.2.1 --- Activation of NF-kB and Related Genes by Both LPS- and PGN-stimulation in CB Neutrophils --- p.111 / Chapter 6.2.2 --- Commonly Expressed Genes - Transcription Factor MAFF --- p.112 / Chapter 6.2.3 --- Commonly Expressed Genes - Novel Gene G0S2 --- p.113 / Chapter 6.2.4 --- Suspected Commonly Expressed Genes - Transcription Factor NR4A3 --- p.114 / Chapter 6.2.5 --- Differentially Expressed Genes - Heat Shock Proteins --- p.115 / Chapter 6.2.6 --- Differentially Expressed Genes 226}0ؤ AP-1 Transcription Factor Complex --- p.118 / Chapter 6.2.7 --- Other Differentially Expressed Genes --- p.121 / Chapter CHAPTER SEVEN --- General Discussion and Conclusion --- p.164 / Bibliography --- p.168

Zhang, Siwei. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 87-98). / Abstracts in English and Chinese. / Chapter 1. --- General introduction --- p.1 / Chapter 1.1. --- "Types, structure and function of human hemoglobin" --- p.1 / Chapter 1.1.1. --- Structure and functions of human hemoglobin --- p.1 / Chapter 1.1.2. --- Types of human hemoglobin --- p.2 / Chapter 1.2. --- Regulatory mechanism of human hemoglobin expression --- p.3 / Chapter 1.2.1. --- The human a and β locus --- p.3 / Chapter 1.2.2. --- Development of globin genes switching concept --- p.4 / Chapter 1.2.3. --- Factors that regulate globin gene expression --- p.5 / Chapter 1.2.3.1. --- The locus control region (LCR) --- p.5 / Chapter 1.2.3.2. --- The cis-regulatory elements --- p.5 / Chapter 1.2.3.3. --- The trans-acting factors --- p.6 / Chapter 1.3. --- The human hemoglobinopathies --- p.8 / Chapter 1.3.1. --- α-thalassemia --- p.8 / Chapter 1.3.2. --- β-thalassemia --- p.9 / Chapter 1.3.3. --- Sickle cell anemia --- p.10 / Chapter 1.4. --- Current approaches towards β-thalassemia treatment --- p.11 / Chapter 1.4.1. --- Blood transfusion --- p.11 / Chapter 1.4.2. --- Bone marrow transplantation --- p.12 / Chapter 1.4.3. --- Drug-induced activation of fetal hemoglobin production --- p.12 / Chapter 1.4.3.1. --- Hydroxyurea --- p.12 / Chapter 1.4.3.2. --- Butyrate and short-chain fatty acids --- p.13 / Chapter 1.4.3.3. --- "Mutagens, DNA methyltransferase inhibitors and other HbF inducible agents" --- p.13 / Chapter 1.4.3.4. --- Cucurbitacin D --- p.14 / Chapter 1.4.4. --- Gene therapy --- p.14 / Chapter 1.5. --- Research Objectives --- p.15 / Chapter 2. --- "Analysis of CuD, Hydroxyurea and other inducers on the induction of α, β, γ, δ， ε，ζ BP-1 genes and fetal hemoglobin induction" --- p.16 / Chapter 2.1. --- Introduction --- p.16 / Chapter 2.1.1. --- Properties of human K562 cell line --- p.16 / Chapter 2.1.2. --- Induction and measurement of fetal hemoglobin --- p.16 / Chapter 2.1.3. --- "Induction of α, β, γ, δ, ε , ζ and BP-1 gene and Real-time RT-PCR analysis" --- p.17 / Chapter 2.2. --- Materials --- p.18 / Chapter 2.2.1. --- Chemicals and reagents --- p.18 / Chapter 2.2.2. --- Kits --- p.19 / Chapter 2.2.3. --- Buffers and solutions --- p.19 / Chapter 2.2.4. --- Cell lines --- p.20 / Chapter 2.3. --- Experimental procedures --- p.20 / Chapter 2.3.1. --- Hemoglobin quantity measurement by HbF ELISA --- p.20 / Chapter 2.3.1.1. --- MTT assay --- p.21 / Chapter 2.3.1.2. --- Preparation of capture-antibody coated ELISA plates --- p.21 / Chapter 2.3.1.3. --- Plate blocking --- p.22 / Chapter 2.3.1.4. --- Sample and standard preparation --- p.22 / Chapter 2.3.1.5. --- HRP antibody and colorimetric detection --- p.23 / Chapter 2.3.1.6. --- Statistical analysis --- p.23 / Chapter 2.3.2. --- Preparation of mRNA extract from K562 cells --- p.23 / Chapter 2.3.3. --- Reverse transcription and Real-time PCR analysis --- p.24 / Chapter 2.4. --- Results --- p.25 / Chapter 2.4.1. --- CuD significantly upregulates HbF expression in K562 cells --- p.25 / Chapter 2.4.2. --- "CuD augments α, β, γ, δ, ε , ζ and BP-1 genes at different level in K562 cells" --- p.28 / Chapter 2.4.3. --- Cucurbitacin D-induced γ-globin gene activation requires12-24 hours in K562 cells --- p.31 / Chapter 2.5. --- Discussion --- p.33 / Chapter 2.5.1. --- Enhancement of fetal hemoglobin production using different chemical compounds --- p.33 / Chapter 2.5.2. --- CuD increased HbF synthesis by increasing γ-globin mRNA amount --- p.35 / Chapter 2.5.3. --- CuD and HU down-regulated the BP-1 gene expression --- p.36 / Chapter 3. --- Determination of potential signal transduction pathways during CuD and HU-mediated fetal hemoglobin production --- p.36 / Chapter 3.1. --- Introductions --- p.36 / Chapter 3.1.1. --- The p38 MAPK family --- p.37 / Chapter 3.1.2. --- The JAK2-STAT3 pathway --- p.38 / Chapter 3.1.3. --- Fundamentals on inhibition assay of p38 MAPK and JAK2-STAT3 pathway --- p.39 / Chapter 3.1.4. --- Fundamentals on nuclear translocation of STAT3 --- p.41 / Chapter 3.2. --- Materials --- p.41 / Chapter 3.2.1. --- Chemicals and reagents --- p.41 / Chapter 3.2.2. --- Kits --- p.44 / Chapter 3.2.3. --- Buffers and solutions --- p.44 / Chapter 3.3. --- Experimental procedures --- p.45 / Chapter 3.3.1. --- Detection of p3 8 MAPK phosphorylation status --- p.46 / Chapter 3.3.1.1. --- Preparation of cytosolic protein extracts --- p.46 / Chapter 3.3.1.2. --- Quantitative measurement of phospho-p38 and pan-p38 by ELIS A method --- p.46 / Chapter 3.3.1.2.1. --- Antigen adsorption and establishment of standard curves --- p.46 / Chapter 3.3.1.2.2. --- Plate washing and application of detection antibody --- p.47 / Chapter 3.3.1.2.3. --- Plate washing and application of secondary antibody --- p.47 / Chapter 3.3.1.2.4. --- Plate washing and chromogen detection --- p.48 / Chapter 3.3.2. --- Detection of signal cascade on JAK2-STAT3 pathway --- p.48 / Chapter 3.3.2.1. --- Preparation of cytosolic protein extracts for Western Blot detection --- p.48 / Chapter 3.3.2.2. --- Gel running and Western Blot detection --- p.48 / Chapter 3.3.3. --- Quantitative measurement of phospho-STAT3-Tyr705 using ELISA method --- p.50 / Chapter 3.3.3.1. --- Preparation of cytosolic protein extracts --- p.50 / Chapter 3.3.3.2. --- Reconstitution and Dilution of STAT3 [pY705] Standard --- p.50 / Chapter 3.3.3.3. --- Measurement of STAT3 [pY705] concentration in cell lysates --- p.51 / Chapter 3.3.4. --- Inhibitor assay of JAK2-STAT3 and p38 MAPK pathway --- p.52 / Chapter 3.3.4.1. --- Establishment of inhibitor assay --- p.52 / Chapter 3.3.4.2. --- HbF ELISA detection --- p.53 / Chapter 3.3.5. --- Detection of STAT3 nuclear translocation and DNA binding affinity --- p.53 / Chapter 3.3.5.1. --- Preparation of nuclear extract from K562 cells --- p.53 / Chapter 3.3.5.2. --- EMS A detection of transcriptional factors binding to γ-promoter region --- p.54 / Chapter 3.3.5.2.1. --- 3´ة end-labeling of EMS A probes --- p.54 / Chapter 3.3.5.2.2. --- Dot blotting for labeling efficiency estimation --- p.56 / Chapter 3.3.5.2.3. --- EMSA binding reaction and non-denaturing gel electrophoresis --- p.57 / Chapter 3.3.5.2.4. --- Membrane development and chemiluminescence detection --- p.58 / Chapter 3.3.5.3. --- Preparation of K562 samples for immunofluorescence detection --- p.60 / Chapter 3.3.5.3.1. --- Slide coating for cell capture --- p.60 / Chapter 3.3.5.3.2. --- Preparation of cell slide --- p.60 / Chapter 3.3.5.3.3. --- Sample fixation and antibody probing treatment --- p.60 / Chapter 3.3.5.3.4. --- Sample imaging and immunofluorescence detection --- p.61 / Chapter 3.4 --- Results --- p.62 / Chapter 3.4.1. --- Activation of p38 MAPK pathway and STAT3 phosphorylation by hydroxyurea --- p.62 / Chapter 3.4.1.1. --- "The p38 MAPK pathway is activated by hydroxyurea, but not activated by Cucurbitacin D" --- p.62 / Chapter 3.4.1.2. --- Increased p38 phosphorylation level elicits STAT3 phosphorylation at Ser727 site --- p.64 / Chapter 3.4.2. --- Activation of JAK2 and STAT3 phosphorylation by Cucurbitacin D --- p.66 / Chapter 3.4.2.1. --- Cucurbitacin D promotes JAK2 activation --- p.66 / Chapter 3.4.2.2. --- Cucurbitacin D and hydroxyurea promote STAT3 phosphorylation at Tyr705 site --- p.66 / Chapter 3.4.3. --- Basal activity of signal transduction pathways is essential for HbF induction --- p.69 / Chapter 3.4.3.1. --- Activation of γ-globin gene requires presence of basal phosphorylation level of p38 MAPK --- p.69 / Chapter 3.4.3.2. --- Inhibition on JAK2-STAT3 pathway results in reduced fetal hemoglobin production --- p.71 / Chapter 3.4.4. --- Translocation and DNA binding of STAT under Cucurbitacin D induction --- p.72 / Chapter 3.4.4.1. --- Cucurbitacin D and hydroxyurea both enhance binding affinity of transcriptional factors to the Gγ/Aγ promoter --- p.72 / Chapter 3.4.4.2. --- Cucurbitacin D and hydroxyurea induces nuclear translocation of STAT3 --- p.75 / Chapter 3.5. --- Discussion --- p.77 / Chapter 3.5.1. --- The role of p38 MAPK activation during γ-globin gene activation --- p.77 / Chapter 3.5.2. --- STAT3 phosphorylation at Ser727 site promotes transcription factor activity and γ-globin gene expression --- p.77 / Chapter 3.5.3. --- The role of JAK2-STAT3 activation during γ-globin gene activation --- p.78 / Chapter 3.5.4. --- Inhibitor assay --- p.79 / Chapter 3.5.5. --- Relations between STAT3 nuclear translocation and enhanced fetal hemoglobin production --- p.82 / Chapter 4. --- Summery and Prospect --- p.83 / Chapter 5. --- References --- p.87

Os efeitos da glicemia materna na regulação da freqüência cardíaca fetal (FCF) constituem aspecto controverso na literatura, principalmente em gestações complicadas pelo diabetes mellitus. O objetivo deste trabalho foi estudar a influência da glicemia materna na FCF analisada pela cardiotocografia computadorizada. Método: Trinta e nove gestantes com diabetes mellitus pré-gestacional foram avaliadas prospectivamente na Clínica Obstétrica do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo, no período compreendido entre julho de 2003 e fevereiro de 2005. As pacientes incluídas possuíam o diagnóstico de diabetes pré-gestacional, gestação única, idade gestacional entre 36 e 40 semanas e ausência de malformações fetais. Para a realização do estudo, cada paciente foi avaliada pela cardiotocografia computadorizada, durante uma hora, sendo analisados os seguintes parâmetros da FCF: freqüência basal, tempo para atingir os critérios de normalidade, freqüência de movimentos fetais, número de contrações uterinas, número de acelerações e desacelerações, episódios de alta e baixa variação, variação de curto prazo. Realizou-se a glicemia capilar imediatamente antes do início da cardiotocografia, 30 e 60 minutos após o começo do exame. Utilizou-se a média glicêmica para análise das relações com os achados cardiotocográficos, com os valores de corte de 100 mg/dL e 120mg/dL. Resultados: Do total de 39 pacientes analisadas, 25 (64,1%) apresentavam média glicêmica &#8805; a 100 mg/dL e 19 (48,7%) &#8805; a 120 mg/dL. A média da FCF mostrou aumento significativo nos grupos com a média glicêmica &#8805; a 100 mg/dL (p<0,05) e a 120mg/dL (p<0,05). Houve correlação positiva significativa (p<0,05 e r=0,57) da FCF com a média glicêmica no exame. Verificou-se correlação negativa significativa (p<0,05) da quantidade de acelerações transitórias acima de 10 bpm (r=-0,32) e de 15 bpm (r=-0,44) com a média glicêmica no exame. A variação de curto prazo da FCF apresentou associação significativa (p<0,05) com a média glicêmica acima de 120mg/dL. Ocorreu correlação negativa significativa (p<0,05) da variação de curto prazo da FCF (r=-0,47) com a média glicêmica no exame. Os demais parâmetros avaliados pela cardiotocografia computadorizada não mostraram diferença significativa com a média glicêmica. Conclusão: os níveis glicêmicos maternos, durante o exame, exercem influência sobre parâmetros da FCF analisada pela cardiotocografia computadorizada. / The effects of maternal glycemia over the of fetal heart rate (FHR) regulation appoint a controversial subject in the literature, mainly in pregnancies affected by diabetes mellitus. The objective of this research was to investigate the influence of maternal glycemia in the FHR indices analysed by computerized cardiotocography. Methods: Thirty nine patients with pre-gestational diabetes mellitus were examined prospectively in the Obstetrics Department of Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo- Brazil in a period of time between July of 2003 and February of 2005. The patients included had pre-gestational diabetes diagnosis, single pregnancy, gestational age between 36 and 40 weeks and absent of fetal malformations. Each patient were evaluated by computerized cardiotocography during 60 minutes, with analysis of these follow FHR parameters: basal FHR, time necessary to reach normality criteria, fetal movements rate, contraction peaks, accelerations and decelerations, episodes of high and low FHR variation, short-term FHR variation. The capillary glycemia were colleted immediately before the cardiotocography be performed, 30 and 60 minutes after the beginning of the exam. The glicemic mean level was used for analysis with the cardiotocografics results, with the cut values of 100 mg/dL and 120mg/dL. Results: From 39 patients studied, 25 (64,1%) presented glicemic mean &#8805; to 100 mg/dL and 19 (48,7%) &#8805; to 120 mg/dL. 1) The mean of FHR showed significant elevation in the groups with the glicemic mean &#8805; to 100 mg/dL and to 120mg/dl (p<0,05); 2) There was significant positive correlation (p<0,05 and r=0,57) between the FHR and the mean glicemic; 3) There was significant negative correlation (p<0,05) between the number of transitory accelerations &#8805; than 10bpm (r=-0,32) and &#8805; than 15 bpm (r=-0,44) and the mean glicemic; 4) The short-term FHR variation presented significant association (p<0,05) with the mean glicemic &#8805; 120mg/dL; 5) There was significant negative correlation (p<0,05) between short-term FHR variation (r=-0,47) and the mean glicemic. The others indices evaluated by computerized cardiotocography didn’t exhibit significant difference with the mean glicemic. Conclusions: The maternal glicemic levels during computerized cardiotocography seem to have influence over these analised FHR parameters.

Orientador: Raquel Fantin Domeniconi / Resumo: O estado nutricional materno desempenha papel crucial na saúde e no bem-estar do feto. Alterações testiculares, prostáticas e espermáticas foram observadas em animais adultos, cujas mães sofreram restrição de proteína. No entanto, não há trabalhos com este modelo experimental que enfoquem o desenvolvimento, padrão de vascularização e seus reflexos na expressão de proteínas no epidídimo. O objetivo deste estudo é investigar o padrão das respostas morfológicas, imuno-histoquímicas e expressão de proteínas do epidídimo da prole de ratos Wistar de mães submetidas à restrição proteica no período de gestação e de lactação. Fêmeas prenhes foram alocadas nos grupos experimentais normoproteico (NP) e hipoproteico (HP) que receberam, respectivamente, dietas contendo 17% e 6% de proteínas durante gestação e lactação. Após o desmame, os ratos receberam dieta padrão para roedores até as idades de 21, 44 e 120 dias pós-natais (DPN) quando foram eutanasiados. Os epidídimos foram coletados e processados segundo técnicas histológicas, imuno-histoquímicas e de western blotting. Nos filhotes HP, o tamanho reduzido e baixo peso observados ao nascimento se mantiveram até o DPN 120, acompanhados da redução dos órgãos do sistema genital masculino para todas as idades analisadas. A dieta hipoproteica materna diminuiu os níveis séricos de testosterona nos animais no DPN 44, aumentou os níveis de aldosterona nos animais no DPN 21 e não alterou os níveis de estradiol em nenhuma das idades. Nos animais ... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Maternal nutrition status plays a crucial role in the health and well-being of the fetus. Changes of testes and prostate as well as spermatic disorders were observed in adult animals whose mothers were subjected to protein restriction. However, there are no studies with this experimental model that focus the development and the vascular pattern of epididymis, as well as their reflexes in the expression of proteins of the epididymis. The aim of this study is to investigate the pattern of morphometric, immunohistochemical and protein expression of the epididymis of the Wistar rat offspring whose mothers were subjected to a low-protein diet during gestation and lactation. Pregnant females were divided into normoprotein (NP) group and low-protein (LP) group that received, respectively, diets containing 17% and 6% of protein during gestation and lactation. After weaning, the LP and NP male pups received the standard diet for rodents until the ages of 21, 44 and 120 days (PND), when they were euthanized. The epididymides were collected and processed according to histological, immunohistochemical and western blotting techniques. In the LP offspring, the smaller body size and low weight observed at birth remained until the PND 120, as well as the reduction of the male genital system organs weight for all analyzed ages. Maternal low-protein diet decreased testosterone serum levels in animals at PND 44, increased aldosterone serum levels in animals at PND 21 and did not altered estradi... (Complete abstract click electronic access below) / Doutor

Bibliography: leaves 284-298 / 4 microfiches (339 fr.) : / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Summary: Shows that foetal acidosis is related to prolongation of the QT interval, a change which cannot be accounted for in foetal heart rate. Describes the configuration and time constants of the foetal electrocardiogram both during labour and at the time of delivery in normal and acidotic subjects. Confirms that foetal acidosis during labour is associated with acidosis at the time of delivery and with clinical depression of the newborn infant / Thesis (M.D.)--Dept. of Obstetrics and Gynaecology, University of Adelaide, 1970

<p>Bird incubation is a natural phenomenon that balances the needs of the parents for nourishment with the needs of the fetus for heat provision and protection. In this context, any means of communication between the fetus and the parents would have an adaptive value. The aim of the study was to investigate whether putative means of fetomaternal communication would correlate to physiological changes caused by environmental alterations. Oxygen consumption was used to measure fetal well being and six independent variables associated with fetal vocalizations and fetal movements were used to evaluate their potential for communicating the fetus statu quo. Broiler fetuses (<em>Gallus gallus domesticus</em>) of three developmental stages (day 18, internally pipped and externally pipped) were challenged by a stepwise reduction in ambient temperature down to 30ºC. A linear drop in oxygen consumption in response to lowered temperatures was found in all three developmental stages indicating that the fetus was affected by the temperature changes. No differences correlating with temperature variations were found in any of the variables associated with fetal vocalization. Fetal vocalizations are consequently not used to communicate the thermal status of the fetus. Movement occurrence, movement intensity and ventilation frequency, however, followed a “maximum peak” trend, with a highest response at the third temperature interval (35.0-35.5ºC). Considering that the lower limit of optimal development is between 35-36ºC, the results suggest that fetal movements can be of potential use to the incubating parent to assess the well-being of the fetus.</p>

Bird incubation is a natural phenomenon that balances the needs of the parents for nourishment with the needs of the fetus for heat provision and protection. In this context, any means of communication between the fetus and the parents would have an adaptive value. The aim of the study was to investigate whether putative means of fetomaternal communication would correlate to physiological changes caused by environmental alterations. Oxygen consumption was used to measure fetal well being and six independent variables associated with fetal vocalizations and fetal movements were used to evaluate their potential for communicating the fetus statu quo. Broiler fetuses (Gallus gallus domesticus) of three developmental stages (day 18, internally pipped and externally pipped) were challenged by a stepwise reduction in ambient temperature down to 30ºC. A linear drop in oxygen consumption in response to lowered temperatures was found in all three developmental stages indicating that the fetus was affected by the temperature changes. No differences correlating with temperature variations were found in any of the variables associated with fetal vocalization. Fetal vocalizations are consequently not used to communicate the thermal status of the fetus. Movement occurrence, movement intensity and ventilation frequency, however, followed a “maximum peak” trend, with a highest response at the third temperature interval (35.0-35.5ºC). Considering that the lower limit of optimal development is between 35-36ºC, the results suggest that fetal movements can be of potential use to the incubating parent to assess the well-being of the fetus.

Background: Fetal alcohol syndrome (FAS) is a devastating disorder whose mechanisms may be best investigated using animal models. Here we present a novel zebrafish FAS model to investigate the effects of low to moderate alcohol exposure during early development on learning. Methods: At 24-hours postfertilization zebrafish embryos were exposed to low doses of ethanol (external concentrations = 0.00, 0.25, 0.50, 0.75 and 1.00% vol/vol) for a very short duration (2 hours). Upon adulthood associative learning in the zebrafish was tested in a plus maze. Results: This exposure led to no gross anatomical abnormalities or increased morbidity or mortality. Overall activity was not significantly affected by embryonic ethanol exposure. A trend towards a dose-dependent decrease in learning and memory performance was observed. Conclusions: We suggest that zebrafish will be an appropriate model with which one can analyze the behavioural effects of embryonic alcohol exposure and the mechanisms of the ensuing abnormalities.