Submitted by Marina Frolini de Moraes null (moraes.marina@gmail.com) on 2016-09-22T22:34:33Z No. of bitstreams: 1 Dissertação - Marina Frolini de Moraes - Definitivo.pdf: 1612264 bytes, checksum: 3a95e8da696237241a24340897c7a2be (MD5) / Approved for entry into archive by Felipe Augusto Arakaki (arakaki@reitoria.unesp.br) on 2016-09-26T18:20:06Z (GMT) No. of bitstreams: 1 moraes_mf_me_bot.pdf: 1612264 bytes, checksum: 3a95e8da696237241a24340897c7a2be (MD5) / Made available in DSpace on 2016-09-26T18:20:06Z (GMT). No. of bitstreams: 1 moraes_mf_me_bot.pdf: 1612264 bytes, checksum: 3a95e8da696237241a24340897c7a2be (MD5) Previous issue date: 2016-08-22 / A vigilância epidemiológica é essencial para o planejamento, implementação e avaliação das práticas de saúde pública. O monitoramento da mortalidade perinatal, dentro do sistema de vigilância epidemiológica, depende da qualidade dos dados dos sistemas de informação. Objetivo: Avaliar o sistema de vigilância de óbitos perinatais de Núcleo Hospitalar de Epidemiologia (NHE) de hospital terciário do interior paulista. Métodos: Trata-se de estudo transversal, realizado a partir dos 80 óbitos ocorridos entre abril de 2014 e abril de 2015 em NHE de hospital terciário do interior paulista. O referencial de avaliação utilizado foi proposto pelo Center for Disease Control americano para uso em saúde pública. Foram abordados os seguintes atributos: utilidade, simplicidade, qualidade dos dados, aceitabilidade, sensibilidade, representatividade, oportunidade, flexibilidade, valor preditivo positivo, estabilidade. Este estudo foi aprovado por Comitê de Ética em Pesquisa local. Resultados: Utilidade: o sistema é pouco utilizado, os dados não são devolvidos aos profissionais, não são utilizados na proposição de ações para evitar a ocorrência de novos casos nem na gestão em saúde. Qualidade dos dados: 6,2% das variáveis estavam em branco nas investigações; a taxa de concordância entre as investigações originais e as realizadas para o estudo foi de 79,1%, sendo que a categoria com maior discordância/ausência de dados foi assistência ao parto (35,8%). Simplicidade: a grande maioria (91,2%) das investigações foi realizada a partir de apenas uma fonte de dados e o tempo médio gasto na investigação de óbitos infantis foi 63,4 minutos com intervalo interquartil de 29,2 minutos e de óbito fetal, 27,3 minutos com intervalo interquartil de 12,5 minutos. Aceitabilidade: nenhum caso de óbito foi notificado pelos atores envolvidos. Sensibilidade: há problemas a serem superados pelo NHE e Núcleo de Vigilância Epidemiológica. Representatividade: 96,8% dos óbitos do município ocorreram no Hospital do estudo. Oportunidade: o tempo entre a ocorrência do óbito e o encaminhamento ao município da investigação completa foi de 15,7 dias. Flexibilidade: o sistema foi considerado flexível. Valor preditivo positivo: todas as investigações de 2015 foram realizadas a partir da Declaração de Óbito e eram compatíveis com os casos. Estabilidade: alguns problemas eventuais foram apontados, tecnológicos ou por deficiências de recursos humanos. Conclusões: Para aprimorar o sistema de vigilância de óbitos perinatais será necessário: implementar ações voltadas aos diferentes profissionais atuantes na área materno infantil do hospital, para que passem a informar os casos ao NHE e para aprimorar a qualidade dos registros de dados nos prontuários; implementar busca ativa de óbitos perinatais em todas as unidades hospitalares que atendem gestantes e recém-nascidos e buscar mecanismos que permitam identificar, além dos óbitos infantis, os óbitos fetais pelo sistema de informática do hospital. / Epidemiological surveillance is essential for the planning, implementation and evaluation of public health practices. The monitoring of perinatal mortality, within the system of epidemiological surveillance, depends on quality of data in information systems. Object: To evaluate the perinatal deaths surveillance system of the Hospital Nucleus of Epidemiology (HNE) of a tertiary referral hospital in the interior of São Paulo state. Methods: A transversal study was conducted based on 80 deaths between April 2014 and April 2015 at the HNE of a tertiary referral hospital in the interior of São Paulo state. Our evaluation references were proposed by the American Center for Disease Control to be used in public health. The following attributes were addressed: utility, simplicity, data quality, acceptability, sensitivity, representativeness, timeliness, flexibility, predictive value positive, and stability. This study was approved by the local Research Ethics Committee. Results: Utility: the system is little used, the data are not returned to professionals, they are used neither in action proposals in order to avoid the emergence of new cases, nor in health management. Data quality: 6.2% of variables were blank in investigations; the agreement rate between original investigations and the ones conducted for the study was 79.1%, of which the category with the greatest disagreement/lack of data was birth assistance (35.8%). Simplicity: the vast majority (91.2%) of investigations was conducted on a single source of data, and the average time spent on investigating infant deaths was 63.4 minutes, with interquartile range of 29.2 minutes and 27.3 minutes with interquartile range of 12.5 minutes on fetal deaths. Acceptability: no death was reported by the individuals involved. Sensitivity: there are issues to be overcome by the NHE and the Epidemiological Surveillance Nucleus. Representativeness: 96.8% of deaths in the municipality occurred in the hospital under study. timeliness: the time span between death and referring the complete investigation to the municipality was 15.7 days. Flexibility: the system was considered flexible. Predictive value positive: all investigations in 2015 were conducted based on the date of death and were compatible with the cases. Stability: some possible problems (of a technological nature or due to lack of staff) were highlighted. Conclusions: To have a better surveillance system for perinatal deaths, it will be necessary to: implement actions geared at different professionals working at the maternity and child ward of the hospital so that they begin to report cases to the HNE and improve the quality of records in patients’ files; to implement an active search of perinatal deaths in all hospital units dealing with pregnant women and newborns and to search for mechanisms that make it possible to identify, besides infant deaths, fetal deaths by means of the hospital’s IT system.

Resumo: Este estudo avaliou o efeito dos níveis de energia (NDT) e proteína bruta (PB) consumidos por vacas de corte durante a gestação sobre o peso dos fetos aos quatro (PF4) e oito meses (PF8) e dos bezerros ao nascimento (PN); sobre o peso pós-natal aos 60 (PV60), 100 (PV100), 180 (PV180) e 205 (PV205) dias de vida; sobre o peso, a idade, o ganho médio diário (GMD), a área de olho de lombo (AOL), o marmoreio e a espessura de gordura (EG) ao abate. Foi conduzida uma revisão sistemática da literatura e uma metanálise (MA) a partir de duas bases de dados eletrônicas (Scopus e Web of Science). A MA para efeitos randomizados foi realizada para cada resultado com as médias do grupo controle e tratado, conduzida com os níveis de NDT e PB em relação às exigências das vacas (NRC, 1996). Foram avaliadas 46 publicações, correspondentes a 50 estudos, 135 ensaios e 9.379 animais. Não houve efeito do consumo de PB e NDT pelas vacas sobre o PF4. Quando as vacas consumiram PB e NDT em níveis iguais às suas exigências, no primeiro (1TRI) e segundo trimestre (2TRI) da gestação, houve redução no PF8 (DM= 2,236kg; P<0,001). O PN reduziu nos bezerros filhos de vacas que consumiram PB igual ou acima das suas exigências no terceiro trimestre da gestação (3TRI) (DM= -2,954kg; P<0,001 e DM= -0,453kg; P=0,045, respectivamente) ou 140% das exigências de NDT (DM= -2,709; P = 0,001). Quando o consumo de NDT foi igual à recomendação do NRC (1996), observou-se aumento de 2,029kg no PN dos bezerros (P<0,001). O PV60 foi inferior quando as vacas consumiram PB (DM= -1,923kg; P<0,001) e NDT (DM= -1,676kg; P<0,001) acima de suas exigências no 3TRI. O PV205 foi 3,122kg inferior (P<0,001) quando as vacas consumiram até 120% de NDT no 2TRI e 3TRI. Os novilhos cujas mães consumiram PB e NDT acima de suas demandas, no 3TRI, foram abatidos 5,5 dias antes (P= 0,015) que os demais. O GMD, o marmoreio e a EG foram superiores em vacas que consumiram NDT e PB acima das suas exigências no 3TRI (P<0,05). Em síntese, os níveis de PB e NDT, consumidos pelas vacas, acima das recomendações do NRC, estão negativamente relacionados com o desenvolvimento dos fetos e com o PN, sendo que esse efeito permanece até os 205 dias de idade. Por outro lado, na fase de terminação o excesso de PB e NDT passa a contribuir com o GMD, o marmoreio e a EG da carcaça das progênies. / Abstract: This study evaluated the effect of total digestible nutrients (TDN) and crude protein (CP) levels, consumed by cows during gestation, on fetal and postnatal development and on carcass traits. We conducted a systematic review of the literature and a metanalysis (MA) on two electronic databases (Scopus and Web of Science). Inclusion criteria were complete studies using pregnant beef cows receiving different diets that analysed fetus weight at four (FW4) and eight months (FW8), and birth weight of calves (BW); postnatal weight at 60 days (PW60), 100 days (PW100), 180 days (PW180) and 205 days (PW205) of life; and on weight, age, mean daily gain (ADG), loin eye area (LEA), marbling and fat thickness (FT) at slaughter. Random effect MA were conducted for each indicator separately with the mean of control and treated group. The analysis was conducted with the TDN and CP levels in relation to cows’ requirements (NRC, 1996). We evaluated 46 publications, reporting 50 studies, 135 trials and 9,379 animals. The CP and TDN level consumed by pregnant cows showed no change on FW4. Cows that consumed CP and TDN in a level equal to their requirements, in the first (1TRI) and second trimester (2TRI) of gestation, showed a reduction in FW8 (DM= 2,236kg; P<0.001). The BW reduced in cows that consumed CP equal to or above their requirements in the third trimester of gestation (3TRI) (DM= -2.954kg, P<0.001 and DM= -0,453kg, P=0.045, respectively), or 140% of TDN requirements (DM= -2.709, P=0.001). The calves of cows that consumed TDN equal to NRC (1996) requirements, increased the BW in 2,029kg (P<0.001). The PW60 was lower when cows consumed CP (DM= -1.923kg; P<0.001) and TDN (DM= -1.676kg; P<0.001) above their requirements in the 3TRI. The PW205 was lower (DM= 3.12kg; P<0.001) when cows consumed 120% of TDN in the 2TRI and 3TRI. Steers whose mothers consumed CP and TDN above their requirements in the 3TRI were slaughtered 5.5 days earlier (P= 0.015) than the others. The ADG, marbling and FT were higher when cows consumed TDN and CP above the NRC (1996) requirements in the 3TRI (P<0.05). In summary, the CP and TDN levels, consumed by cows, above the NRC recommendations, are negatively associated to fetal development and birth calf weight, and this effect remains until 205 days of age. Alternatively, in the feeder phase, CP and TDN in excess contribute to average daily gain, marbling and fat thickness of the progeny.

Introdução: Alterações no ambiente fetal conferem um risco aumentado para doenças crônicas como obesidade, doença cardiovascular, hipertensão arterial e diabetes tipo 2. As evidências sugerem que a restrição de crescimento intrauterino (RCIU) pode programar de forma persistente as preferências alimentares, e acredita-se que esse tipo de alteração comportamental, pode explicar, pelo menos em parte, o aumento do risco para essas doenças em indivíduos que sofreram RCIU. Portanto, torna-se importante entender os fatores associados e mecanismos envolvidos nesse comportamento. O objetivo deste trabalho foi investigar o efeito da RCIU no comportamento alimentar em animais e humanos, assim como os possíveis mecanismos envolvidos na sua programação. Métodos: Ratas Sprague Dawley prenhes foram randomizadas para o grupo controle (Adlib), que recebeu dieta padrão ad libitum ou grupo restrição 50% (FR), que recebeu 50% do consumo habitual de genitoras alimentadas ad libitum. As dietas foram oferecidas a partir do dia 10 de gestação até o dia 21 de lactação. Em até 24h após o nascimento, foi realizada a adoção cruzada formando os grupos: Adlib_Adlib, FR_Adlib, FR_FR, Adlib_FR. O consumo de ração padrão foi comparado entre todos os grupos. A preferência alimentar, a preferência condicionada por lugar induzida por alimento palatável, assim como a fosforilação da enzima tirosina hidroxilase e os níveis de receptores D2 no núcleo acumbens foram comparados entre os grupos de interesse (Adlib_Adlib e FR_Adlib). Nos humanos, 75 jovens, classificados quanto à RCIU, participaram de avaliação antropométrica, bioquímica e de comportamento alimentar (teste de escolha alimentar, no qual todos recebiam um valor monetário para compra de um lanche, e Dutch Eating Behaviour Questionnaire, DEBQ). Dados de neuroimagem funcional em repouso entre regiões relacionadas à recompensa de 28 indivíduos foram processados e analisados, de um total de 43 exames realizados. Resultados: No estudo experimental, viu-se que o consumo de ração padrão não foi diferente entre os grupos. Ratos restritos apresentaram preferência pela dieta palatável, mas menor condicionamento de preferência ao lugar associado ao alimento palatável. A fosforilação da tirosina hidroxilase no núcleo acumbens foi maior nestes animais no estado basal, mas após exposição ao doce essa diferença entre os grupos permaneceu apenas nos machos. A RCIU também se associou a menores níveis de receptores D2 no núcleo acumbens. No estudo clínico, encontrou-se que a menor razão de crescimento fetal (indicativo de maior RCIU) e alto índice de massa corporal predizem um estilo alimentar restritivo visto pelo DEBQ. Pessoas nascidas com RCIU também usaram menor quantidade do um recurso financeiro oferecido no teste de escolha alimentar após um período de jejum. Os dados de neuroimagem funcional sugerem que os indivíduos restritos apresentam um padrão de conectividade em repouso alterado entre o córtex orbito-frontal, o estriado ventral/dorsal e a amígdala. Conclusão: A RCIU esteve associada com preferência por alimentos palatáveis e alterações no sistema dopaminérgico no estudo experimental e alterações da conectividade em repouso entre áreas do sistema mesocorticolímbico no estudo clínico. As alterações observadas no sistema dopaminérgico dos animais restritos indicam que esse sistema estaria envolvido na programação da preferência alimentar nesses indivíduos. Além disso, o padrão de conectividade em repouso observado nos indivíduos restritos sugere que alterações em determinadas regiões do sistema de recompensa poderiam estar associadas com mudanças no comportamento alimentar. As alterações neurocomportamentais observadas confirmam a existência de programação fetal do comportamento alimentar pela RCIU, apontando modificações persistentes no sistema de recompensa do cérebro, o que pode ser visto como um fator de risco para o desenvolvimento de obesidade e suas comorbidades. / Introduction: Fetal environment changes can lead to adaptations that are associated with increased risk for obesity, cardiovascular disease, hypertension and diabetes in adult life. Evidence suggests that intrauterine growth restriction (IUGR) can persistently program the subject’s preference for palatable foods. It is believed that feeding behavior alterations can explain, at least in part, the increased risk for chronic diseases in IUGR individuals. Therefore, it becomes important to understand the factors and mechanisms involved in this behavior. The aim of this study was to explore how IUGR affects feeding behavior of animals and humans, as well as to verify the potential mechanisms related to this behavioral programming. Methods: Time-mated pregnant Sprague-Dawley rats were randomly allocated to Control (receiving standard chow ad libitum) or 50% food restricted (FR), receiving 50% of the ad libitum-fed dam’s habitual intake. These diets were provided from day 10 of pregnancy throughout day 21 of lactation. Within 24 hours after birth, pups were crossfostered, forming four groups: Adlib_Adlib, FR_Adlib, FR_FR, Adlib_FR. Standard chow consumption was compared between all groups. Food preference, conditioned place preference to a palatable diet, and the nucleus accumbens tyrosine hydroxylase phosphorylation and D2 receptor levels were analyzed focusing on two groups of interest (Adlib_Adlib and FR_Adlib). In humans, 75 youths were classified regarding IUGR and had anthropometric data, biochemical data, and feeding behavior (food choice task, in which everyone received a monetary value to purchase a snack, and Dutch Eating Behaviour Questionnaire) assessed. Forty three neuroimaging exams were performed and resting state functional connectivity between brain regions related to reward of 28 individuals were processed and analyzed. Results: In the experimental study, standard chow consumption was not different between groups. IUGR adult rats had increased preference for palatable food, but showed less conditioned place preference to a palatable diet compared to controls. At baseline, the accumbal tyrosine hydroxylase phosphorylation was increased in IUGR rats compared to controls. After sweet food exposure, the difference between groups remained only in males. Accumbal D2 receptors levels were decreased in IUGR rats. In the clinical study, it was found that low birth weight ratio (indicative of higher IUGR) and high body mass index predict a restrained eating style as seen by the DEBQ. IUGR individuals used a smaller quantity of a financial resource offered in the food choice task after a fasting period. Resting state functional connectivity data suggest that IUGR individuals had an altered pattern of connectivity between the orbitofrontal cortex, the ventral/dorsal striatum and the amygdala. Conclusion: IUGR was associated with a preference for palatable foods and alterations in the dopaminergic system in the experimental study, as well as changes in the resting state functional connectivity between regions of the mesocorticolimbic pathway in the clinical study. Alterations in the mesolimbic dopaminergic system observed in IUGR rats indicate an important role in the programming of food preferences. Moreover, the IUGR pattern of brain connectivity observed suggests that alterations in certain regions involved in reward processing and evaluation could be associated with changes in eating behavior. Neurobehavioral changes observed confirmed the existence of a fetal programming of feeding behavior associated with IUGR, pointing out to persistent modifications in the brain reward system, which can be seen as a risk factor for the development of obesity and its comorbidities.

Fatores como o tempo de espera ou o risco a ser enfrentado por uma recompensa maior têm um efeito importante e, por vezes, paradoxal no processo de tomada de decisão. O Capítulo I apresenta uma discussão teórica sobre a tomada de decisão mediada por esses dois fatores, bem como sua interligação com o conceito de impulsividade e o Transtorno de Déficit de Atenção e Hiperatividade (TDAH). O Capítulo II oferece uma aplicação desses conceitos em um modelo animal do espectro da Síndrome Alcoólica Fetal, condição que apresenta algumas características semelhantes ao TDAH. Utilizando ratos expostos ao álcool no período pré-natal, duas tarefas de tomada de decisão mediada por tempo ou probabilidade foram usadas para testar se esses animais apresentam diferenças em seus padrões de escolha. Não foram encontradas diferenças significativas entre grupos controle e experimentais. Futuros estudos com outros protocolos, espécies e/ou linhagens são desejáveis. / Factors such as the time to wait, or the risk to be faced for a bigger reward have an important and, sometimes, paradoxical effect on the decision making process. Chapter I presents a theoretical discussion about decision making mediated by these two factors, as well as how they relate to the concept of impulsivity and the Attention-Deficit Hyperactivity Disorder (ADHD). Chapter II offers a practical application of these concepts in an animal model of the spectrum of Fetal Alcohol Syndrome, a condition that presents some similar characterstics in comparison to ADHD. Using rats prenatally exposed to ethanol, two decision making tasks, mediated by time or probability, were used to test if these animals show any difference in their patterns of choice. No significant differences were found between control and experimental groups. Future studies with other protocols, species and/or strains are desirable.

Glycodelin, a homodimeric glycoprotein belonging to the lipocalin superfamily, is synthesised predominantly by the cells of the reproductive system of certain primates including humans. Of the four different known glycoforms of the molecule, glycodelin A (GdA), secreted by the glandular epithelial cells of the endometrium in response to progesterone, is involved in the immunosuppression of the maternal immune response to the semi-allograft fetus. GdA secretion onsets few days after ovulation. In the absence of fertilization, GdA levels drop, but subsequent to a successful fertilization, the concentrations peak till the 12th week of pregnancy and fall steadily to low levels. The importance of GdA has been implicated in implantation, endometrial receptivity, trophoblast invasion and differentiation, and modulating the functions of almost all immune cells. GdA has profound influence on the activity of T cells. It inhibits the proliferation of T cells, induces apoptosis in activated T cells, inhibits the IL-2 production and leads to skewing of the Th-1/Th-2 balance towards Th-2 type of immune response. Cytotoxic T lymphocytes are more resistant to the induction of apoptosis by GdA, but, it suppresses their cytolytic activity Additionally, GdA induces apoptosis in monocytes and natural killer (NK) cells, inhibits the proliferation of B cells and induces tolerogenic phenotype in dendritic cells. Clinical studies showing that women undergoing recurring spontaneous abortions have low levels of GdA supports its role in prevention of fetus rejection. The immunomodulatory activity of Gd resides in the protein backbone, however, apart from GdA and GdF which have similar oligosaccharide chains, other glycoforms do not possess this activity. Glycosylation seems to dictate the stability, folding and activity of Gd. In absence of glycosylation, the expression of the recombinant Gd is compromised and the protein is improperly folded while over-mannosylation of Gd impairs its immunomodulatory function. Additionally, sialylation seen on the glycan chain regulates the activity. Therefore, in order to obtain adequate amounts of active recombinant Gd (rGd), expression of the protein was attempted in three different systems, insect, yeast and bacteria (Chapter 1). In all of the described systems, the rGd protein was found apoptotically active. The protein expressed in the Sf21 insect cells was demonstrated to be differentially glycosylated compromising the activity. Hence, a genetically modified yeast strain, Pichia pastoris SuperMAN5 was explored for expression. Though presence of a single glycosylated protein species was observed in small-scale cultures, similar to the case of Sf21 cell expression, differentially glycosylated proteins were detected in large-scale fermentation and even the yield was low. Eventually, mutant Gd, modified to increase the stability and aid in proper protein folding, was expressed in E.coli and demonstrated to be able to induce apoptosis in Jurkat cells (T cell leukemia cell line). This active rGd was used for further studies. The immunomodulatory function of GdA during pregnancy protects the semi-allograft fetus from rejection by the maternal immune system. In the process, GdA tweaks the T cell immune response from pro-inflammatory to anti-inflammatory in a specific and localized manner. Allograft rejection seen during mis-match transplantations is basically a pro-inflammatory condition which is mediated by the activation of cellular immune response, NK cell cytotoxicity and antibody-dependent immune response, the same processes that are suppressed for a successful pregnancy. Chapter 2 discusses whether it is feasible to use Gd to prevent allograft rejection. Killing of target graft cells by the cytotoxic T lymphocytes (CTLs) predominantly presides acute graft rejection. GdA treatment has been shown to suppress the cytotoxicity of in vitro generated CTLs. On this basis, the earlier study was translated to in vivo conditions by establishing an allograft nude mouse model. The tumor rejection mediated by the action of in vitro generated cytotoxic alloactivated PBMCs in the nude mouse imitated the allograft rejection. A heterogenous population of immune cells with the predominance of CTLs was chosen to accommodate a more interactive immune response in the tumor microenvironment and enabled the study of other cells which may contribute to the rejection. Reactivation and proliferation of CD4+ and CD8+ T cells following their infiltration in the tumor validated our hypothesis. On treatment with rGd, the cytotoxicity of the alloactivated PBMCs was suppressed, thereby inhibiting the tumor rejection in the nude mouse. Real time PCR analysis showed that rGd treatment was able to affect the functions of the immune cells in vivo. It decreased the T cell population most probably by inducing apoptosis. As expected, the reduction was more prominent in case of CD4+ T cells than CD8+ T cells. The their expression of key molecules responsible for the cytotoxicity such as IL-2, granzyme B and EOMES, was observed to be downregulated by rGd. Concomitantly, decreased levels of pro-inflammatory cytokines, TNFα and IL-6 were also seen. Expression of Foxp3, marker for regulatory T cells, was upregulated in the tumor infiltrating immune cells suggesting an expansion of the concerned population upon rGd treatment. Overall, rGd seems to suppress the cellular immune response to the tumor by modulating the T cell population and their functions. Since, T cell-dependent immune response is central to allograft rejection, the ability of rGd to regulate it could be of therapeutic use in the management of allograft rejection. NK cells are essential for the maintenance of pregnancy, evident from their abundance (70% of total leukocytes) at the first trimester decidua. The third chapter focuses on how Gd regulates the NK cell function. The cytokine production from CD56bright subset of NK cells and their interaction with the HLA antigens expressed by the trophoblast cells helps in creating a favourable environment for the growth of the fetus. It is important to note that the NK cell population present in the decidua exclusively express Gd, implicating a role of Gd in their differentiation from the peripheral CD56bright cells. However, an increased number of CD56dimCD16+ cells in the peripheral blood dictates a negative outcome for the pregnancy. The study, presented in Chapter 3, demonstrated that rGd treatment induces caspase-dependent apoptosis in the activated CD56dimCD16+ cells and reduces their cytotoxicity by downregulating granzyme B and IFNγ production. Similar effect of rGd is also seen on the NKT cells characterised as CD3+CD56dimCD16-. Furthermore, in YT-Indy cells, an activated NK cell line, it was shown that the induction of apoptosis by rGd involves Ca2+ signalling which could explain why Gd affects activated immune cells only. This study therefore reinforces the role of Gd in modulating the NK cell activity during pregnancy. Cytotoxicity of NK and NKT cells also plays an important role during allograft rejection. Decrease in the mRNA levels of CD56 upon rGd treatment in the allograft mouse model indicates that the effect of Gd on NK cells observed in cell culture system can be translated to in vivo conditions. In conclusion, suppression of the cellular immune response and NK cell mediated cytotoxicity by rGd could potentiate its’ probable use in the management of allograft rejection. .

Neste trabalho foi desenvolvido um sistema on-line para a avaliação de doses fetais em radiologia diagnóstica, chamado Dose Fetal Web. A metodologia empregada no sistema estima a dose fetal a partir de dados referentes a exames radiológicos realizados pela paciente gestante, ou a partir da monitoração pessoal de uma IOE gestante. Os dados de entrada deste programa são associados às tabelas de coeficientes de conversão de dose uterina para dose fetal, NUD, geradas por meio de simulações pelo método de Monte Carlo. Este sistema incorpora informações de operação de equipamentos de hospitais e clínicas radiológicas acompanhados por um programa de garantia de qualidade, bem como dados reais de parâmetros fetais e maternos coletados durante exames de ultra-sonografia obstétrica. A dose fetal médica, a dose fetal ocupacional e os riscos radiológicos associados a estas exposições foram avaliados por meio de estudos de casos simulados usando o sistema on-line. No caso hipotético de uma gestante ocupacionalmente exposta (IOE), foi avaliada uma base de dados reais de serviços de hemodinâmica. Além disso, a distribuição de cargas de trabalho de salas de cateterismo cardíaco foi incorporada ao modelo matemático. Estas duas metodologias foram implementadas na avaliação da dose fetal ocupacional, a fim de avaliar estes casos particulares de exposição, que oferecem níveis mais altos de doses ocupacionais aos profissionais expostos. / In this work, an online system called Dose Fetal Web was developed for the evaluation of fetal doses in diagnostic radiology. The methodology used in this system estimates the fetal dose from data related to radiologic diagnostic examinations of a pregnant patient, or from the routine individual monitoring dosimetry of a pregnant worker. The input data of this program are associated to the conversion-coefficients tables of uterine to fetal dose, NUD, generated by means of Monte Carlo simulations. This system incorporates operational information of equipment in hospitals and radiological facilities which are attended by a quality assurance program, as well as real maternal and fetal parameter data collected during obstetric ultrasound.The fetal dose from both medical and occupational exposures of the pregnant woman, as well as the risks associated with these exposures, were evaluated by means of simulated case studies using the on-line system. In the hypothetical case of an occupationally-exposed pregnant worker, a real database regarding routine individual monitoring dosimetry in Cath Lab facilities was evaluated. Moreover, the workload spectrum of a cardiac angiographic room was incorporated into the mathematical model. These two methodologies were used in the evaluation of the occupational fetal dose, in order to evaluate these particular cases of exposure, which offer higher occupational dose levels to the exposed staff.

Orientador: Fernando Ferreira Costa / Tese (doutorado) - Universidade Estadual de Campinas, Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-10T05:30:27Z (GMT). No. of bitstreams: 1 Lanaro_Carolina_D.pdf: 2434106 bytes, checksum: 653633a8c42bec18d4e48b182ef0e289 (MD5) Previous issue date: 2007 / Resumo: A anemia falciforme (AF) é causada pela substituição de uma única base no códon 6 do gene da globina beta (GAG - GTG), levando à produção de hemoglobina S. Estudos recentes têm mostrado que a ativação de guanilato ciclase (GCs) em linhagens de células eritrocíticas e em cultura primária de precursores da série vermelha, causa um aumento na transcrição do gene gama globina e conseqüentemente aumento na produção de hemoglobina fetal (Hb F) (Ikuta et al., 2001). Os nucleotídeos cíclicos, AMPc e GMPc são segundos mensageiros responsáveis por vários sinais extracelulares, incluindo hormônios e neurotransmissores e provavelmente têm papel importante na anemia falciforme regulando a produção de HbF. Este trabalho teve como objetivo, primeiramente, estabelecer um sistema de cultura de células CD 34+ e posteriormente investigar a existência de uma via NO-GMPc/AMPc envolvida na produção de Hb F nos eritrócitos de pacientes com anemia falciforme e verificar se haveria aumento da expressão de GCs e de gama globina nestes mesmos pacientes. Amostras de sangue foram coletadas de voluntários sadios e pacientes com anemia falciforme. Foi utilizado cultura de células CD34+ hematopoéticas de sangue periférico, e ao longo da diferenciação eritróide (7°, 10° e 13° dia) foram coletadas células para citometria de fluxo, citospin, dosagem de GMPc e AMPc e avaliação da expressão gênica de gama globina, GCs alfa e beta subunidades, eNOS, PDE3B, CD36, CD29B e CD49. Observamos que gama globina encontra-se significativamente mais expressa nas culturas falciformes no 13° dia de diferenciação eritróide em relação ao mesmo ponto das culturas controle (2,945 ± 0,303 x 1,445 ± 0,165, respectivamente ,p=0,0095, n=6). A expressão de GCs alfa e beta subunidades, eNOS, PDE3B, CD36, CD29B e CD49 não apresentou diferenças significativas durante a diferenciação eritróide nas culturas falciformes e controles. Os níveis de GMPc nas culturas de pacientes com anemia falciforme estavam significativamente diminuídos ao longo da eritropoese in vitro em comparação com as culturas controles (dia 7: 0,2765 ± 0,0527 x 0,639 ± 0,1221 (p=0,0260); dia 10: 0,1166 ± 0,0219 x 0,268 ± 0,1181; dia 13: 0,037 ± 0,0061 x 0,093 ± 0,0236 (p=0,0152), respectivamente), enquanto o AMPc, encontrava-se elevado no dia 7 das culturas falciformes em relação às culturas controles (dia 7: 72,017 ± 14,849 x 24,987 ± 13,497 (p=0,048); dia 10: 36,3384 ± 16,708 x 8,663 ± 4,591; dia 13: 1,7138 ± 1,093 x 0,680 ± 0,370, respectivamente). O tratamento com BAY 41-2272 (ativador de GCs) em única cultura controle aumentou aproximadamente 3x os níveis de gama globina após 72 h de incubação. Nossos resultados sugerem que as células tronco hematopoéticas dos pacientes com AF são diferentes às dos controles, uma vez que ambas, sob as mesmas condições in vitro expressaram níveis significativamente diferentes de gama globina. Um cross talk entre o GMPc e o AMPc poderia regular a expressão de gama globina ao longo da diferenciação eritróide. Estes dados resultam no primeiro trabalho a demonstrar a expressão basal de gama globina ao longo da diferenciação eritróide de pacientes com anemia falciforme. Desta maneira, o esclarecimento desta via pode contribuir com entendimento da fisiopatologia da anemia falciforme e identificar novos alvos para a regulação farmacológica de hemoglobina fetal / Abstract: Sickle cell disease (SCD) is caused by the substitution of a single base in codon 6 of the beta globin gene (GAG-GTG), causing the production of S hemoglobin. Recent studies have demonstrated that the activation of soluble guanylate cyclase (sGC) in erythrocytic cell lines and in primary erythroid precursor culture causes an increase in the transcription of the gamma globin gene and, consequently, an augmented production of fetal hemoglobin (HbF) (Ikuta et al., 2001). The cyclic nucleotides, cAMP and cGMP, are second messengers that are responsible for mediating the signaling of various extracellular signals, including those of hormones and neurotransmitters and, probably, have an important role in SCD by regulating the production of HbF. This study aimed to investigate the existence of a NO-cAMP/cAMP pathway involved in the production of HbF in the erythroid cells of patients with SCD and to determine whether there is an increase in the expression of sGC and of gamma globin in these same patients. Blood samples were collected from healthy volunteers and SCD patients. Peripheral blood hematopoietic CD34+ cell culture was used for the investigation; cells were differentiated into erythroid lineage and cells collected (7th, 10th and 13th day) for flow cytometry, citospin, measurement of cGMP/cAMP and evaluation of the gene expression of gamma globin, the sGC alpha and beta subunits, eNOS, PDE3B, CD36, CD29B and CD49. We demonstrate that gamma globin was significantly more expressed in the cultures of SCD cells on the 13th day of erythroid differentiation compared to the same time point in control cells (2.95 ± 0.30 vs. 1.45 ± 0.17, respectively, p=0.0095, n=6). The expression of sGC alpha and beta subunits, eNOS, PDE3B, CD36, CD29B and CD49 did not present significant differences during erythroid differentiation in control or SCD cell cultures. Levels of cGMP were significantly decreased during erythropoiesis in the SCD cultures compared to control cultures (day 7: 0.277 ± 0.053 vs. 0.639 ± 0.122 (p=0.026); day 10: 0.117 ± 0.022 vs. 0.268 ± 0,118; day 13: 0.037 ± 0.006 vs. 0.093 ± 0.024 (p=0.015), respectively). In contrast, levels of cAMP were significantly increased in the SCD cultures compared to the control cultures (day 7: 72.02 ± 14.85 vs. 24.99 ± 13.50 (p=0.048); day 10: 36.34 ± 16.71 vs. 8.66 ± 4.59; day 13: 1.74 ± 1.09 vs. 0.680 ± 0.370, respectively). Treatment with BAY 41-2272 (sGC inducer) in a single control cell culture increased the levels of gamma globin by approximately 3 times after 72h of incubation. Results suggest that the hematopoietic stem cells of SCD patients are significantly and constitutively different to those of controls, since under the same in vitro culture conditions, differentiation of the two cell types results in consistently significant different levels of gamma globin. A cross talk between cGMP and cAMP may regulate the expression of gamma globin during erythroid differentiation in patients with SCD. Thus, further investigation of this pathway may contribute to the understanding of the physiopathology of the disease and identify new targets for the pharmacological regulation of HbF production / Doutorado / Medicina Experimental / Doutor em Fisiopatologia Medica

Foram utilizados 10 sistemas genitais femininos de mocós (Kerodon rupestris) para estabelecer parâmetros de referência relativos à osmolaridade, pH, cálcio, fósforo, uréia, creatinina, glicose e proteínas totais. Os animais foram criados em cativeiro no CEMAS(Centro de Criação de Animais Silvestres), Mossoró – RN. As fêmeas estavam da metade (30-45 dias) para o final da gestação (65-70 dias). As bolsas amnióticas e alantoideanas foram puncionadas individualmente para colheita dos líquidos fetais, que foram centrifugados e analisados posteriormente. Para o líquido amniótico, as concentrações médias em mg/dl foram: glicose = 45,87 ± 22,38; cálcio = 6,31 ± 1,24; fósforo = 1,64 ± 0,72; creatinina = 0,45 ± 0,12; uréia = 34,03 ± 5,96; proteínas totais = 31,24 ± 16,67. Para o líquido alantoideano, as concentrações médias em mg/dl foram: glicose = 59,17 ± 10,85; cálcio = 5,58 ± 0,59; fósforo = 1,27 ± 0,73; creatinina = 0,38 ± 0,38; uréia = 31,49 ± 2,28; proteínas totais = 30,70 ± 18,39. Foram observadas pequenas oscilações entre as concentrações dos parâmetros bioquímicos do fluido amniótico e alantoideano. Estas concentrações são determinadas, provavelmente, pela atividade metabólica materno-fetal. Para os estudos colpocitológicos foram utilizadas 8 fêmeas, sexualmente maduras, mantidas em cativeiro sob condições climáticas tropicais. Foram realizados esfregaços vaginais a cada 72 horas, durante 40 dias. Os animais mostraram ciclo poliestral contínuo, com duração média de 24,25 ± 3,99 dias, não havendo portanto sazonalidade. Durante o proestro houve prevalência de células superficiais anucleadas, intermediárias e parabasais. A fase de estro apresentou índices significativamente mais altos de células superficiais anucleadas e ausência de leucócitos. Alto índice de células intermediárias foi observado no metaestro, com abundância de leucócitos e vacúolos citoplasmáticos (células de Foam). O anestro mostrou índices significativamente mais altos de células parabasais quando comparados com as outras fases do ciclo. Os leucócitos apareceram em número reduzido. / The aim of the present work was to study some reproductives aspects of mocó female (Kerodon rupestris), by assesment of the following parameters: certain biochemical characteristics of fetal fluids and characterization of the estrous cycle by exfoliative colpocytology. In order to establish the levels of the following parameters: osmolarity, pH, calcium, phosphorous, total protein, urea, creatinina, glycose, presents in the amniotic and allantoic fetal fluids, 10 genital systems of mocós were sampled (gestation = 30-45 and 65-70 days). The amniotic and allantoic sacs were punctured and the fetal fluid obtained for posterior analysis. It was observed the following concentrations (mg/ml) in the amniotic fluid: glycose= 45,87 ± 22,38; calcium = 6,31 ± 1,24; phosphorus = 1,64 ± 0,72; creatinine = 0,45 ± 0,12; urea = 34,03 ± 5,96; total protein = 31,24 ± 16,67. In the allantoic fluid the concentrations were: : glycose = 59,17 ± 10,85; calcium = 5,58 ± 0,59; phosphorous = 1,27 ± 0,73; creatine = 0,38 ± 0,38; urea = 31,49 ± 2,28; total protein = 30,70 ± 18,39. Results obtained from the analisys of the composition of fetal fluids showed small oscillations between the amniotic and the allantoic fluids. Their concentrations, probably, are determined by the materno-fetal metabolic activity. Colpocytology studies were realized using 8 adult females kept in captivity, in climatologic conditions of humid tropic. Vaginal smears, prepared by means of swabs, were made each 72 hours, during 40 days. Animals showed a polyestrus continuos cycle (on an average 24,25 ± 3,99 days), so no sazonality was observed. During proestrus phase the exfoliative arrangement showed high frequency of anucleated superficial, intermediate and parabasal cells, respectively. Oestrus phase had elevated rates of superficial cells (both anucleated and nucleated) and absence of leukocytes. In the metestrus phase it was found highest indices of intermediate cells and increase of leukocytes. During the anestrus phase the predominant cells type was parabasal, and leukocytes were reduced.

INTRODUÇÃO: Embora todos os indivíduos com anemia falciforme (AF) apresentem o mesmo defeito molecular nos genes da beta-globina, existe uma considerável variabilidade fenotípica entre eles. A síntese continuada da hemoglobina fetal (HbF) é o mais potente modificador da morbimortalidade da AF. A HbF diminui a polimerização da desoxi-HbS, reduzindo a intensidade da anemia hemolítica crônica e dos fenômenos vaso-oclusivos, consequentemente, as complicações sistêmicas da AF. Há uma grande variação na taxa de síntese de HbF (1% a 30%) em indivíduos com AF. Vários estudos demonstraram que o tratamento com hidroxiureia (HU) diminui a morbimortalidade desta hemoglobinopatia, principalmente, por estimular a síntese de HbF. Na dose máxima tolerada (DMT), a HU geralmente aumenta os níveis de HbF entre 10% e 40%. Contudo, há uma grande variabilidade de resposta, a DMT é muito variável, cerca de 25% dos portadores de AF não respondem e não há preditores de resposta definidos à HU. Estudos polimorfismos de nucleotídeo único (SNPs) demonstraram a influência de modificadores epigenéticos na regulação da expressão da HbF. Estes elementos são, principalmente, o oncogene BCL11A, a região intergênica HMIP e o polimorfismo Xmn1-HBG2. Estes 3 quantitative trait loci (QTLs) detêm 20% a 50% do controle da expressão dos genes HBG (genes envolvidos na síntese da HbF). OBJETIVOS: Avaliar o comportamento epidemiológico e a associação dos SNPs Xmn1- HBG2; BCL11A rs7482144, rs4671393 e rs11886868; HMIP-2 rs9399137 e rs9402686 com a expressão da HbF e com o comportamento dos parâmetros hematimétricos em portadores de AF tratados com HU. PACIENTES E MÉTODOS: Neste estudo pioneiro de coorte prospectivo foram incluídos sequencialmente indivíduos com AF, em uso regular de HU por pelo menos 6 meses, que não receberam transfusão sanguínea 3 meses antes de ingressar no estudo e que não faziam uso de drogas que estimulassem a síntese de HbF. Foram coletados 4 ml de sangue venoso periférico para extração do DNA genômico. A genotipagem dos polimorfismos foi realizada por meio da reação da cadeia de polimerase em tempo real. RESULTADOS: Foram avaliados 121 pacientes, entre 1 ano e 9 meses-54 anos (19 ± 14) anos idade, que estavam recebendo doses regulares de HU entre 8,6-42,8 (23 ± 7,6) mg/kg/dia, durante 6- 254 (102± 67) meses. Não encontramos correlação entre a contagem de leucócitos, de neutrófilos e de reticulócitos; hemoglobina total; volume corpuscular médio e a concentração de hemoglobina corpuscular média, com os valores basais da HbF. A HbF basal (r=0,40; P < 0,001), a hemoglobina total basal (r=0,26; P = 0,005) e o tempo de exposição (r = -0,30; P = 0,001) foram associadas significativamente com maiores taxas de HbF ao final do estudo. Não houve correlação dos polimorfismos com os parâmetros hematimétricos, com o tempo de exposição e com a DMT de HU. Os SNPs HMIP-2 rs9399137 e rs9402686 foram responsáveis por 5,7% e 8,4% do total de variação da HbF basal (P= 0,01 e P=0,002). Não houve correlação, porém, entre os demais polimorfismos com a variação dos níveis basais de HbF. Os SNPs BCL11A rs1427407, rs4671393 e rs11886868 foram responsáveis, respectivamente, por uma variação de 7,6%, 4,5% e 4,3% nos níveis de HbF final (P=0,017; P=0,025 e P=0,029). Ainda, houve uma associação do rs1427407 (B = 0,29; P = 0,035) e do rs4671393 (B = 0,28; P = 0,036) em relação aos valores do delta HbF (variação da HbF final menos a HbF inicial). CONCLUSÃO: Estes dados sugerem que os indivíduos com AF com SNP BCL11A rs1427407 respondem mais favoravelmente ao tratamento com HU, no incremento dos níveis de HbF. São necessários estudos com populações maiores para validarmos estes achados. / INTRODUCTION: Although all individuals with sickle cell anemia (SCA) have the same molecular defect in the beta-globin genes, considerable phenotypic variability exists between them. Continued synthesis of fetal hemoglobin (HbF) is the most powerful SCA morbimortality modifier. HbF decreases the polymerization of deoxy-Hb, reducing the intensity of chronic hemolytic anemia and vaso-occlusive phenomena, and consequently systemic complications of SCA. There is a large variation in the HbF synthesis rate (1 to 30%) in patients with SCA. Several studies show that treatment with hydroxyurea (HU) decreases the morbimortality of this hemoglobinopathy, mainly by stimulating HbF synthesis. At the maximum tolerated dose (MTD), HU generally increases HbF levels from 10 to 40%. However, there is great variability in response as the MTD is highly variable, about 25% of SCA patients do not respond and there are no response predictors set for HU. Single nucleotide polymorphism studies (SNPs) demonstrate the influence of epigenetic modifiers in the regulation of HbF expression. These elements are, mainly, the BCL11A oncogene, the HMIP intergenic region and the Xmn1-HBG2 polymorphism. These 3 quantitative trait loci (QTLs) hold 20 to 50% of HBG gene expression control (genes involved in HbF synthesis). OBJECTIVE: To study the epidemiological behaviors and the association of SNPs Xmn1- HBG2; BCL11A rs7482144, rs4671393 and rs11886868; HMIP-2 rs9399137 and rs9402686 with HbF expression and with the behavior of hematimetric parameters in SCA patients treated with HU. PATIENTS AND METHODS: In this pioneering prospective cohort study, we included SCA patients, in regular treatment with HU at least for 6 months, who had not received blood transfusions in the 3 months prior to entering in the study and who didn‘t use drugs that stimulate HbF synthesis. We collected 4 ml of venous blood to proceed with the genomic DNA extraction. The polymorphism genotyping was done by real-time polymerase chain reaction. RESULTS: We evaluated 121 individuals with SCA aged between 1 year 9 months and 54 years (19 ± 14) who were receiving HU doses between 8.6 and 42.8 (23 ± 7.6) mg/kg/day for 6 to 254 (102 ± 67) months. No correlation was found between total leukocyte, neutrophils and reticulocytes counts; total hemoglobin; mean corpuscular volume and the concentration of mean corpuscular hemoglobin with baseline values of HbF. Basal HbF (r = 0.40; P <0.001), total baseline hemoglobin (r = 0.26; P = 0.005) and exposure time to HU (r = -0.30; P = 0.001) were significantly associated with higher HbF rates at the end of the study. There was no correlation of polymorphisms with the hematological parameters, exposure time and the MTD of HU. The SNPs HMIP-2, rs9399137 and rs9402686 accounted for 5.7% and 8.4% of the total variation of baseline HbF (P = 0.01 and P = 0.002). There was no correlation, however, between the other polymorphisms and variation in baseline HbF levels. The SNPs BCL11, rs1427407, rs4671393 and rs11886868 were responsible, respectively, for a variation of 7.6%, 4.5% and 4.3% in the final HbF levels (P = 0.017, P = 0.025 and P = 0.029). Still, there was an association of rs1427407 (B = 0.29; P = 0.035) and rs4671393 (B = 0.28; P = 0.036) in relation to delta HbF values (final minus initial HbF variation). CONCLUSIONS: These data suggest that individuals with SCA who have SNP rs1427407 BCL11A respond more favorably to HU treatment, with increased HbF levels. Studies with larger populations are necessary to validate these findings.

Fatores como o tempo de espera ou o risco a ser enfrentado por uma recompensa maior têm um efeito importante e, por vezes, paradoxal no processo de tomada de decisão. O Capítulo I apresenta uma discussão teórica sobre a tomada de decisão mediada por esses dois fatores, bem como sua interligação com o conceito de impulsividade e o Transtorno de Déficit de Atenção e Hiperatividade (TDAH). O Capítulo II oferece uma aplicação desses conceitos em um modelo animal do espectro da Síndrome Alcoólica Fetal, condição que apresenta algumas características semelhantes ao TDAH. Utilizando ratos expostos ao álcool no período pré-natal, duas tarefas de tomada de decisão mediada por tempo ou probabilidade foram usadas para testar se esses animais apresentam diferenças em seus padrões de escolha. Não foram encontradas diferenças significativas entre grupos controle e experimentais. Futuros estudos com outros protocolos, espécies e/ou linhagens são desejáveis. / Factors such as the time to wait, or the risk to be faced for a bigger reward have an important and, sometimes, paradoxical effect on the decision making process. Chapter I presents a theoretical discussion about decision making mediated by these two factors, as well as how they relate to the concept of impulsivity and the Attention-Deficit Hyperactivity Disorder (ADHD). Chapter II offers a practical application of these concepts in an animal model of the spectrum of Fetal Alcohol Syndrome, a condition that presents some similar characterstics in comparison to ADHD. Using rats prenatally exposed to ethanol, two decision making tasks, mediated by time or probability, were used to test if these animals show any difference in their patterns of choice. No significant differences were found between control and experimental groups. Future studies with other protocols, species and/or strains are desirable.