Using a tailored dose distribution for personalized radiotherapy with the help of positron emission tomography (PET) might give an edge for successful tumour elimination. One of the main determinants for tumour radioresistance in several solid tumours has been investigated as hypoxia, including head and neck cancer (HNC). Using novel methods of converting radiotracer uptake into partial oxygen pressure distribution in the form of partial pressure maps, it is possible to delineate the hypoxic region of a target to further escalate the treatment dose there, aiming at an increase in tumour control. However, the registration between functional and structural images may have an impact on the effectiveness of dose escalation, and choosing the correct registration method could be imperative. In this master’s thesis, the impact of choosing rigid or deformable image registration between planning-CT and PET/CT images on the characterization of the hypoxic compartment, as well as on the treatment evaluation in terms of tumour control and normal tissue complication, was assessed. This was achieved by, using hypoxic patients of a cohort of 22 HNC patients, creating a separate plan for each registration method, for each patient, and comparing them quantitatively. The results showed that both methods would yield distinctly different dose distributions when planned using the same objectives and constraints in terms of dose level and shape. Furthermore, they both give a distribution of similar quality. However, using rigid registration together with the deformed PET did not render lower results overall in tumour control. Thus, no advantage could be seen in choosing deformable registration over rigid registration when aiming at tumour control.

Doctor of Philosophy / Graduate Biochemistry Group / Dolores J. Takemoto / Eukaryotic cells possess a unique way to communicate with each other by passing metabolites and small molecules through protein pores that connect adjacent cells. Although there are many types and families of protein pores, connexins comprise a unique family. Six connexin monomers assemble into a hemichannel, which is transported to the cell membrane. An opposing cell membrane containing compatible connexin hemichannels is located and connected, forming an intercellular dodecameric protein complex. This results in a protein channel that connects two separate cytoplasmic compartments to each other. This type of channel is known as a gap junction. Connexin expression and function is commonly tissue specific. Of the 21 known human connexins, less than half are currently well characterized. Three connexins are expressed in the lens, connexin 43 (Cx43), 46 (Cx46), and 50 (Cx50). Of these three, Cx46 and Cx50 both have major functions in the mature lens. Cx46 functions as a major gap junction channel, which maintains mature lens homeostasis, while Cx50 possesses growth control properties in the lens. Cx46 expression is modulated in breast and bone tumors, and during ischemia. It is hypothesized that Cx46 provides resistance to hypoxia mediated cell death by prolonging survival. In this study, Cx46 expression was detected in human Y79 retinoblastoma cells. Decreasing the expression of Cx46 in nude mice carrying Y79 xenografts slowed early stage tumor growth. Y79 cells in culture survive for over 72 hours in 1% oxygen in vitro. C46 was upregulated in cultured lens cells when grown under hypoxia. Human lens epithelial cells, rabbit N/N1003A lens cells, and Y79 cells proliferated in 1% oxygen until Cx46 expression was depleted by use of siRNA. Protection from hypoxia-induced cell death was provided by transfection with the C-terminus of Cx46. We further determined that the promoter activity of Cx46 was increased in 1% oxygen. These results indicate that Cx46 would increase in response to hypoxia and suggest a role for Cx46 in protection from hypoxia. The studies demonstrate a novel function for Cx46 in cell survival during hypoxia.

We studied Heart Rate Variability (HRV) evolution during therapeutic hypothermia in newborns with hypoxic ischemic encephalopathy (HIE) using spectral analysis. We hypothesized that HRV measures are predictive of neurological outcome in babies with HIE. Non-stationarity in the data causes inaccurate quantification of the spectral power. A modification was proposed to power spectral analysis approach to mitigate the effect of non-stationarity. The modified and the standard approaches were applied to cardiac beat-to-beat intervals of newborns receiving hypothermia treatment. The performance of the approaches in distinguishing the RRi dynamics of two groups of newborns was assessed using area under the receiver operating characteristic (ROC) curve. Our results showed that the modified spectral analysis distinguished the two groups of neonates better than the standard approach. These results may be useful in identifying the deteriorating physiology of the infants receiving hypothermia treatment early in time and strategize alternate interventions for them.

INTRODUÇÃO: A encefalopatia hipóxico-isquêmica (EHI) corresponde a uma das maiores causas de morbidade e mortalidade neonatal. Ocorre em consequência à asfixia perinatal aguda, representada por baixo escore de Apgar e evidências de distúrbios neurológicos ao nascimento. A hipotermia terapêutica (HT) tem mostrado benefícios relevantes no prognóstico neurológico a longo prazo, por reduzir o metabolismo cerebral, retardando o início da despolarização hipóxica celular. Os efeitos da HT no sistema cardiovascular foram pouco estudados, suscitando questionamentos quanto à adequada interpretação dos achados ecocardiográficos nesta condição terapêutica. OBJETIVO: avaliar o comportamento hemodinâmico e da função ventricular de recém-nascidos com EHI na vigência de HT, utilizando-se técnicas ecocardiográficas convencionais e avançadas. MÉTODO: trata-se de um estudo observacional desenvolvido em três instituições, em que 22 recém-nascidos com EHI foram avaliados por meio da ecocardiografia nas duas fases da HT (durante a hipotermia e após o reaquecimento). O grupo controle foi composto por 22 recém-nascidos saudáveis. Os bebês foram submetidos a HT seguindo critérios do protocolo de hipotermia de cada um dos serviços. RESULTADOS: Função ventricular esquerda: as frações de ejeção (FE) e de encurtamento foram maiores após o reaquecimento (74 ± 5% e 41 ± 5% respectivamente) em relação ao grupo controle (70 ± 5%, p = 0,003 e 37 ± 4%, p = 0,002). O índice de performance miocárdica (IPM) do ventrículo esquerdo (VE) avaliado pelo Doppler pulsado se manteve constante nas duas fases da HT (0,51 ± 0,13, hipotermia = reaquecimento) e foi menor na comparação destas com o grupo controle (0,63 ± 0,18, p = 0,02). Os valores do strain circunferencial e radial, do twist, da torção e do strain longitudinal global do VE (STLGLVE) foram semelhantes entre o grupo controle e o grupo estudo, tanto durante a hipotermia quanto após o reaquecimento. Função ventricular direita: Observou-se incremento da velocidade da onda s´ do ventrículo direito (VD) após o reaquecimento (de 0,07 ± 0,02 m/s durante a hipotermia para 0,09 ± 0,01 m/s, p < 0,001), sendo esta também mais elevada quando comparada aos valores do grupo controle (0,07 ± 0,01 m/s, p < 0,001). Houve queda dos valores da variação fracional das áreas (FAC) do VD após o reaquecimento (38 ± 11% durante a hipotermia, 36 ± 11% após o reaquecimento e 43 ± 10% grupo controle), com diferenças significativas entre esses dois últimos (p = 0,03). Quanto ao IPM do VD, o grupo controle apresentou médias menores (0,29 ± 0,13) que o grupo caso durante a hipotermia (0,46 ± 0,33, p = 0,03). O strain longitudinal global do VD (STLGLVD) foi significativamente pior tanto durante a hipotermia (-18 ± -5%, p = 0,02) quanto após o reaquecimento (-18 ± 4%, p = 0,01) quando comparados ao grupo controle (-21 ± 2%). Parâmetros hemodinâmicos: A pressão sistólica na artéria pulmonar foi mais elevada no grupo estudo durante as duas fases do tratamento (hipotermia 45 ± 24 mmHg, p = 0,02 e reaquecimento 53 ± 34 mmHg, p = 0,01 versus grupo controle 29 ± 11 mmHg). A FC foi significativamente mais baixa durante a hipotermia comparada ao período após o reaquecimento (FC 111 ± 19 bpm versus 144 ± 20 bpm, p < 0,001) e ao grupo controle (FC 130 ± 16 bpm, p < 0,001). Durante o reaquecimento, observou-se elevação do débito cardíaco (DC) esquerdo e direito em relação ao período de hipotermia (DC esquerdo 214 ± 39 ml/kg/min versus 155 ± 47 ml/kg/min, p < 0,001; DC direito 369 ± 141 ml/kg/min versus 269 ± 113 ml/Kg/min, p = 0,005) sendo significativamente mais elevado que no grupo controle (DC Esquerdo 174 ± 47 ml/kg/min, p = 0,004 e DC direito 288 ± 74 ml/Kg/min, p = 0,02). CONCLUSÕES: A função ventricular esquerda permanece estável nas duas fases da HT, demonstrando o baixo comprometimento cardíaco esquerdo do resfriamento induzido. Os valores da FE, da fração de encurtamento e da onda s´ do VD, maiores após o reaquecimento, podem ser consequentes a um estado hiperdinâmico do coração. Disfunção ventricular direita foi observada nos momentos em que a pressão pulmonar estava elevada. O STLGLVD foi a única ferramenta capaz de identificar o comprometimento da função sistólica do VD durante a HT. / INTRODUCTION: The hypoxic-ischemic encephalopathy (HIE) corresponds to one of the biggest causes of neonatal morbidity and mortality. It occurs in consequence to acute perinatal asphyxia, represented by low Apgar score and evidences of neurological disorders in birth. The therapeutic hypothermia (TH) has shown significant benefits in long term neurological prognosis, by reducing the cerebral metabolism, delaying the onset of the hypoxic depolarization in cellular level. The TH effects in cardiovascular system have been insufficiently researched, raising questions regarding the adequate reading of the echocardiographic results in this condition. OBJECTIVE: to evaluate the hemodynamic and the ventricular performance of neonates with HIE submitted to TH, using conventional and advanced echocardiographic techniques. METHODS: this research is an observational study developed in three institutions, in which 22 neonates with HIE were evaluated by echocardiography in the two phases of TH (during hypothermia and after rewarming). The control group was composed by 22 healthy neonates. The infants were submitted to TH following hypothermia protocol criteria of each services. RESULTS: Left ventricular function: the ejection fraction (EF) and the shortening fraction were higher after rewarming (74 ± 5% and 41 ± 5% respectively) compared to the control group (70 ± 5%, p = 0.003 and 37 ± 4%, p = 0.002). The myocardial performance index (MPI) of the left ventricle (LV), evaluated by pulsed wave Doppler, remained constant in the two phases of TH (0.51 ± 0.13, hypothermia = rewarming) and this MPI was lower in comparison to the control group (0.63 ± 0.18, p = 0.02). The values of the circumferential and radial strain, the twist, the torsion and the global longitudinal strain (GLS) of the LV were similar between the control group and the study group, as during hypothermia as after rewarming. Right ventricular function: it was noted increment of the right ventricle (RV) s´ wave velocity after rewarming (from 0.07 ± 0.02 m/s during hypothermia to 0.09 ± 0.01 m/s, p < 0.001), also it was higher when compared to the control group (0.07 ± 0.01 m/s, p < 0.001). There was decrease of the RV fractional area change (FAC) values after rewarming (38 ± 11% during hypothermia, 36 ± 11% after rewarming and 43 ± 10% in control group), with significant differences between these two last values (p = 0.03). Regarding RV\'s MPI, the control group presented lower averages (0.29 ± 0.13) than the case group during hypothermia (0.46 ± 0.33, p = 0.03). The RV GLS was worse as during hypothermia (-18 ± -5%, p = 0.02) as after rewarming (-18 ± 4%, p = 0.01) when compared to the control group (-21 ± 2%). Hemodynamic parameters: The pulmonary artery systolic pressure was higher in the study group during the two phases of the treatment (hypothermia 45 ± 24 mmHg, p = 0.02 and rewarming 53 ± 34 mmHg, p = 0.01 versus control group 29 ± 11 mmHg). The heart rate (HR) was significantly lower during hypothermia compared to the after rewarming period (HR 111 ± 19 bpm versus 144 ± 20 bpm, p < 0.001) and to the control group (HR 130 ± 16 bpm, p < 0.001). After rewarming it was seen increase of the left and right cardiac output (CO) compared to the hypothermia period (left CO 214 ± 39 ml/kg/min versus 155 ± 47 ml/kg/min, p < 0.001; right CO 369 ± 141 ml/kg/min versus 269 ± 113 ml/Kg/min, p = 0.005), remaining significantly higher than in the control group (left CO 174 ± 47 ml/kg/min, p = 0.004 and right CO 288 ± 74 ml/Kg/min, p = 0.02). CONCLUSIONS: The LV function remains stable in the two phases of TH, showing low left cardiac impairment of the induced cooling. The values of EF, shortening fraction and RV s´ wave were higher after rewarming, possibly due to a hyperdynamic heart state. A right ventricular dysfunction was observed when the pulmonary artery systolic pressure was high. The RV GLS was the only tool able to identify the RV systolic impairment during TH.

INTRODUÇÃO: A asfixia perinatal é uma das principais causa de óbito nos recémnascidos (RN) de termo acima de 2500g no Brasil, sendo também a causa mais importante de encefalopatia e lesão cerebral permanente em crianças. Não existindo ainda um consenso acerca de qual seria o melhor critério para seu diagnóstico. OBJETIVOS: Verificar a prevalência de asfixia e de encefalopatia hipóxico-isquêmica segundo dois critérios diagnósticos, avaliar influência do tipo de parto e a evolução neurológica. MÉTODO: Corte transversal prospectivo, onde foram incluídos 30 recém-nascidos que apresentaram asfixia segundo dois critérios diagnósticos: critério 1 foi preconizado pela AAP/ACOG de 1996 (pH de cordão 7,0, disfunção múltipla de órgãos, manifestações neurológicas na primeira semana de vida além do Apgar entre 0-3 no quinto minuto); o critério 2 foi de Buonocore em 2002, modificado (pH de cordão 7,2, Apgar de 4-6 no quinto minuto e necessidade de fração de oxigênio inspirada 0,40 manter saturação de 86%); num período de dois anos (2004/2006) sendo excluídos aqueles que pudessem apresentar encefalopatia por outras causas como malformações, infecções congênitas, erro inato do metabolismo. Para realizar o diagnóstico foram colhida gasometria de cordão dos recémnascidos a termo que apresentaram Apgar de quinto minuto 6 e feitas provas de função cardíaca, hepática, renal e controle hematológico além da avaliação neurológica pelos critérios clínicos de Sarnat e Sarnat de 1976, para verificar o grau de encefalopatia. RESULTADOS: Durante este período a prevalência observada de asfixia foi de 3,2 por 1000 nascimentos a termo (IC a 95% - [2,1 por mil; 4,5 por mil]) e de encefalopatia de 1,7 por 1000 nascimentos a termo (IC a 95% - [0,8 por mil; 2,5 por mil]). A taxa de mortalidade foi de 16,7% e 36,7% evoluíram com encefalopatia grave. Não houve correlação estatística da asfixia e nem da encefalopatia quanto às características maternas, exceto uma tendência maior nas nulíparas e primíparas com parto normal. Quanto à indicação do parto 46,7% apresentava trabalho de parto sem intercorrências, mas pelo critério 1 houve maior número com sofrimento fetal relacionado à maior gravidade da asfixia. O sexo mais freqüente foi o masculino e em ambos os grupos apresentaram acidose metabólica e respiratória e alterações enzimáticas principalmente cardíacas e hepáticas e, função renal com aumento da creatinina. Foi observado que para Sarnat estágios 1 e 2, leve e moderada, houve uma maior proporção de recém-nascidos no critério 2 enquanto que para o Sarnat estágio 3, grave, a maior proporção foi com o critério 1 Resumo (p = 0,016). Enquanto o Apgar de primeiro minuto não mostrou correlação com a gravidade da encefalopatia, 85% dos recém-nascidos com encefalopatia leve/moderada tiveram Apgar de quinto minuto entre 4-6 e a maioria com quadro grave o Apgar foi entre 0-3 (p = 0,018). Houve uma tendência de acordo com o aumento da gravidade da encefalopatia a uma redução do dióxido de carbono sanguíneo, bicarbonato e aumento negativo do excesso de base além do aumento de enzima cardíaca (creatina fosfoquinase), mas não foi estatisticamente significante. CONCLUSÃO: Não houve correlação estatística entre asfixia e a gravidade da encefalopatia com fatores maternos. Todos os recém-nascidos apresentaram acidose respiratória e metabólica e entre as alterações enzimáticas cardíacas foram as mais importante. Em relação ao índice de Apgar, a nota de quinto minuto mostrou melhor correlação com a gravidade evolutiva dos pacientes. Com o critério 1 (Academia Americana de Pediatria) houve melhor correlação com a mortalidade, no entanto por ser muito rigoroso acaba por excluir recém-nascidos que evoluem com quadros de encefalopatia grave / INTRODUCTION: The perinatal asphyxia is one of the main causes of death in newborns and also the most important cause of encephalopathy and permanent cerebral lesion in children. OBJECTIVES: To check the prevalence of asphyxia and of hypoxic-ischemic encephalopathy in term newborns, using two diagnostic criteria; to assess whether the diagnostic criterion used and the type of obstetric assistance are related to the grade of seriousness of the asphyxia and of the encephalopathy. Methods: Prospective transversal cut study carried out in a public hospital in the East Zone of São Paulo, in which 30 term newborns with perinatal asphyxia were included and classified in two groups, according to two diagnostic criteria adopted: criterion 1 recommended by American Academy of Pediatrics (1996), and which considers as bearer of perinatal asphyxia the newborn presenting: cord pH 7.0, multiple organ dysfunction, neurological manifestations in the first week of life and Apgar value in the fifth minute of life between 0-3. Criterion 2 defined by Buonocore in 2002 and which consists in: cord pH 7.2, Apgar value in the fifth minute of life between 4-6 and fraction inspired of oxygen need 0.40 to maintain a saturation of 86%. To confirm the diagnosis, the following laboratorial examinations were carried out: gasometry, hepatic, renal and cardiac function tests, besides the hematological control. To assess the neurological function and verify the grade of hypoxic-ischemic encephalopathy, the clinical criteria of Sarnat and Sarnat were used. RESULTS: The prevalence of perinatal asphyxia observed in this case was of 3.2 per 1,000 term births (IC at 95% - [2.1 per one thousand; 4.5 per one thousand]) and of hypoxic-ischemic encephalopathy was of 1.7 per 1,000 term births (IC at 95% - [0.8 per one thousand; 2.5 per one thousand]). As regards the criteria used, the newborns of criterion 1 statistically presented more fetal suffering when compared to those of criterion 2, and this fact was also related to the grade of seriousness of the asphyxia. The newborns of the two groups presented cardiac changes with elevation of the specific enzyme, hepatic changes with elevation of the glutamic pyruvic and oxaloacetic transaminases and renal changes proven by elevation of creatinine, besides the relevant respiratory and metabolic acidosis. The newborns with serious metabolic acidosis and high levels of creatine phosphokinase had a greater degree of neurological impairment. In 85% of newborns with light/moderate encephalopathy was verified an Apgar value at fifth minute of life between 4-6, and in newborns with serious encephalopathy this value was between 0-3 (p = 0.018). A positive trend for Summary the presence of asphyxia and encephalopathy was found in children of primiparous mothers and born during normal parturition. When assessing the degree of neurological impairment through the criteria of Sarnat and Sarnat, A greater proportion of newborns of criterion 2 were found in the lighter degrees. In degree 3, which is the most serious, a greater proportion of newborns of criterion 1 (p = 0,016) was found. The mortality rate in these cases was of 16.7%, and most of the newborn were of criterion 1. CONCLUSION: The prevalence of perinatal asphyxia and hypoxic -ischemic encephalopathy is as mentioned in the world literature, and smaller than found in Brazil. Criterion 1 was the one that showed a better correlation with the mortality of patients. However, as it is too rigorous, it may exclude the newborn that survive and develop hypoxic-ischemic encephalopathy. As regards the type of obstetric assistance, despite the fact that no statistically significant difference was observed, there was a positive trend to the presence of asphyxia and encephalopathy in children of primiparous mothers born during normal parturition

Les lymphocytes « Natural Killer » (NK) sont des effecteurs de l’immunité innée, capables de lyser les cellules cancéreuses grâce au relargage de la protéase cytotoxique Granzyme B (GzmB). Récemment, de nouvelles stratégies anti-cancéreuses, basées sur l’utilisation des cellules NK, ont émergé et se sont révélées très prometteuses. Il est maintenant clairement établi que le microenvironnement tumoral hypoxique influence la réponse immunitaire et constitue, de ce fait, un obstacle majeur pour établir des protocoles d’immunothérapies efficaces. Des études récentes ont montré que l’autophagie est un régulateur important de l’immunité innée dans le microenvironnement tumoral, mais les mécanismes de régulation impliqués restent encore peu connus. Nous avons montré in vitro que l'hypoxie diminue la sensibilité des cellules de carcinome mammaire à la lyse dépendante des cellules NK par un mécanisme impliquant l'activation de l'autophagie. De manière intéressante, cette diminution de lyse est reversée par l’inhibition de l’autophagie. Nous avons démontré que la résistance des cellules tumorales hypoxiques à la lyse par les cellules NK n'est liée ni à un défaut de reconnaissance des cellules cibles, ni à une altération de l’activité cytotoxique des effecteurs. Nous avons mis en évidence que l'activation de l’autophagie conduit à la dégradation de GzmB dans les lysosomes des cellules hypoxiques. Ainsi, ces cellules deviennent résistantes à l’apoptose, qui est normalement induite par GzmB, transféré par les cellules NK. L’invalidation génétique et pharmacologique de l'autophagie permet de restaurer le niveau intracellulaire de GzmB et réduit la résistance des cellules cibles hypoxiques in vitro. Nos résultats mettent en évidence que l'autophagie est un régulateur primordial de la réponse immunitaire anti-tumorale dépendante des cellules NK. Nous avons validé ce concept in vivo en montrant que l’inhibition de l'autophagie favorise de manière significative la prise en charge de la tumeur par les cellules NK dans des modèles murins de mélanome et de carcinome mammaire. Cette étude contribue à l’avancé des connaissances sur la manière dont l'autophagie, induite par l'hypoxie, affecte la lyse dépendante des cellules NK et ouvre la voie à la formulation de nouvelles stratégies thérapeutiques anti-tumorales combinant l’utilisation des cellules NK à des inhibiteurs d'autophagie. / Natural killer (NK) cells are effectors of the antitumor immunity, able to kill cancer cells through the release of the cytotoxic protease granzyme B. NK-based therapies have recently emerged as promising anticancer strategies. However, it is well established that hypoxic microenvironment interferes with the function of antitumor immune cells and constitutes a major obstacle for cancer immunotherapies. Recent studies demonstrated that autophagy is an important regulator of innate immune response in this microenvironment, but the mechanism by which autophagy regulates NK cell-mediated antitumor immune responses remains elusive. Here, we demonstrate that hypoxia impairs breast cancer cell susceptibility to NK-mediated lysis in vitro via the activation of autophagy. This impairment was not related to a defect in target cell recognition by NK cells but to the degradation of NK-derived granzyme B in autophagosomes of hypoxic cells. Inhibition of autophagy by targeting beclin1 (BECN1) restored granzyme B levels in hypoxic cells in vitro and induced tumor regression in vivo by facilitating NK-mediated tumor cell killing. Together, our data highlight autophagy as a mechanism underlying the resistance of hypoxic tumor cells to NK-mediated lysis and provides a cutting-edge advance in our understanding of the underlying mechanism. This study might pave the way for the formulation of more effective NK cell-based antitumor therapies.

INTRODUÇÃO: A asfixia perinatal é uma das principais causa de óbito nos recémnascidos (RN) de termo acima de 2500g no Brasil, sendo também a causa mais importante de encefalopatia e lesão cerebral permanente em crianças. Não existindo ainda um consenso acerca de qual seria o melhor critério para seu diagnóstico. OBJETIVOS: Verificar a prevalência de asfixia e de encefalopatia hipóxico-isquêmica segundo dois critérios diagnósticos, avaliar influência do tipo de parto e a evolução neurológica. MÉTODO: Corte transversal prospectivo, onde foram incluídos 30 recém-nascidos que apresentaram asfixia segundo dois critérios diagnósticos: critério 1 foi preconizado pela AAP/ACOG de 1996 (pH de cordão 7,0, disfunção múltipla de órgãos, manifestações neurológicas na primeira semana de vida além do Apgar entre 0-3 no quinto minuto); o critério 2 foi de Buonocore em 2002, modificado (pH de cordão 7,2, Apgar de 4-6 no quinto minuto e necessidade de fração de oxigênio inspirada 0,40 manter saturação de 86%); num período de dois anos (2004/2006) sendo excluídos aqueles que pudessem apresentar encefalopatia por outras causas como malformações, infecções congênitas, erro inato do metabolismo. Para realizar o diagnóstico foram colhida gasometria de cordão dos recémnascidos a termo que apresentaram Apgar de quinto minuto 6 e feitas provas de função cardíaca, hepática, renal e controle hematológico além da avaliação neurológica pelos critérios clínicos de Sarnat e Sarnat de 1976, para verificar o grau de encefalopatia. RESULTADOS: Durante este período a prevalência observada de asfixia foi de 3,2 por 1000 nascimentos a termo (IC a 95% - [2,1 por mil; 4,5 por mil]) e de encefalopatia de 1,7 por 1000 nascimentos a termo (IC a 95% - [0,8 por mil; 2,5 por mil]). A taxa de mortalidade foi de 16,7% e 36,7% evoluíram com encefalopatia grave. Não houve correlação estatística da asfixia e nem da encefalopatia quanto às características maternas, exceto uma tendência maior nas nulíparas e primíparas com parto normal. Quanto à indicação do parto 46,7% apresentava trabalho de parto sem intercorrências, mas pelo critério 1 houve maior número com sofrimento fetal relacionado à maior gravidade da asfixia. O sexo mais freqüente foi o masculino e em ambos os grupos apresentaram acidose metabólica e respiratória e alterações enzimáticas principalmente cardíacas e hepáticas e, função renal com aumento da creatinina. Foi observado que para Sarnat estágios 1 e 2, leve e moderada, houve uma maior proporção de recém-nascidos no critério 2 enquanto que para o Sarnat estágio 3, grave, a maior proporção foi com o critério 1 Resumo (p = 0,016). Enquanto o Apgar de primeiro minuto não mostrou correlação com a gravidade da encefalopatia, 85% dos recém-nascidos com encefalopatia leve/moderada tiveram Apgar de quinto minuto entre 4-6 e a maioria com quadro grave o Apgar foi entre 0-3 (p = 0,018). Houve uma tendência de acordo com o aumento da gravidade da encefalopatia a uma redução do dióxido de carbono sanguíneo, bicarbonato e aumento negativo do excesso de base além do aumento de enzima cardíaca (creatina fosfoquinase), mas não foi estatisticamente significante. CONCLUSÃO: Não houve correlação estatística entre asfixia e a gravidade da encefalopatia com fatores maternos. Todos os recém-nascidos apresentaram acidose respiratória e metabólica e entre as alterações enzimáticas cardíacas foram as mais importante. Em relação ao índice de Apgar, a nota de quinto minuto mostrou melhor correlação com a gravidade evolutiva dos pacientes. Com o critério 1 (Academia Americana de Pediatria) houve melhor correlação com a mortalidade, no entanto por ser muito rigoroso acaba por excluir recém-nascidos que evoluem com quadros de encefalopatia grave / INTRODUCTION: The perinatal asphyxia is one of the main causes of death in newborns and also the most important cause of encephalopathy and permanent cerebral lesion in children. OBJECTIVES: To check the prevalence of asphyxia and of hypoxic-ischemic encephalopathy in term newborns, using two diagnostic criteria; to assess whether the diagnostic criterion used and the type of obstetric assistance are related to the grade of seriousness of the asphyxia and of the encephalopathy. Methods: Prospective transversal cut study carried out in a public hospital in the East Zone of São Paulo, in which 30 term newborns with perinatal asphyxia were included and classified in two groups, according to two diagnostic criteria adopted: criterion 1 recommended by American Academy of Pediatrics (1996), and which considers as bearer of perinatal asphyxia the newborn presenting: cord pH 7.0, multiple organ dysfunction, neurological manifestations in the first week of life and Apgar value in the fifth minute of life between 0-3. Criterion 2 defined by Buonocore in 2002 and which consists in: cord pH 7.2, Apgar value in the fifth minute of life between 4-6 and fraction inspired of oxygen need 0.40 to maintain a saturation of 86%. To confirm the diagnosis, the following laboratorial examinations were carried out: gasometry, hepatic, renal and cardiac function tests, besides the hematological control. To assess the neurological function and verify the grade of hypoxic-ischemic encephalopathy, the clinical criteria of Sarnat and Sarnat were used. RESULTS: The prevalence of perinatal asphyxia observed in this case was of 3.2 per 1,000 term births (IC at 95% - [2.1 per one thousand; 4.5 per one thousand]) and of hypoxic-ischemic encephalopathy was of 1.7 per 1,000 term births (IC at 95% - [0.8 per one thousand; 2.5 per one thousand]). As regards the criteria used, the newborns of criterion 1 statistically presented more fetal suffering when compared to those of criterion 2, and this fact was also related to the grade of seriousness of the asphyxia. The newborns of the two groups presented cardiac changes with elevation of the specific enzyme, hepatic changes with elevation of the glutamic pyruvic and oxaloacetic transaminases and renal changes proven by elevation of creatinine, besides the relevant respiratory and metabolic acidosis. The newborns with serious metabolic acidosis and high levels of creatine phosphokinase had a greater degree of neurological impairment. In 85% of newborns with light/moderate encephalopathy was verified an Apgar value at fifth minute of life between 4-6, and in newborns with serious encephalopathy this value was between 0-3 (p = 0.018). A positive trend for Summary the presence of asphyxia and encephalopathy was found in children of primiparous mothers and born during normal parturition. When assessing the degree of neurological impairment through the criteria of Sarnat and Sarnat, A greater proportion of newborns of criterion 2 were found in the lighter degrees. In degree 3, which is the most serious, a greater proportion of newborns of criterion 1 (p = 0,016) was found. The mortality rate in these cases was of 16.7%, and most of the newborn were of criterion 1. CONCLUSION: The prevalence of perinatal asphyxia and hypoxic -ischemic encephalopathy is as mentioned in the world literature, and smaller than found in Brazil. Criterion 1 was the one that showed a better correlation with the mortality of patients. However, as it is too rigorous, it may exclude the newborn that survive and develop hypoxic-ischemic encephalopathy. As regards the type of obstetric assistance, despite the fact that no statistically significant difference was observed, there was a positive trend to the presence of asphyxia and encephalopathy in children of primiparous mothers born during normal parturition

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Neonatal hypoxic-ischemic injury (HI) is the direct complication to severe choking and may cause brain damage. HI may be found in different stages and clinical manifestations contributing to neonatal morbidity and mortality. The neuropathology of neonatal HI insult is multi-factorial and complex. Hypoxic-ischemic brain damage begins during the insult and extends during the recovery period after reperfusion, thus, it is an evolutionary process. Adenosine deaminase (ADA) is an aminohidrolase actively involved in the metabolism of purines catalyzing irreversibly adenosine and 2'desoxiadenosine into inosine and 2'desoxinosine, respectively. The objectives of this study were to evaluate the activity of ADA in the cortex of rats subjected to neonatal HI at different post-insult time points. Effects of thiobarbituric acid reactive species (TBARS) levels were also assessed in cortex. The histological analysis was evaluated using hematoxylin eosin (HE) and glial fibrillary acidic protein (GFAP) in the cortex of these animals. The ADA activity was significantly increased 8 days after the insult in the left hemisphere in the cortex. In this period, TBARS levels were significantly increased in the cortex of these animals. HE revealed the presence of ischemic area in the cerebral cortex 8 days after HI. A moderate lymphocytic infiltration was also evidenced in the cortex during this period. A proliferation and an increase in the expression of GFAP in the periphery of the ischemic area was observed, resulting in astrocytosis in the cortex of these animals. In conclusion, an activation of the immune system was observed due to the inflammatory process caused by the HI insult that may be correlated with astrocytosis and lymphocytic infiltration observed in the cerebral cortex of animals that suffered insult 8 days after neonatal HI. / A lesão hipóxico-isquêmica (HI) neonatal é a complicação imediata à asfixia grave e pode causar dano cerebral. A HI pode apresentar-se em diferentes estágios e manifestações clínicas contribuindo assim intensamente na morbidade e mortalidade neonatal. A neuropatologia do insulto HI neonatal é multi-fatorial e complexa. O dano cerebral hipóxicoisquêmico inicia durante o insulto e estende-se no período de recuperação após a reperfusão, portanto é um processo evolutivo. A adenosina deaminase (ADA) é uma aminohidrolase que participa ativamente do metabolismo das purinas catalisando irreversivelmente a adenosina e 2 desoxiadenosina em inosina e 2 desoxinosina, respectivamente. Os objetivos deste estudo foram avaliar em ratos submetidos à HI neonatal a atividade da ADA no córtex destes animais em diferentes tempos pós-insulto. Também foram avaliados em córtex os efeitos dos níveis de espécies reativas ao ácido tiobarbitúrico (TBARS). A análise histológica foi avaliada através da hematoxilina eosina (HE) e proteína glial fibrilar ácida (GFAP) no córtex destes animais. A atividade da ADA aumentou significativamente 8 dias após o insulto no hemisfério esquerdo no córtex. Neste período os níveis de TBARS mostraram-se significativamente aumentados no córtex destes animais. A HE revelou presença de área isquêmica no córtex cerebral 8 dias após a HI. Também evidenciou uma moderada infiltração linfocitária no córtex neste período. Houve proliferação e aumento na expressão da GFAP na periferia da área isquêmica, resultando em astrocitose no córtex dos animais submetidos à HI. Conclui-se que houve uma ativação do sistema imune em decorrência do processo inflamatório causado pelo insulto HI que pode estar correlacionada com a astrocitose e a infiltração linfocitária observada no córtex cerebral dos animais que sofreram o insulto 8 dias após a HI neonatal.

This thesis is concerned with a very common disorder of iron homeostasis: iron deficiency. The specific focus is the manner in which iron deficiency influences physiological responses to hypoxia in humans. This work is predicated on observations made over many decades in vitro and in vivo, suggesting that variations in the bioavailability of iron have important consequences for certain biological processes known to depend on oxygen availability. Three separate but related studies together form the basis for this thesis. The first two, Study A and Study B, adopt a similar approach in recruiting healthy volunteers who differ according to iron status, yielding iron-deficient and iron-replete groups in both cases. In Study A, the behaviour of the pulmonary circulation is investigated during a sustained hypoxic exposure, before and after an intravenous infusion of iron. In Study B, skeletal muscle metabolism is explored, both at the level of high-energy phosphate metabolism and the integrated physiological responses to exercise on a cycle ergometer. In the third study, Study C, a different approach is taken, recruiting patients with chronic obstructive pulmonary disease (COPD), and exploring the prevalence and associations of iron deficiency in this condition. Chapters 2 and 3 describe experiments using sustained hypoxia in a normobaric chamber, during which the pulmonary circulation is assessed non-invasively using Doppler echocardiography. These reveal augmented hypoxic pulmonary vasoconstriction (HPV) in iron-deficient individuals, who also exhibit greater sensitivity to the effects of an infusion of intravenous iron. Additionally, the way in which certain circulating mediators important for iron haemostasis change over the course of these hypoxic exposures, and how iron status influences these responses, is explored. Chapter 4 reports the findings of experiments using 31P-magnetic resonance spectroscopy and cardiopulmonary exercise testing, which demonstrate abnormal whole-body metabolism in iron-deficient individuals during large muscle-mass exercise, despite the absence of a clear defect in mitochondrial oxidative phosphorylation. Intravenous iron is found to have significant effects to alter the lactate threshold in healthy individuals, but the effects are more striking in iron-deficient individuals. Collectively, these experiments imply that iron deficiency promotes a more glycolytic phenotype. Chapter 5 explores iron deficiency in COPD, a condition in which pulmonary vascular disease, hypoxia and skeletal muscle dysfunction coexist, and examines some of the difficulties in assessing iron status in the setting of a chronic inflammatory disorder. Iron deficiency is found to be common, and unexpectedly associated with significantly more severe hypoxaemia, in patients with COPD. Possible reasons for these findings, and their clinical implications, are considered. Chapter 6 provides a summary of the main conclusions to be drawn from the studies presented in this thesis.

This thesis is concerned with the role of iron in modulating right ventricular (RV) afterload during exercise in healthy people aged between 50 and 80 years. This is predicated on the requirement of the hypoxia-inducible factor (HIF) pathway for ferrous iron. A secondary objective is to examine the reactive oxygen species (ROS) hypothesis in human hypoxic pulmonary vasoconstriction (HPV) using exposure to hyperoxia. Chapters 3 and 4 describe basal relationships that may affect the HIF pathway and exercise capacity during ageing. These were explored in 113 participants using blood tests and exercise tests. Age and inflammatory factors, C-reactive protein, and ferritin were associated with impaired exercise capacity. In addition, ageing did not significantly affect haematological variables or iron status indicators. Chapters 5 and 6 describe the effect of a single intravenous iron infusion on the haematological variables in 32 participants in a randomised, placebo-controlled and double-blinded study. The effects of iron infusion on RV afterload during light exercise, and exercise capacity during heavy exercise, were examined in these participants. With iron infusion, erythropoietin production, and the increase in RV afterload during light exercise were blunted, potentially indicating involvement of the HIF pathway. However, blunting of RV afterload neither influenced the cardiac output during light exercise nor exercise capacity. Chapter 7 describes a study of 11 healthy volunteers, which investigated the ROS hypothesis in HPV using acute isocapnic hypoxia following an 8-hour exposure to hyperoxia. This sustained hyperoxic exposure did not influence the hypoxic behavior of the pulmonary vasculature. This thesis demonstrates the complex relationship between iron status and exercise capacity in older adults. It shows that the decrease in RV afterload during exercise caused by intravenous iron supplementation does not lead to an augmented cardiac output or exercise capacity. Finally, it calls into question the role of ROS in HPV.