Previously it has been shown that cultures of Plasmodium falciparum died following exposure to a febrile temperature of 40°C, as demonstrated by a decrease in parasitemia of the following generation. In the current study, the effect of 40°C treatment on culture media, erythrocytes, and parasite glucose consumption, were ruled out as possible influences on parasite death, demonstrating that 40°C impacted the parasites directly. Metabolic profiling of DNA synthesis, protein synthesis, and glucose utilization during exposure to 40°C clearly indicated that febrile temperatures had direct effect on major metabolic pathways and parasite development, beginning 20-24 hr after erythrocyte invasion. The ring stages were relatively refractory to heat and recovered completely if returned to 37°C. The mechanism of parasite death was investigated for evidence of an apoptosis-like pathway in cells treated with 40°C, chloroquine, and staurosporine. Lack of typical physiological hallmarks, namely, caspase activation, characteristic mitochondrial membrane potential changes, and DNA degradation as indicated by DNA laddering, eliminated ‘classical’, apoptosis as a mechanism of parasite death. Parasites dying under the influence of 40°C, staurosporine, and chloroquine initially appeared pyknotic in light and electron microscopy, as in apoptosis, but eventual swelling and lysis of the food vacuole membrane led to secondary necrosis. Initially, chloroquine did induce DNA laddering, but it was later attributed to occult white blood cell contamination. While not apoptosis, the results do not rule out other forms of temperature-induced programmed cell death.

No description available.

INTRODUÇÃO: Neutropenia febril (NF) é frequente complicação quimioterapia para tumores sólidos e é de suma importância a identificação dos pacientes de alto risco para o seu desenvolvimento. OBJETIVOS: Caracterização clínica, laboratorial e dos fatores de risco para NF em pacientes admitidos para antibioticoterapia. MATERIAL E MÉTODOS: Estudo retrospectivo de todos os pacientes consecutivamente internados com NF no ICESP (Instituto do Câncer do Estado de São Paulo) entre maio de 2008 e maio de 2012. Critérios de inclusão: idade >= 16 anos, diagnóstico de NF (temperatura axilar >= 37,8ºC e neutrófilos < 500/mm3 ou entre 500-1000/mm³ com tendência à queda) em pacientes portadores de tumor sólido. Dados clínico-laboratoriais e de evolução foram coletados; realizada análise univariada e multivariada a fim de investigar a relação entre os fatores de risco e o desenvolvimento de complicações. RESULTADOS: 333 episódios de NF em 295 pacientes com tumores sólidos foram avaliados. Idade mediana de 57 anos (16-88), 150 do sexo feminino (51%). Os sítios primários das neoplasias mais frequentes foram mama (15%), pulmão (14%), sarcomas (13%), colorretal (10%), estômago (9%), cabeça e pescoço (8%) e testículo (5%). 31 pacientes (10%) apresentaram mais de um episódio de NF. À admissão, a mediana de contagem de neutrófilos foi 690/mm3, e a mediana de MASCC atribuído 19 (7-26). Sítios de infecção mais comumente identificados foram pulmão (19%), trato urinário (15%), corrente sanguínea (13%), abdominal (10%) e partes moles (8%); quanto à etiologia, bacilos Gram-negativos isolados em 36 (11%) episódios e cocos Gram-positivos em 15 (9%). Mediana de internação de 10 dias (0-106 dias). Alguma complicação grave foi identificada em 248 (74%) episódios, sendo que hipotensão (47%), admissão em UTI (35%), insuficiência renal (30%), insuficiência respiratória (19%) e alteração do estado mental (17%) as mais comuns (> 10%). A mortalidade foi 14% (46 pacientes). A análise univariada revelou como fatores de risco para complicações idade >= 60 anos (OR 3.1, 95%CI 1.75-5.47, p 0.0001), controle sistêmico da neoplasia (OR 0.51, 95%CI 0.31-0.85, p 0.01), DPOC (OR 4.45, CI95% 1.71 - 11.54, p 0.0016), presença de sintomas ao diagnóstico (OR 2.16, CI95% 1.26-3.69, p 0.0063), desidratação (OR 4.63, CI95% 2.57-8.31, p<0.0001) e regular ou mau estado geral (OR 3.31, CI95% 1.93-5.68, p<0.0001). Na análise multivariada, permaneceram como fatores de risco a desidratação (OR 3.7, CI95% 2.09-6.78, p 0.000009), DPOC (OR 3.7, CI 95% 1.27-11.04, p 0.0166) e idade >= 60 anos (OR 2.5, CI95% 1.37-4.58, p 0.0029). O modelo multivariado corretamente classificou os episódios como de alto risco em 75% dos eventos. Elaboramos um novo escore de risco baseado nos valores de OR, onde pacientes desidratados receberam quatro pontos, aqueles com DPOC três pontos e aqueles com idade >= 60 anos, dois pontos. O escore final corresponde à soma das parcelas acima. Consideramos os pacientes como de alto risco com escore > 5 pontos (sensibilidade 72%, especificidade 64%). CONCLUSÕES: Complicações clínicas graves são comuns durante os episódios de NF, em pacientes com tumores sólidos. DPOC, idade >= 60 anos e desidratação representam fatores de risco para o desenvolvimento de complicações. Um novo escore de fácil execução foi proposto, o qual deverá ser validado prospectivamente / BACKGROUND: Febrile neutropenia (FN) is a frequent complication during chemotherapy in solid tumors, and to identify those patients (pts) with higher risk of developing complications during FN episodes is important. Here we aimed to characterize those risk factors for severe complications during FN episodes in pts with solid tumors, admitted for intravenous antibiotics. MATERIAL AND METHODS: It is a retrospective study of all consecutive pts admitted with FN at ICESP (Instituto do Câncer do Estado de São Paulo) between May/2008 and May/2012. Eligibility criteria included: age >= 16y, the diagnosis of FN (documented axillary temperature greater than 37.8°C, and neutrophil count < 500/mm3 or expected to fall below 500/mm3) as an adverse event of chemotherapy for a solid tumor. Potentially life-threatening complications during FN episodes were collected and univariate and multivariate logistic regression analyses were performed to assess the relationship between risk factors and these complications. RESULTS: 333 FN episodes in 295 pts with solid tumors were studied. Median age was 57 y (16-88), 150 female (51%). Most frequent primary sites included: breast (15%), lung (14%), bone/soft tissues (13%), colorectal (10%), stomach (9%), head & neck (8%) and testis (5%). 31 pts (10%) presented more than 1 FN episode. At admission, median neutrophil count was 690/mm3, and the median MASCC score was 19 (7-26). Infection sites were identified as pulmonary (19%), urinary tract (15%), bloodstream (13%), abdominal (10%) and soft tissues (8%), and regarding etiology, Gram-negative bacilli could be isolated in 36 (11%) and Gram-positive cocci in 15 FN episodes (9%). All pts were admitted with a median duration of hospital stay of 10 d (0-106 d). Overall, a severe complication as a consequence of FN was detected in 248 episodes (74%), being hypotension (47%), ICU admission (35%), renal failure (30%), respiratory failure (19%) and altered mental state (17%) the most common (> 10%), and 46 pts died (14%). A univariate analysis revealed age >= 60y (OR 3.1, 95%CI 1.75-5.47, p 0.0001), controlled cancer (OR 0.51, 95%CI 0.31-0.85, p 0.01), previous COPD (OR 4.45, CI95% 1.71 - 11.54, p 0.0016), presence of symptoms (OR 2.16, CI95% 1.26-3.69, p 0.0063) or dehydration (OR 4.63, CI95% 2.57-8.31, p < 0.0001) and regular or bad general condition (OR 3.31, CI95% 1.93-5.68, p < 0.0001) as risk factors for complications. On multivariate analysis, only dehydration (OR 3.7, CI95% 2.09-6.78, p 0.000009), previous COPD (OR 3.7, CI 95% 1.27-11.04, p 0.0166) and age >= 60y (OR 2.5, CI95% 1.37-4.58, p 0.0029) were associated with severe complications. The multivariate model correctly classified 75% of all FN episodes as complicated. We elaborated a new risk score based on the OR, where dehydrated pts scored 4 points, those with COPD 3 points and those with age >= 60y 2 points. The final score was calculated by the sum of all above. We have considered as high risk pts those who scored > 5 points (sensitivity 72%, specificity 64%). CONCLUSIONS: Severe complications were common during febrile neutropenic episodes in pts with solid tumors. COPD, age >= 60 y and dehydration represent clinically significant risk factors for severe complications in FN pts. A new score was proposed, though it should be prospectively validated

INTRODUÇÃO: Neutropenia febril (NF) é frequente complicação quimioterapia para tumores sólidos e é de suma importância a identificação dos pacientes de alto risco para o seu desenvolvimento. OBJETIVOS: Caracterização clínica, laboratorial e dos fatores de risco para NF em pacientes admitidos para antibioticoterapia. MATERIAL E MÉTODOS: Estudo retrospectivo de todos os pacientes consecutivamente internados com NF no ICESP (Instituto do Câncer do Estado de São Paulo) entre maio de 2008 e maio de 2012. Critérios de inclusão: idade >= 16 anos, diagnóstico de NF (temperatura axilar >= 37,8ºC e neutrófilos < 500/mm3 ou entre 500-1000/mm³ com tendência à queda) em pacientes portadores de tumor sólido. Dados clínico-laboratoriais e de evolução foram coletados; realizada análise univariada e multivariada a fim de investigar a relação entre os fatores de risco e o desenvolvimento de complicações. RESULTADOS: 333 episódios de NF em 295 pacientes com tumores sólidos foram avaliados. Idade mediana de 57 anos (16-88), 150 do sexo feminino (51%). Os sítios primários das neoplasias mais frequentes foram mama (15%), pulmão (14%), sarcomas (13%), colorretal (10%), estômago (9%), cabeça e pescoço (8%) e testículo (5%). 31 pacientes (10%) apresentaram mais de um episódio de NF. À admissão, a mediana de contagem de neutrófilos foi 690/mm3, e a mediana de MASCC atribuído 19 (7-26). Sítios de infecção mais comumente identificados foram pulmão (19%), trato urinário (15%), corrente sanguínea (13%), abdominal (10%) e partes moles (8%); quanto à etiologia, bacilos Gram-negativos isolados em 36 (11%) episódios e cocos Gram-positivos em 15 (9%). Mediana de internação de 10 dias (0-106 dias). Alguma complicação grave foi identificada em 248 (74%) episódios, sendo que hipotensão (47%), admissão em UTI (35%), insuficiência renal (30%), insuficiência respiratória (19%) e alteração do estado mental (17%) as mais comuns (> 10%). A mortalidade foi 14% (46 pacientes). A análise univariada revelou como fatores de risco para complicações idade >= 60 anos (OR 3.1, 95%CI 1.75-5.47, p 0.0001), controle sistêmico da neoplasia (OR 0.51, 95%CI 0.31-0.85, p 0.01), DPOC (OR 4.45, CI95% 1.71 - 11.54, p 0.0016), presença de sintomas ao diagnóstico (OR 2.16, CI95% 1.26-3.69, p 0.0063), desidratação (OR 4.63, CI95% 2.57-8.31, p<0.0001) e regular ou mau estado geral (OR 3.31, CI95% 1.93-5.68, p<0.0001). Na análise multivariada, permaneceram como fatores de risco a desidratação (OR 3.7, CI95% 2.09-6.78, p 0.000009), DPOC (OR 3.7, CI 95% 1.27-11.04, p 0.0166) e idade >= 60 anos (OR 2.5, CI95% 1.37-4.58, p 0.0029). O modelo multivariado corretamente classificou os episódios como de alto risco em 75% dos eventos. Elaboramos um novo escore de risco baseado nos valores de OR, onde pacientes desidratados receberam quatro pontos, aqueles com DPOC três pontos e aqueles com idade >= 60 anos, dois pontos. O escore final corresponde à soma das parcelas acima. Consideramos os pacientes como de alto risco com escore > 5 pontos (sensibilidade 72%, especificidade 64%). CONCLUSÕES: Complicações clínicas graves são comuns durante os episódios de NF, em pacientes com tumores sólidos. DPOC, idade >= 60 anos e desidratação representam fatores de risco para o desenvolvimento de complicações. Um novo escore de fácil execução foi proposto, o qual deverá ser validado prospectivamente / BACKGROUND: Febrile neutropenia (FN) is a frequent complication during chemotherapy in solid tumors, and to identify those patients (pts) with higher risk of developing complications during FN episodes is important. Here we aimed to characterize those risk factors for severe complications during FN episodes in pts with solid tumors, admitted for intravenous antibiotics. MATERIAL AND METHODS: It is a retrospective study of all consecutive pts admitted with FN at ICESP (Instituto do Câncer do Estado de São Paulo) between May/2008 and May/2012. Eligibility criteria included: age >= 16y, the diagnosis of FN (documented axillary temperature greater than 37.8°C, and neutrophil count < 500/mm3 or expected to fall below 500/mm3) as an adverse event of chemotherapy for a solid tumor. Potentially life-threatening complications during FN episodes were collected and univariate and multivariate logistic regression analyses were performed to assess the relationship between risk factors and these complications. RESULTS: 333 FN episodes in 295 pts with solid tumors were studied. Median age was 57 y (16-88), 150 female (51%). Most frequent primary sites included: breast (15%), lung (14%), bone/soft tissues (13%), colorectal (10%), stomach (9%), head & neck (8%) and testis (5%). 31 pts (10%) presented more than 1 FN episode. At admission, median neutrophil count was 690/mm3, and the median MASCC score was 19 (7-26). Infection sites were identified as pulmonary (19%), urinary tract (15%), bloodstream (13%), abdominal (10%) and soft tissues (8%), and regarding etiology, Gram-negative bacilli could be isolated in 36 (11%) and Gram-positive cocci in 15 FN episodes (9%). All pts were admitted with a median duration of hospital stay of 10 d (0-106 d). Overall, a severe complication as a consequence of FN was detected in 248 episodes (74%), being hypotension (47%), ICU admission (35%), renal failure (30%), respiratory failure (19%) and altered mental state (17%) the most common (> 10%), and 46 pts died (14%). A univariate analysis revealed age >= 60y (OR 3.1, 95%CI 1.75-5.47, p 0.0001), controlled cancer (OR 0.51, 95%CI 0.31-0.85, p 0.01), previous COPD (OR 4.45, CI95% 1.71 - 11.54, p 0.0016), presence of symptoms (OR 2.16, CI95% 1.26-3.69, p 0.0063) or dehydration (OR 4.63, CI95% 2.57-8.31, p < 0.0001) and regular or bad general condition (OR 3.31, CI95% 1.93-5.68, p < 0.0001) as risk factors for complications. On multivariate analysis, only dehydration (OR 3.7, CI95% 2.09-6.78, p 0.000009), previous COPD (OR 3.7, CI 95% 1.27-11.04, p 0.0166) and age >= 60y (OR 2.5, CI95% 1.37-4.58, p 0.0029) were associated with severe complications. The multivariate model correctly classified 75% of all FN episodes as complicated. We elaborated a new risk score based on the OR, where dehydrated pts scored 4 points, those with COPD 3 points and those with age >= 60y 2 points. The final score was calculated by the sum of all above. We have considered as high risk pts those who scored > 5 points (sensitivity 72%, specificity 64%). CONCLUSIONS: Severe complications were common during febrile neutropenic episodes in pts with solid tumors. COPD, age >= 60 y and dehydration represent clinically significant risk factors for severe complications in FN pts. A new score was proposed, though it should be prospectively validated

Les neurones des couches superficielles du collicule supérieur et du cortex visuel primaire du rat adulte sont sensibles à de basses fréquences spatiales de haut contraste défilant à des vitesses élevées. Entre les jours post-nataux 27-30 et l’âge adulte, les fréquences temporelles optimales des neurones du cortex visuel primaire augmentent, tandis que leurs seuils de contraste diminuent. Cependant, les fréquences spatiales optimales, les valeurs de résolution spatiale et les bandes passantes spatiales de ces neurones sont, dès l’ouverture des paupières, similaires à celles observées chez le rat adulte. Ces profils de réponse neuronale suggèrent que les projections rétino-colliculaires et rétino-géniculo-corticales sont essentiellement issues de neurones ganglionnaires rétinofuges magnocellulaires et koniocellulaires. Les neurones du cortex visuel primaire du rat ayant subi des convulsions hyperthermiques présentent, dès l’ouverture des paupières, de basses fréquences spatiales optimales, de larges bandes passantes directionnelles et temporelles ainsi que des seuils de contraste élevés par rapport aux neurones du rat normal. À l’âge adulte, de basses fréquences temporelles optimales et de larges bandes passantes spatiales sont également observées chez le rat ayant subi des convulsions hyperthermiques. L’altération des profils de réponse des neurones du cortex visuel primaire du rat ayant subi de convulsions hyperthermiques suggère un déséquilibre entre les mécanismes neuronaux excitateurs et inhibiteurs de cette aire corticale. Ces résultats suggèrent également qu’un épisode unique de convulsions fébriles infantiles suffit à altérer le développement des propriétés spatio-temporelles des champs récepteurs des neurones du cortex visuel primaire. / Neurons in superficial layers of the rat superior colliculus and primary visual cortex are sensitive to highly contrasted low spatial frequencies drifting at fast speeds. Between post-natal days 27-30 and adulthood, the optimal temporal frequencies of neurons in the primary visual cortex increase, whereas their contrast thresholds decrease. However, the optimal spatial frequencies, spatial resolution values and spatial bandwidths of these neurons are, soon after eyelid opening, similar to those observed in the adult rat. These neuronal response profiles suggest that the retino-collicular and retino-geniculo-cortical projections are mainly innervated by magnocellular and koniocellular retinal ganglion cells. Neurons in the primary visual cortex of rats having experienced hyperthermic seizures are, soon after eyelid opening, sensitive to low optimal spatial frequencies and show broad directional and temporal bandwidths, as well as elevated contrast thresholds when compared to neurons of normal rats. At adulthood, low optimal temporal frequencies and broad spatial bandwidths are also observed in rats having experienced hyperthermic seizures. The alteration of response profiles of neurons in the primary visual cortex of rats having experienced hyperthermic seizures suggests an unbalance between excitatory and inhibitory mechanisms in this cortical structure. These results also suggest that a single episode of febrile seizures could be sufficient to impede the development of the spatio-temporal receptive field properties of neurons in the primary visual cortex.

Les neurones des couches superficielles du collicule supérieur et du cortex visuel primaire du rat adulte sont sensibles à de basses fréquences spatiales de haut contraste défilant à des vitesses élevées. Entre les jours post-nataux 27-30 et l’âge adulte, les fréquences temporelles optimales des neurones du cortex visuel primaire augmentent, tandis que leurs seuils de contraste diminuent. Cependant, les fréquences spatiales optimales, les valeurs de résolution spatiale et les bandes passantes spatiales de ces neurones sont, dès l’ouverture des paupières, similaires à celles observées chez le rat adulte. Ces profils de réponse neuronale suggèrent que les projections rétino-colliculaires et rétino-géniculo-corticales sont essentiellement issues de neurones ganglionnaires rétinofuges magnocellulaires et koniocellulaires. Les neurones du cortex visuel primaire du rat ayant subi des convulsions hyperthermiques présentent, dès l’ouverture des paupières, de basses fréquences spatiales optimales, de larges bandes passantes directionnelles et temporelles ainsi que des seuils de contraste élevés par rapport aux neurones du rat normal. À l’âge adulte, de basses fréquences temporelles optimales et de larges bandes passantes spatiales sont également observées chez le rat ayant subi des convulsions hyperthermiques. L’altération des profils de réponse des neurones du cortex visuel primaire du rat ayant subi de convulsions hyperthermiques suggère un déséquilibre entre les mécanismes neuronaux excitateurs et inhibiteurs de cette aire corticale. Ces résultats suggèrent également qu’un épisode unique de convulsions fébriles infantiles suffit à altérer le développement des propriétés spatio-temporelles des champs récepteurs des neurones du cortex visuel primaire. / Neurons in superficial layers of the rat superior colliculus and primary visual cortex are sensitive to highly contrasted low spatial frequencies drifting at fast speeds. Between post-natal days 27-30 and adulthood, the optimal temporal frequencies of neurons in the primary visual cortex increase, whereas their contrast thresholds decrease. However, the optimal spatial frequencies, spatial resolution values and spatial bandwidths of these neurons are, soon after eyelid opening, similar to those observed in the adult rat. These neuronal response profiles suggest that the retino-collicular and retino-geniculo-cortical projections are mainly innervated by magnocellular and koniocellular retinal ganglion cells. Neurons in the primary visual cortex of rats having experienced hyperthermic seizures are, soon after eyelid opening, sensitive to low optimal spatial frequencies and show broad directional and temporal bandwidths, as well as elevated contrast thresholds when compared to neurons of normal rats. At adulthood, low optimal temporal frequencies and broad spatial bandwidths are also observed in rats having experienced hyperthermic seizures. The alteration of response profiles of neurons in the primary visual cortex of rats having experienced hyperthermic seizures suggests an unbalance between excitatory and inhibitory mechanisms in this cortical structure. These results also suggest that a single episode of febrile seizures could be sufficient to impede the development of the spatio-temporal receptive field properties of neurons in the primary visual cortex.

No description available.

Les différences sexuelles ne se limitent pas uniquement aux organes de reproduction, elles sont aussi très marquées dans plusieurs pathologies humaines. De ce fait, les études impliquant un seul sexe ne pourraient jamais permettre d’élucider les mécanismes qui sous-tendent ces pathologies. De plus, l’exclusion des femelles/filles/femmes des protocoles de recherche a des impacts négatifs sur la qualité de vie des patients, plus spécifiquement celle des filles et femmes. Des études récentes ont suggéré que la testostérone et ses métabolites affectent le développement de l’hippocampe aux niveaux biochimique, morphologique et fonctionnel. En revanche, les données ne sont pas aussi extensives que celles de leurs rôles chez les adultes. Ainsi, une meilleure compréhension des mécanismes par lesquels l’hormone stéroïdienne influence le développement du cerveau facilitera l’identification des cibles thérapeutiques de plusieurs maladies neurodéveloppementales qui affectent le fonctionnement de l’hippocampe. Afin de se développer adéquatement, le cerveau mâle requiert une exposition aux hormones sexuelles mâles pendant une période de temps donnée. En revanche, le cerveau femelle possède une phase critique peu après la naissance au cours de laquelle une exposition aux hormones sexuelles mâles le masculinise en produisant des caractéristiques comparables à celles rencontrées chez des mâles biologiques. Ainsi, la capacité de manipuler les cerveaux femelles dans le but de les masculiniser représente un outil expérimental important pour investiguer les différences sexuelles. Du fait que les hormones sexuelles telles que la testostérone et l’estradiol représentent respectivement l’élément caractéristique de chacun des sexes, cette thèse a pour objectif de disséquer l’implication de la testostérone dans le développement et le fonctionnement du cerveau en étudiant en plus des rats mâles et femelles, des femelles traitées avec la testostérone ainsi que des mâles rendus insensibles à la testostérone. En premier lieu, nous avons investigué sur un système de neurotransmission spécifique, à savoir le système GABAergique, qui est important pour le contrôle des convulsions communément observées dans l’épilepsie. Ce système possède des particularités notables en fonction du sexe, particularités qui pourraient être l’une des causes de la prédisposition des mâles à l’épilepsie. En effet, notre étude révèle qu’au niveau basal, les femelles ainsi que les mâles insensibles à la testostérone montrent très tôt au cours de leur développement une localisation à la membrane du co-transporteur KCC2 qui régule la force de la neurotransmission inhibitrice. Par ailleurs, nous avons aussi détecté des niveaux élevés du neurotrophine BDNF qui est un puissant modulateur du fonctionnement des cellules GABAergiques, ceci, au cours de la première semaine postnatale. Par ailleurs, chez les adultes, nous avons trouvé que les femelles ainsi que les mâles insensibles à la testostérone montrent une augmentation de la transmission GABergique spontanée comparativement aux mâles et aux femelles qui ont été exposées à la testostérone. En somme, ces données démontrent que le fonctionnement de la circuiterie GABAergique est modulé par le niveau de testostérone périnatal, ce qui suggère d’un rôle des hormones sexuelles dans la régulation de l’excitabilité cellulaire. De plus, les différences sexuelles dans le cerveau sont largement déterminées par des facteurs extrinsèques. Parmi ces derniers, le stress du début de la vie est un facteur extrinsèque puissant qui altère l’habileté à contrôler la rétroaction négative des glucocorticoïdes sur l’axe hypothalamo-hypophyso-surrénalien (HHS). Le stress est également connu pour affecter différentiellement les rats mâles comparativement aux femelles. Nous démontrons alors que la corticostérone rend l’hippocampe vulnérable à une seconde insulte, telle que les épilepsies induites par l’hyperthermie. En effet, chez les rats traités à la corticostérone, la latence d’induction des épilepsies par hyperthermie est réduite, le temps de récupération plus long et le nombre d’évènements épileptiques plus nombreux. En outre, nous avons trouvé que tous ces effets sont plus proéminents chez les mâles que chez les femelles. Ces données confirment l’existence d’un lien entre le stress du début de la vie et la susceptibilité aux convulsions hyperthermiques chez les rats mâles et femelles. Une meilleure compréhension des conséquences des convulsions fébriles pourrait aider dans le pronostic et le traitement des patients souffrant d’épilepsie. Somme toute, cette thèse met en lumière le rôle complexe des hormones sexuelles dans la régulation des circuits GABAergiques, des réponses au stress et de l’hyperexcitabilité du cerveau en développement. Une meilleure compréhension des mécanismes pathologiques propres aux modèles animaux mâles et femelles résulterait en de meilleures interventions et thérapies aussi bien chez les hommes que les chez les femmes. / Sex differences extend far beyond reproductive health — there is a widespread prevalence of sex differences in many human diseases and conditions. Therefore, studies limited to a single-sex cannot fully give a comprehensive picture of the underlying disease mechanisms, and the neglect of females/girls/women in biological research negatively impacts patients' quality of life, especially women. Recent data suggest that testosterone and its metabolites affect the hippocampus during development at the biochemical, morphological, and functional levels, although the data are not nearly as extensive as what is known in adults. Therefore, a better understanding of these effects will elucidate steroid hormone-dependent mechanisms of brain development and, possibly, help identifying ways to mitigate the burden of the many neurodevelopmental disorders that involve hippocampal function. The male brain is unique in that it must be exposed to male sex hormones for a fixed period of time, which is so-called critical period. This is deemed a critical period because if androgens levels do not rise at this time in males, the brain will fail to be masculinized. The female brain, on the other hand, has a sensitive period shortly after birth, during which exposure to male sex hormones may masculinize the brain and produce features comparable to those seen in biological males. This capacity to manipulate females toward more masculinized brains represent an important experimental tool to investigate sex differences. Because sexual hormones, such as testosterone and estradiol, are a distinct point of divergence between sexes, my thesis proposes to study the implication of testosterone by using, in addition to male and female animals, females treated with testosterone as well as testosterone-insensitive male rats. First, we investigated a specific neurotransmitter system, the GABAergic system, which contributes to the control of seizures commonly observed in epilepsies. This system shows robust differences between males and females, which may be involved with the predisposition to epilepsy observed in males. Our study revealed that at baseline conditions female and testosterone-insensitive male rats show an earlier localization at the membrane of the chloride co-transporter KCC2, which regulates the strengths of inhibitory neurotransmission, and higher levels of the neurotrophin BDNF, which is a powerful modulator of GABAergic cell function, during the first postnatal week. In addition, we found that female and testosterone-insensitive male rats show enhanced spontaneous GABA synaptic transmission when compared to males and testosterone-exposed females in adults. Overall, these data show that perinatal testosterone levels modulate GABAergic circuit function, suggesting a role of sex hormones in regulating cell excitability. Second, sex differences in the brain are largely determined by extrinsic factors. Early-life stress is one such powerful extrinsic factor that impairs the ability to control glucocorticoid negative feedback on the HPA axis. Stress is also known to differentially affect male and female rats. Here, we show that corticosterone alone renders the hippocampus vulnerable to a second insult, namely hyperthermia-induced seizures, in fact in corticosterone-treated rats the latency to hyperthermia-induced seizures was shorter, the recovery time longer, and a larger number of hyperthermia-induced seizures. Further, these effects were a lot more prominent in males than in females. These findings support a link between early-life stress and hyperthermic seizure susceptibility in both male and female rats. A better understanding of the consequences of febrile seizures could help improve the prognosis and treatment of patients with epilepsy. Altogether, these findings shed light on the complex roles of sex hormones in regulating GABAergic circuits, stress responses and circuit hyper-excitability in the developing brain. A better understanding of disease-mechanisms underlying male and female animal models could lead to better interventions and therapeutics in both men and women.

Le co-transporteur KCC2 spécifique au potassium et chlore a pour rôle principal de réduire la concentration intracellulaire de chlore, entraînant l’hyperpolarisation des courants GABAergic l’autorisant ainsi à devenir inhibiteur dans le cerveau mature. De plus, il est aussi impliqué dans le développement des synapses excitatrices, nommées aussi les épines dendritiques. Le but de notre projet est d’étudier l’effet des modifications concernant l'expression et la fonction de KCC2 dans le cortex du cerveau en développement dans un contexte de convulsions précoces. Les convulsions fébriles affectent environ 5% des enfants, et ce dès la première année de vie. Les enfants atteints de convulsions fébriles prolongées et atypiques sont plus susceptibles à développer l’épilepsie. De plus, la présence d’une malformation cérébrale prédispose au développement de convulsions fébriles atypiques, et d’épilepsie du lobe temporal. Ceci suggère que ces pathologies néonatales peuvent altérer le développement des circuits neuronaux irréversiblement. Cependant, les mécanismes qui sous-tendent ces effets ne sont pas encore compris. Nous avons pour but de comprendre l'impact des altérations de KCC2 sur la survenue des convulsions et dans la formation des épines dendritiques. Nous avons étudié KCC2 dans un modèle animal de convulsions précédemment validé, qui combine une lésion corticale à P1 (premier jour de vie postnatale), suivie d'une convulsion induite par hyperthermie à P10 (nommés rats LHS). À la suite de ces insultes, 86% des rats mâles LHS développent l’épilepsie à l’âge adulte, au même titre que des troubles d’apprentissage. À P20, ces animaux presentent une augmentation de l'expression de KCC2 associée à une hyperpolarisation du potentiel de réversion de GABA. De plus, nous avons observé des réductions dans la taille des épines dendritiques et l'amplitude des courants post-synaptiques excitateurs miniatures, ainsi qu’un déficit de mémoire spatial, et ce avant le développement des convulsions spontanées. Dans le but de rétablir les déficits observés chez les rats LHS, nous avons alors réalisé un knock-down de KCC2 par shARN spécifique par électroporation in utero. Nos résultats ont montré une diminution de la susceptibilité aux convulsions due à la lésion corticale, ainsi qu'une restauration de la taille des épines. Ainsi, l’augmentation de KCC2 à la suite d'une convulsion précoce, augmente la susceptibilité aux convulsions modifiant la morphologie des épines dendritiques, probable facteur contribuant à l’atrophie de l’hippocampe et l’occurrence des déficits cognitifs. Le deuxième objectif a été d'inspecter l’effet de la surexpression précoce de KCC2 dans le développement des épines dendritiques de l’hippocampe. Nous avons ainsi surexprimé KCC2 aussi bien in vitro dans des cultures organotypiques d’hippocampe, qu' in vivo par électroporation in utero. À l'inverse des résultats publiés dans le cortex, nous avons observé une diminution de la densité d’épines dendritiques et une augmentation de la taille des épines. Afin de confirmer la spécificité du rôle de KCC2 face à la région néocorticale étudiée, nous avons surexprimé KCC2 dans le cortex par électroporation in utero. Cette manipulation a eu pour conséquences d’augmenter la densité et la longueur des épines synaptiques de l’arbre dendritique des cellules glutamatergiques. En conséquent, ces résultats ont démontré pour la première fois, que les modifications de l’expression de KCC2 sont spécifiques à la région affectée. Ceci souligne les obstacles auxquels nous faisons face dans le développement de thérapie adéquat pour l’épilepsie ayant pour but de moduler l’expression de KCC2 de façon spécifique. / The potassium-chloride cotransporter KCC2 decreases intracellular Cl- levels and renders GABA responses inhibitory. In addition, it has also been shown to modulate excitatory synapse development. In this project, we investigated how alterations of KCC2 expression levels affect these two key processes in cortical structures of a normal and/or epileptic developing brain. First, we demonstrate that KCC2 expression is altered by early-life febrile status epilepticus. Febrile seizures affect about 5% of children during the first year of life. Atypical febrile seizures, particularly febrile status epilepticus, correlate with a higher risk of developing cognitive deficits and temporal lobe epilepsy as adults, suggesting that they may permanently change the developmental trajectory of neuronal circuits. In fact, the presence of a cerebral malformation predisposes to the development of atypical febrile seizures and temporal lobe epilepsy. The mechanisms underlying these effects are not clear. Here, we investigated the functional impact of this alteration on subsequent synapse formation and seizure susceptibility. We analyzed KCC2 expression and spine density in the hippocampus of a well-established rodent model of atypical febrile seizures, combining a cortical freeze lesion at post-natal day 1 (P1) and hyperthermia-induced seizure at P10 (LHS rats). 86% of these LHS males develop epilepsy and learning and memory deficits in adulthood. At P20, we found a precocious increase in KCC2 protein levels, accompanied by a negative shift of the reversal potential of GABA (EGABA) by gramicidin-perforated patch. In parallel, we observed a reduction in dendritic spine size by DiI labelling and a reduction of miniature excitatory postsynaptic current (mEPSC) amplitude in CA1 pyramidal neurons, as well as impaired spatial memory. To investigate whether the premature expression of KCC2 played a role in these alterations in the LHS model, and on seizure susceptibility, we reduced KCC2 expression in CA1 pyramidal neurons by in utero electroporation of shRNA using a triple-probe electrode. This approach lead to reduced febrile seizure susceptibility, and rescued spine size shrinkage in LHS rats. Our results show that an increase of KCC2 levels induced by early-life insults affect seizure susceptibility and spine development and may be a contributing factor to the occurrence of hippocampal atrophy and associated cognitive deficits in LHS rats. Second, we investigated whether KCC2 premature overexpression plays a role in spine alterations in the hippocampus. We overexpressed KCC2 in hippocampal organotypic cultures by biolistic transfection and in vivo by in utero electroporation. In contrast to what was previously published, we observed that both manipulations lead to a decrease in spine density in the hippocampus, as well as an increase in spine head size in vivo. In fact, it has been previously shown that overexpressing KCC2 leads to an increase of spine density in the cortex in vivo. To prove that this discrepancy is due to brain regional differences, we overexpressed KCC2 in the cortex by in utero electroporation, and similarly found an increase in spine density and length. Altogether, our results demonstrate for the first time, that alterations of KCC2 expression are brain circuit-specific. These findings highlights the obstacles we will face to find adequate pharmacological treatment to specifically modulate KCC2 in a region-specific and time-sensitive manner in epilepsy.

Introdução: No presente estudo foram avaliados biomarcadores na ocorrência de febre em pacientes neutropênicos após transplante de células tronco hematopoiéticas (TCTH). Objetivo: O objetivo principal foi avaliar os valores séricos de biomarcadores: proteína C reativa (PCR), procalcitonina (PCT) e IL-6 (interleucina-6) que possam identificar precocemente infecção em TCTH. Outro objetivo foi fatores de risco para óbito nessa população. Métodos: Os biomarcadores foram avaliados em um estudo prospectivo que incluiu 296 pacientes neutropênicos, submetidos a TCTH autólogo ou alogênico. Os biomarcadores PCT, PCR e IL-6 foram dosados nos seguintes momentos:dia da neutropenia constatada sem febre, evento febril ou hipotermia (T < 35ºC), 24 h após a febre ou hipotermia, 72 horas após a febre ou hipotermia e febre prolongada ou seja 48 horas após a coleta no momento anterior ou na persistência da febre, cinco dias após a coleta no momento anterior. Os dados clínicos e laboratoriais, foram avaliados até a evolução para alta ou o óbito, em uma planilha Excel® 2003 e foram processados pelos programas SPSS e STATA. Os pacientes foram classificados nos seguintes grupos (I- afebril; II- febre de origem indeterminada FOI e III- febre clinica ou microbiologicamente comprovada) em relação a cada marcador estudado (PCT, PCR e IL-6). Foram feitos cálculos para estabelecer área sob a curva ROC, sensibilidade, especificidade, para avaliação da febre e óbito. Para avaliar o desfecho óbito foi realizada análise multivariada com regressão logística stepwise. Resultados: Dos 296 pacientes, 190 apresentaram febre. Duzentos e dezesseis (73%) foram submetidos a transplantes autólogos e 80 (27,0%) alogênicos. Dos 80 casos de TCTH alogênicos 74 (92,6%) eram aparentados e apenas 6 (7,4%) aparentados. Dos 80 casos alogênicos 69 (86,3%) eram fullmatch e 11(13,7%) mismatch. Em relação aos grupos já citados acima, temos a seguinte distribuição: grupo I: 106 pacientes (35,8%); grupo II: 112 pacientes (37,8%) e grupo III: 78 (26,4%). Os valores de média e mediana da IL-6 no momento afebril no grupo I em relação ao grupo II (p = 0,013), apresentando valor significativamente maiores. Os níveis da PCR no grupo I diferiram de forma significativa dos encontrados no grupo III (p < 0,05). Os grupos diferiram em relação aos níveis de IL-6 e de PCR no momento febril. O grupo II apresentou concentrações de IL-6 e de PCR significativamente menores que o grupo III. Os melhores valores de corte de PCT para os momentos de coleta: febre, 24 horas após a febre, 72 horas de febre, e febre prolongada foram respectivamente: 0,32; 0,47; 0,46 e 0,35?g/L. No momento da febre a sensibilidade foi 52,3 e a especificidade 52,6 para o diagnóstico de infecção. Os melhores valores de corte de PCR para os momentos de febre, 24 horas após, 72 horas após e febre prolongada foram, respectivamente: 79, 120, 108 e 72 mg/L. No momento da febre a sensibilidade foi 55,4 e especificidade foi 55,1. Os melhores valores de corte de IL-6 para os momentos de febre, 24 h após, 72 horas após a febre e febre prolongada foram respectivamente: 34, 32, 16 e 9 pg/mL. A sensibilidade e especificidade no momento da febre foram respectivamente: 59,8 e 59,7. Na análise dos três biomarcadores no grupo de pacientes autólogos, verifica-se que só a IL-6 apresenta valores significativos nos momentos iniciais (afebril, febre e 24 horas após a febre). Os seguintes fatores de risco independentes foram identificados na análise multivariada: doador aparentado, doador não aparentado, infecção por Gram-negativo, DHL >= 390 (UI/L), ureia >= 25 (mg/dL) e PCR >= 120 (mg/L). Conclusões: IL-6 e PCR têm associação com diagnóstico precoce de infecção clinica ou microbiologicamente confirmada em neutropenia febril após TCTH. A associação dos três marcadores não apresentou nenhuma vantagem, e não melhorou a acurácia diagnóstica. A IL-6 foi o único biomarcador significativamente associado de forma precoce com infecção quando avaliado apenas pacientes submetidos a TCTH autólogos As variáveis independentes associadas com óbito foram: transplante alogênico, infecção por Gram-negativos, DHL >= 390UI/L no momento da febre e ureia >= 25 mg/dL no momento da febre e PCR >= 120 (mg/L) / Introduction: In the present study, biomarkers were assessed in the occurrence of fever in neutropenic patients upon hematopoietic stem cell transplantation (HSCT). Objective: The main objective was to assess the serum values of biomarkers: C-reactive protein (CRP), procalcitonin (PCT) and IL-6 (interleukin-6) which can early identify infection in HSCT. Another objective was risk factors for death in that population. Methods: The biomarkers were assessed in a prospective study which comprised 296 neutropenic patients submitted to autologous or allogeneic HSCT. The biomarkers PCT, CRP and IL-6 were dosed at the following moments: day of afebrile neutropenia, febrile event or hypothermia (T < 35ºC), 24 h upon fever or hypothermia, 72 hours upon fever or hypothermia and long-standing fever, that is, 48 hours upon the last sampling or at fever persistence, five days upon the last sampling. The clinical and laboratory data were assessed up to the evolution to discharge or death, in an Excel® 2003 spreadsheet and were processed by the SPSS and STATA software. Patients were classified in the following groups (I- afebrile; II- fever of unknown origin FUO and III- clinically or microbiologically proven fever) in regard to each biomarker studied (PCT, CRP and IL-6). Calculations were made to establish the area under the ROC curve, sensitivity, specificity, for the assessment of the evolution and death. In order to assess the death outcome, a multivariate analysis with stepwise logistic regression was conducted. Results: Out of the 296 patients, 190 had fever. Two hundred and sixteen (73%) were submitted to autologous transplantations and 80 (27.0%) to allogeneic ones. Out of the 80 cases of allogeneic HSCT, 74 (92.6%) were related and only 6 (7.4%) were unrelated. Out of the 80 allogeneic cases, 69 (86.3%) were fullmatch and 11(13.7%) were mismatch. In regard to the groups mentioned above, we have the following distribution: group I: 106 patients (35.8%); group II: 112 patients (37.8%) and group III: 78 patients (26.4%). The mean and median values of IL-6 at fever onset in group I in regard to group II (p = 0.013), presenting significantly higher values. The levels of CRP in group I differed significantly from those found in group III (p < 0.05). The groups differed in regard to the levels of IL-6 and CRP at fever onset. Group II presented IL-6 and CRP concentrations significantly lower than group III. The best cut-off values of PCT for sampling: fever onset, 24 hours upon fever, 72 hours of fever, and long-standing fever were, respectively: 0.32; 0.47; 0.46 and 0.35?g/L. At fever onset, sensitivity was 52.3 and specificity 52.6 for infection diagnosis. The best cut-off values of CRP for fever onset, 24 hours upon fever, 72 hours upon fever and long-standing fever were, respectively: 79, 120, 108 and 72 mg/L. At fever onset, sensitivity was 55.4 and specificity was 55.1. The best cut-off values of IL-6 for fever onset, 24 hours upon fever, 72 hours upon fever and long-standing fever were, respectively: 34, 32, 16 and 9 pg/mL. At fever onset, sensitivity and specificity were, respectively: 59.8 and 59.7. In the analysis of the three biomarkers in the group of autologous patients, it is observed that only IL-6 presents significant values at initial moments (afebrile, fever and 24 hours upon fever). The following independent risk factors were identified in the multivariate analysis: related donor, unrelated donor, Gram-negative infection, DHL >= 390 (UI/L), urea >= 25 (mg/dL) and CRP>=120 (mg/L). Conclusions: IL-6 and CRP are associated to the early diagnosis of clinically or microbiologically confirmed infection in post-HSCT febrile neutropenia. The association of the three biomarkers did not present any advantage, nor did it improve diagnostic accuracy. IL-6 was the only biomarker significantly associated at an early stage with infection when assessed only in patients submitted to autologous HSCT. The independent variables associated with death were: allogeneic transplantation, Gram-negative infection, DHL >= 390UI/L at fever onset and urea >= 25 mg/dL at fever onset and PCR >= 120 (mg/L)