Children who are more fearful and inhibited during early childhood are at greater risk for social problems (e.g., loneliness, social isolation) and clinically significant internalizing disorders during adolescence and adulthood (e.g., Rubin, Chen, McDougall, Bowker, & McKinnon, 1995; Williams et al., 2009). While the impact of fearful temperament on adjustment indices are regularly the focus of study, less well understood are biological and social processes that may affect the development of fearful temperament. The present study considered the role of the 5-HTTLPR polymorphism and parenting on change in fearful and inhibited temperamental characteristics during early childhood. The s/s genotype was expected to be associated with elevated and sustained levels of fearful temperament. Moreover, supportive parenting was expected to be associated with less fearful temperament while more harsh parenting would be associated with more fearful temperamental characteristics, especially for children with the s/s 5-HTTLPR genotype. Study hypotheses were tested using 165 families (i.e., biological mothers and fathers, 3-5 year old children) who participated in the Family Transitions Project (FTP: R. D. Conger & K. J. Conger, 2002). Children were genotyped using cheek swabs. Parents reported on children’s temperamental characteristics at ages 3, 4, and 5. Independent observations of mothers and fathers completing a puzzle with their 3 and 4 year old children were used to measure parenting. Results were partially supportive of predictions. Parenting interacted with the 5-HTTLPR genotype to predict trajectories of shyness and soothability dimensions of fearful temperament, but the pattern of findings varied for mothers and fathers. Results are discussed in terms of differential susceptibility and the conceptualization of risk and resilience.

Die Fitness-limitierende Ressource für die meisten männlichen Primaten ist die unteilbare Befruchtung von Weibchen. Daher herrscht in der Regel ein aggressives Konkurrenzverhalten unter Männchen, um sich den Zugang zu fertilen Weibchen zu sichern. Jedoch zeigen Männchen unterschiedlicher Arten eine erhebliche Variation in ihrer Aggressivität. In manchen Arten kann sogar ein kooperatives Verhalten zwischen Männchen beobachten werden, das meist mit weniger angespannten und toleranteren Sozialbeziehungen verbunden ist. Die proximaten und ultimaten Ursachen für diese interspezifische Variation werden durch verschiedene Aspekte des Sozialsystems einer Art bedingt, jedoch sind die zugrundeliegenden Ursachen noch nicht vollständig geklärt. Paviane (Papio spp.) stellen ein gut geeignetes Modell dar, um die Grundlage von männlicher Aggressivität und Toleranz zu untersuchen, da die unterschiedlichen Arten dieser Gattung eine erhebliche Variation in männlichem Konkurrenzverhalten und in anderen Aspekten ihrer Sozialsysteme zeigen. Männliche Bärenpaviane (P. ursinus) im südlichen und männliche Guineapaviane (P. papio) im westlichen Afrika scheinen dabei entgegengesetzte Extrema von Aggressivität und Toleranz darzustellen. Das Ziel meiner Promotion war daher, verschiedene Aspekte der männlichen Dominanzbeziehungen in den beiden Arten zu untersuchen und zu vergleichen. Bärenpaviane leben in stabilen Gruppen mit mehreren Männchen und mehreren Weibchen, in denen geschlechtsreife Männchen in benachbarte Gruppen abwandern. Dort versuchen sie mit Hilfe von aggressivem Verhalten einen hohen Rang zu erlangen, da ihnen dieser ein Vorrecht („Priority of access“) auf fertile Weibchen verschafft und über den reproduktiven Erfolg eines Männchens bestimmt. Im Gegensatz dazu leben Guineapaviane in einer mehrschichtigen (“multi-level“) Gesellschaft, in der nahverwandte Männchen häufig in der gleichen Gruppe verbleiben. Die Sozialbeziehungen zwischen Männchen scheinen dabei vor allem durch eine niedrige Frequenz agonistischer Interaktionen und eine hohe räumliche Toleranz sowie Kooperation gekennzeichnet zu sein. Daher stellen Aggressionen in dieser Art scheinbar nicht das primäre Mittel in der Konkurrenz um den Zugang zu Weibchen dar. Bisher gab es noch keine vergleichbaren Daten, um diese Vermutung über Verhaltensunterschiede zwischen den beiden Arten zu überprüfen. Daher war ein Ziel meiner Dissertation die Aggressivität und räumliche Toleranz zwischen männlichen Bärenpavianen im Moremi Game Reserve, Botsuana, und männlichen Guineapavianen im Parc National de Niokolo Koba, Senegal, zu vergleichen. Unterschiede in männlicher Konkurrenz spiegeln sich auch in unterschiedlichen altersabhängigen Verläufen des Reproduktionserfolgs wider. Bei Primaten ist dieser Verlauf wiederum mit Variation im Testosteronspiegel verbunden. Neben diesen Langzeitverläufen wurde gezeigt, dass zusätzliche, kurzfristige Anstiege in Testosteronspiegeln ein aggressives Verhalten während der Konkurrenz um Paarungspartner fördern (‚Challenge hypothesis‘). Das zweite Ziel meiner Dissertation war daher, den Zusammenhang zwischen Testosteron und dominanzbezogener Aggression zu untersuchen, indem ich die Variation in Testosteronspiegeln in Bezug auf Alter, Aggression, und Dominanzbeziehungen analysiert habe. Die Art und Weise, wie Individuen konkurrieren, beeinflusst auch, wie physiologische Kosten (oder ‚allostatic load‘) zwischen Individuen unterschiedlichen Dominanzstatus verteilt sind. Diese Kosten spiegeln sich in inter-individuellen Differenzen in Glucocorticoidspiegeln wider. Das dritte Ziel meiner Arbeit war daher die Effekte von männlichen Sozialbeziehungen auf physiologische Kosten zu untersuchen, indem ich die Variation in Glucocorticoidspiegeln in Relation zu Dominanzstatus zwischen Bären- und Guineapavianen analysiert habe. Inter-individuelle Unterschiede in Aggressivität sind stark erblich bedingt. Daher ist zu vermuten, dass auch Unterschiede zwischen Arten eine genetische Grundlage haben. Die beiden Längenpolymorphismen 5-HTTLPR - im Gen des Serotonintransporters - und MAOALPR - im Gen der monoaminen Oxidase A - beeinflussen die Aktivität des serotonergen Neurotransmittersystems und wurden mit Variation von Aggressivität in Verbindung gebracht. Verschiedene Allele dieser beiden Loci könnten daher auch mit Artunterschieden bei Pavianen gekoppelt sein. Das vierte Ziel meiner Dissertation war daher, Allele dieser beiden Loci zwischen fünf Pavianarten zu vergleichen. Durch die Erhebung der ersten unmittelbar vergleichbaren Verhaltensdaten für männliche Bären- und Guineapaviane konnte ich zeigen, dass männliche Bärenpaviane häufiger in agonistische Interaktionen verwickelt sind als Guineapaviane. Im Gegensatz dazu zeigen Guineapaviane eine höhere räumliche Toleranz gegenüber anderen Männchen und tauschen manchmal sogar affiliatives Verhalten aus. Des Weiteren zeigten männliche Bärenpaviane konsistente Dominanzbeziehungen und eine lineare Hierarchie, während die Linearität der Hierarchien von Guineapavianen allgemein niedriger war. Diese Beobachtungen stimmen mit vorherigen Beobachtungen über männliche Sozialbeziehungen in beiden Arten überein. Dies bedeutet, dass die beiden Arten sich tatsächlich in der Intensität von männlicher Wettbewerbskonkurrenz („contest competition“) unterscheiden. In keiner der beiden Arten konnte ein Zusammenhang zwischen Testosteronspiegel und Alter oder Dominanzrang gefunden werden. Es gab jedoch einen statistischen Trend, dass in Bärenpavianen der Testosteronspiegel mit Aggressivität korreliert. Dies wurde zuvor nur in Perioden beobachtet, in denen Männchen hoher Dominanzränge von anderen Männchen herausgefordert wurden (d.h. „unstabile Perioden“). Dies war in der vorliegenden Studie nicht der Fall. Jedoch könnten eine kurz zuvor beobachtete Teilung der Gruppe und darauf folgende regelmäßige Begegnungen der beiden neu entstandenen Gruppen eine ähnliche Situation verursacht haben. In Guineapavianen waren Testosteronspiegel nicht mit der Häufigkeit von agonistischen Verhalten korreliert. Dies könnte daran liegen, dass solche Verhalten in dieser Art keine bedeutende Rolle in der Konkurrenz um Paarungsmöglichkeiten spielen. Hochrangige männliche Bärenpaviane zeigten höhere Glucocorticoidspiegel als niedrigrangigere Männchen. Auch dies wurde bei Bärenpavianen bisher nur während „unstabiler Perioden“ beobachtet und weist wieder darauf hin, dass die unübliche Teilung der Gruppe eine ähnliche Situation hervorgerufen haben kann. In Guineapavianen korrelierten Glucocorticoidspiegel nicht mit Dominanzpositionen, was die Vermutung unterstütz, dass Dominanzränge in dieser Art wenig oder keine Bedeutung haben. Ein Vergleich von 5-HTTLPR und MAOALPR Allelen zwischen fünf Arten von Pavianen lässt vermuten, dass der 5-HTTLPR-Genotyp nicht mit interspezifischer Variation im Verhalten in Verbindung steht; dieser Locus war in fast allen Arten monomorph. Ein erweiterter Vergleich zwischen mehreren Arten von Pavianartigen (Papionini) deutete aber an, dass dieser Locus während der historischen Ausbreitung von Makaken nach Asien sehr wahrscheinlich unter Selektionsdrücken stand. Die Bedeutung dieser Variation muss noch untersucht werden, könnte aber eventuell mit Unterschieden in der Umwelt der jeweiligen Arten zusammenhängen. Im Gegensatz dazu war der Locus MAOALPR polymorph und das Muster verschiedener Allele passte überwiegend mit den vermuteten Verhaltensunterschieden zwischen Pavianarten zusammen. Die beobachtete Variation in diesem Locus bietet daher eine gute Möglichkeit, um genetisch bedingte Verhaltensunterschiede zwischen verschiedenen Pavianen genauer zu untersuchen und dadurch die genetischen Grundlagen der Variation in Aggressivität bei Primaten besser zu verstehen. Zusammengefasst hat meine Dissertation grundlegende Einblicke in die proximaten Ursachen und Kosten der Unterschiede in männlicher Dominanzbeziehungen zwischen Pavianarten geliefert. Erste Daten weisen darauf hin, dass Verhaltensunterschiede in Bezug auf Aggressivität zwischen Pavianen mit dem MAOALPR-Genotyp verbunden sind. Eine Untersuchung dieses Locus im Zusammenhang mit Neurotransmitter-Aktivität und Verhalten könnte daher weitere Aufschlüsse über die proximaten Mechanismen geben, die unterschiedlichen Aggressivitätsmustern in Primaten unterliegen. Verhaltensbeobachtungen und Glucocorticoid-Messungen lassen vermuten, dass die reproduktive Strategie von männlichen Guineapavianen mit weniger „offensichtlichen“ Kosten verbunden ist. Daher könnten eine Untersuchung der Mechanismen, die den Zugang zu Weibchen bei Guineapavianen regeln, und ein Vergleich des reproduktiven Erfolgs zwischen unterschiedlichen Männchen weitere Einblicke in die Evolution von reproduktiven Strategien und damit von Aggressivitäts- und Toleranzunterschiede zwischen männlichen Primaten liefern.

A depressão pós-parto (DPP) é um importante problema de saúde pública podendo provocar uma ruptura do vínculo entre a mãe e o bebê e até estar associada com respostas trágicas, como suicídio materno e infanticídio. A DPP é multifatorial e o seu surgimento pode ser favorecido por componentes hormonais, genéticos e ambientais. O ciclo gravídico-puerperal é considerado um período de risco, pois algumas mulheres possuem uma sensibilidade particular as alterações hormonais. O risco de DPP é aumentado em mulheres que possuem histórico de depressão na família, logo, um componente genético determina maior suscetibilidade. Segundo o DMS-IV, existe uma relação entre a sintomatologia depressiva e as alterações na concentração cerebral de neurotransmissores, com destaque para serotonina. O transportador de serotonina (SERT) controla a intensidade e duração da re-captação da serotonina nas sinapses serotonérgicas. Diversos trabalhos associam os polimorfismos do SERT com transtornos mentais, como unipolar, bipolar, depressão e esquizofrenia. Nosso objetivo foi analisar as concentrações salivares de cortisol (CORT), as freqüências alélicas e genotípicas dos polimorfismos 5-HTTVNTR e 5- HTTLPR no gene SLC6A4 entre mulheres que desenvolveram ou não DPP. A amostra foi constituída por 128 mulheres brancas da cidade de Pelotas/RS, triadas em ambulatórios do SUS. A avaliação diagnóstica foi realizada através de entrevista psiquiátrica e diagnóstica usando como instrumento o Beck Depression Inventory entre 30 a 45 dias após o nascimento das crianças. A coleta de material biológico (leucócitos e saliva) foi realizada no turno da manhã, respeitando o período de duas horas de jejum. A região promotora do gene contendo o polimorfismo 5-HTTLPR (inserção/deleção) e a região do segundo íntron contendo o polimorfismo 5- HTTVNTR (repetições em tandem) foram amplificadas através da reação em cadeia da polimerase. A dosagem do CORT foi realizada a partir da saliva por técnica de ELISA utilizando kit específico. A mediana e o intervalo interquartil das concentrações salivares do CORT entre os portadores dos diferentes genótipos foram comparados entre os grupos estudados usando o teste de Kruskal-Wallis e Mann-Whitney. A comparação das freqüências alélicas e genotípicas dos polimorfismos estudados entre as mulheres que apresentarem ou não DPP foram feitas pelo teste do Qui-quadrado com correção de Yates (p£ 0,05). A análise da distribuição das freqüências genotípicas dos polimorfismos 5-HTTLPR e 5- HTTVNTR do SERT permitiu verificar que a população está sob Equilíbrio Hardy– Weinberg. Quando os polimorfismos foram analisados isoladamente, não foi observada associação entre os polimorfismos 5-HTTLPR (p=0,48) e 5-HTTVNTR (p=0,77) e o diagnóstico para DPP. Porém, a análise combinada dos haplótipos dos polimorfismos 5-HTTLPR e 5-HTTVNTR demonstraram que mulheres portadoras do diplótipo L-12/L-12 apresentaram escores menores de sintomas depressivos (mediana: 0,5; intervalo inter-quartil: 0,00-4,00, p=0,04) quando comparadas com mulheres portadoras de outros diplótipos (mediana: 4,0; intervalo inter-quartil: 1,00- 10,00). O polimorfismo 5-HTTVNTR foi associado com as concentrações salivares de CORT (p=0,03), já o polimorfismo 5-HTTLPR não foi associado (p=0,41). Nossos achados são inovadores, visto que até a presente data a associação dos genótipos 5-HTTLPR e 5-HTTVNTR com DPP e concentrações salivares de CORT ainda não haviam sido investigados. / The postpartum depression (PPD) is an important public health problem that may cause a rupture of the bond between the mother and the baby and may even be associated with tragic responses such as maternal suicide and infanticide. The DPP is multifactorial and its appearance can be favored by hormonal components, genetic and environmental factors. The pregnancy and childbirth is considered a risk period, because some women have a particular sensitivity to hormonal changes. The rate of DPP is increased in women who have a record of depression in the family, so a genetic component determines higher susceptibility. According to the DSM-IV, there is a relationship between depressive symptoms and brain concentration changes of neurotransmitters, particularly serotonin. The serotonin transporter (SERT) controls the intensity and duration of re-uptake of serotonin in serotonergic synapses. Several studies linking polymorphisms of SERT with mental disorders such as unipolar, bipolar, depression, and schizophrenia. Our objective was to analyze the concentrations of salivary cortisol (CORT), the allele and genotype frequencies of polymorphisms 5-HTTLPR and 5-HTTVNTR SLC6A4 gene in women who developed or not DPP. The sample consisted of 128 white women from Pelotas, RS, sorted out from public health clinics. The diagnostic evaluation was conducted through interviews and psychiatric diagnostic instrument as using the Beck Depression Inventory among 30 to 45 days after the birth of children. The collection of biological materials (leukocytes and saliva) was performed in the morning, observing the twohour period of fasting. The promoter region of the gene containing the 5-HTTLPR polymorphism (insertion/deletion) and the region containing the second intron polymorphism 5-HTTVNTR (tandem repeats) were amplified by polymerase chain reaction. The dose of CORT was performed from the saliva by ELISA using the specific kit. The median and interquartile interval of salivary concentrations of CORT among patients of different genotypes were compared between groups using the Kruskal-Wallis and Mann-Whitney. The comparison of allele and genotype frequencies of polymorphisms among women who developed or not DPP were made by chi-square test with Yates correction (p <0.05). The analysis of the distribution of genotype frequencies of polymorphisms 5-HTTLPR and 5-HTTVNTR SERT showed that the population is under Hardy-Weinberg Equilibrium. When the polymorphisms were analyzed alone, no association was observed between the 5-HTTLPR (p=0.48) and 5-HTTVNTR (p=0.77) polymorphisms and the PPD diagnosis. But, the information from these analyses combined with information regarding the haplotypes of the 5-HTTLPR and 5-HTTVNTR polymorphisms demonstrated that women carriers of diplotype L-12/L-12 have a lower depression symptoms score (median: 0.5; interquartile range: 0.00-4.00; p=0.04) than women with other diplotypes (median: 4.0; inter-quartile range: 1.00-10.00). The 5-HTTVNTR polymorphism was associated with the salivary concentrations of CORT (p=0.03), whereas the 5-HTTLPR polymorphism was not associated (p=0.41). Our findings are innovative since the association of 5- HTTLPR genotypes and 5-HTTVNTR with DPP and salivary concentrations of CORT had not been investigated before.

Introdução: Os transtornos de ansiedade (TA) são prevalentes na infância e na adolescência e geram prejuízos significativos, podendo persistir na vida adulta. Os traços de personalidade e temperamento associados à ansiedade, como o comportamento inibido (CI) e a evitação de danos, também devem ser considerados. O neurotransmissor serotonina possui um papel crítico no desenvolvimento e na plasticidade do encéfalo. O gene do seu transportador (5-HTT) é um forte candidato para estudos de associação genéticos e psiquiátricos. O polimorfismo na região promotora do gene do 5-HTT (5-HTTLPR) é funcional e, portanto, de relevância para estudos de associação na psiquiatria. Objetivos: Investigar a associação entre o 5-HTTLPR, através da sua classificação bialélica e trialélica, e os TA e fenótipos relacionados à ansiedade (CI e evitação de danos), em uma amostra de adolescentes ansiosos e não-ansiosos e seus familiares. Metodologia: Um total de 510 indivíduos participou do estudo. Os participantes foram 225 adolescentes (129 casos e 96 controles para TA) e seus familiares biológicos (194 mães, 63 pais e 22 irmãos). Foi realizado o diagnóstico psiquiátrico através de entrevista clínica e do Schedule for Affective Disorders and Schizophrenia for School-Age Children – Present and Lifetime Version Diagnostic Interview (K-SADS-PL). A escala Temperament and Character Inventory (TCI) e uma adaptação da escala Retrospective Self-report Scale of Behavioral Inhibition foram usadas para mensurar a evitação de danos e o comportamento inibido, respectivamente. As análises moleculares resultaram da extração de DNA da amostra de saliva dos adolescentes e seus familiares, seguida de amplificação do DNA por PCR e digestão enzimática com enzima MspI. Os genótipos foram agrupados pelo nível de expressividade: baixo (SS, LGS, LGLG); intermediário (LALG, LAS) e alto (LALA). A análise estatística foi realizada com o software PLINK e nível de significância α < 0.05. Resultados: Não foi encontrada associação entre o 5-HTTLPR, considerando a classificação bialélica e trialélica, e os transtornos de ansiedade, comportamento inibido e a evitação, tanto no caso-controle quanto no estudo de trios. Conclusões: Nossos resultados não sugerem a presença de associação entre o 5-HTTLPR e os transtornos de ansiedade e seus fenótipos relacionados (comportamento inibido e evitação de danos) em adolescentes. Diante de resultados controversos descritos na literatura, estudos de meta-análises podem ser necessários para auxiliar no esclarecimento sobre essa questão. / Introduction: Anxiety disorders (AD) are prevalent in childhood and adolescence and results in significant impairments. It usually persists into adulthood. Anxiety traits such as behavioral inhibition and harm avoidance may also be considered. The serotonin neurotransmitter plays an important role in the development and the plasticity of the brain. The serotonin transporter gene (5-HTT) is considered a strong candidate and the Serotonin Transporter Gene-linked Polymorphic Region (5-HTTLPR) functional and, therefore, relevant in studies concerning the association between genetic and psychiatric disorders. Objectives: To investigate the association between 5-HTTLPR (biallelic and triallelic classification) and AD and anxiety related phenotypes (behavioral inhibition and harm avoidance) in a sample of adolescents and their families. Methodology: A total of 510 subjects participated in this study. Participants were 225 adolescents (129 anxiety cases and 96 community controls) and their biological families (194 mothers, 66 fathers and 22 siblings). We assessed psychiatric diagnosis throughout a clinical interview and using the Schedule for Affective Disorders and Schizophrenia for School-Age Children – Present and Lifetime Version Diagnostic Interview (K-SADS-PL). The Temperament and Character Inventory (TCI) and an adaptation of the Resnick Behavioral Inhibition Scale were used in order to measure harm avoidance and behavioral inhibition, respectively. The molecular analysis resulted in the extraction of DNA from saliva sample of the adolescents and their families, followed by DNA amplification by PCR and enzymatic digestion with the MspI. The genotypes were grouped by level of expression: low (SS, LGS, LGLG); intermediated (LALG, LAS) and high (LALA). Statistical analysis was performed with the software PLINK, with the significance level of α < 0.05. Results: No association was found between 5-HTTLPR, considering the biallelic or triallelic analysis, and anxiety disorders, behavioral inhibition and harm avoidance in both case-control and trios studies. Conclusion: Our results do not support a major role of 5-HTTLPR in anxiety disorders and anxiety-related phenotypes (behavioral inhibition and harm avoidance) in adolescents. In the face of mixed results, further investigations and meta-analytic studies are needed in order to clarify this research question.

A depressão pós-parto (DPP) é um importante problema de saúde pública podendo provocar uma ruptura do vínculo entre a mãe e o bebê e até estar associada com respostas trágicas, como suicídio materno e infanticídio. A DPP é multifatorial e o seu surgimento pode ser favorecido por componentes hormonais, genéticos e ambientais. O ciclo gravídico-puerperal é considerado um período de risco, pois algumas mulheres possuem uma sensibilidade particular as alterações hormonais. O risco de DPP é aumentado em mulheres que possuem histórico de depressão na família, logo, um componente genético determina maior suscetibilidade. Segundo o DMS-IV, existe uma relação entre a sintomatologia depressiva e as alterações na concentração cerebral de neurotransmissores, com destaque para serotonina. O transportador de serotonina (SERT) controla a intensidade e duração da re-captação da serotonina nas sinapses serotonérgicas. Diversos trabalhos associam os polimorfismos do SERT com transtornos mentais, como unipolar, bipolar, depressão e esquizofrenia. Nosso objetivo foi analisar as concentrações salivares de cortisol (CORT), as freqüências alélicas e genotípicas dos polimorfismos 5-HTTVNTR e 5- HTTLPR no gene SLC6A4 entre mulheres que desenvolveram ou não DPP. A amostra foi constituída por 128 mulheres brancas da cidade de Pelotas/RS, triadas em ambulatórios do SUS. A avaliação diagnóstica foi realizada através de entrevista psiquiátrica e diagnóstica usando como instrumento o Beck Depression Inventory entre 30 a 45 dias após o nascimento das crianças. A coleta de material biológico (leucócitos e saliva) foi realizada no turno da manhã, respeitando o período de duas horas de jejum. A região promotora do gene contendo o polimorfismo 5-HTTLPR (inserção/deleção) e a região do segundo íntron contendo o polimorfismo 5- HTTVNTR (repetições em tandem) foram amplificadas através da reação em cadeia da polimerase. A dosagem do CORT foi realizada a partir da saliva por técnica de ELISA utilizando kit específico. A mediana e o intervalo interquartil das concentrações salivares do CORT entre os portadores dos diferentes genótipos foram comparados entre os grupos estudados usando o teste de Kruskal-Wallis e Mann-Whitney. A comparação das freqüências alélicas e genotípicas dos polimorfismos estudados entre as mulheres que apresentarem ou não DPP foram feitas pelo teste do Qui-quadrado com correção de Yates (p£ 0,05). A análise da distribuição das freqüências genotípicas dos polimorfismos 5-HTTLPR e 5- HTTVNTR do SERT permitiu verificar que a população está sob Equilíbrio Hardy– Weinberg. Quando os polimorfismos foram analisados isoladamente, não foi observada associação entre os polimorfismos 5-HTTLPR (p=0,48) e 5-HTTVNTR (p=0,77) e o diagnóstico para DPP. Porém, a análise combinada dos haplótipos dos polimorfismos 5-HTTLPR e 5-HTTVNTR demonstraram que mulheres portadoras do diplótipo L-12/L-12 apresentaram escores menores de sintomas depressivos (mediana: 0,5; intervalo inter-quartil: 0,00-4,00, p=0,04) quando comparadas com mulheres portadoras de outros diplótipos (mediana: 4,0; intervalo inter-quartil: 1,00- 10,00). O polimorfismo 5-HTTVNTR foi associado com as concentrações salivares de CORT (p=0,03), já o polimorfismo 5-HTTLPR não foi associado (p=0,41). Nossos achados são inovadores, visto que até a presente data a associação dos genótipos 5-HTTLPR e 5-HTTVNTR com DPP e concentrações salivares de CORT ainda não haviam sido investigados. / The postpartum depression (PPD) is an important public health problem that may cause a rupture of the bond between the mother and the baby and may even be associated with tragic responses such as maternal suicide and infanticide. The DPP is multifactorial and its appearance can be favored by hormonal components, genetic and environmental factors. The pregnancy and childbirth is considered a risk period, because some women have a particular sensitivity to hormonal changes. The rate of DPP is increased in women who have a record of depression in the family, so a genetic component determines higher susceptibility. According to the DSM-IV, there is a relationship between depressive symptoms and brain concentration changes of neurotransmitters, particularly serotonin. The serotonin transporter (SERT) controls the intensity and duration of re-uptake of serotonin in serotonergic synapses. Several studies linking polymorphisms of SERT with mental disorders such as unipolar, bipolar, depression, and schizophrenia. Our objective was to analyze the concentrations of salivary cortisol (CORT), the allele and genotype frequencies of polymorphisms 5-HTTLPR and 5-HTTVNTR SLC6A4 gene in women who developed or not DPP. The sample consisted of 128 white women from Pelotas, RS, sorted out from public health clinics. The diagnostic evaluation was conducted through interviews and psychiatric diagnostic instrument as using the Beck Depression Inventory among 30 to 45 days after the birth of children. The collection of biological materials (leukocytes and saliva) was performed in the morning, observing the twohour period of fasting. The promoter region of the gene containing the 5-HTTLPR polymorphism (insertion/deletion) and the region containing the second intron polymorphism 5-HTTVNTR (tandem repeats) were amplified by polymerase chain reaction. The dose of CORT was performed from the saliva by ELISA using the specific kit. The median and interquartile interval of salivary concentrations of CORT among patients of different genotypes were compared between groups using the Kruskal-Wallis and Mann-Whitney. The comparison of allele and genotype frequencies of polymorphisms among women who developed or not DPP were made by chi-square test with Yates correction (p <0.05). The analysis of the distribution of genotype frequencies of polymorphisms 5-HTTLPR and 5-HTTVNTR SERT showed that the population is under Hardy-Weinberg Equilibrium. When the polymorphisms were analyzed alone, no association was observed between the 5-HTTLPR (p=0.48) and 5-HTTVNTR (p=0.77) polymorphisms and the PPD diagnosis. But, the information from these analyses combined with information regarding the haplotypes of the 5-HTTLPR and 5-HTTVNTR polymorphisms demonstrated that women carriers of diplotype L-12/L-12 have a lower depression symptoms score (median: 0.5; interquartile range: 0.00-4.00; p=0.04) than women with other diplotypes (median: 4.0; inter-quartile range: 1.00-10.00). The 5-HTTVNTR polymorphism was associated with the salivary concentrations of CORT (p=0.03), whereas the 5-HTTLPR polymorphism was not associated (p=0.41). Our findings are innovative since the association of 5- HTTLPR genotypes and 5-HTTVNTR with DPP and salivary concentrations of CORT had not been investigated before.

Introdução: Os transtornos de ansiedade (TA) são prevalentes na infância e na adolescência e geram prejuízos significativos, podendo persistir na vida adulta. Os traços de personalidade e temperamento associados à ansiedade, como o comportamento inibido (CI) e a evitação de danos, também devem ser considerados. O neurotransmissor serotonina possui um papel crítico no desenvolvimento e na plasticidade do encéfalo. O gene do seu transportador (5-HTT) é um forte candidato para estudos de associação genéticos e psiquiátricos. O polimorfismo na região promotora do gene do 5-HTT (5-HTTLPR) é funcional e, portanto, de relevância para estudos de associação na psiquiatria. Objetivos: Investigar a associação entre o 5-HTTLPR, através da sua classificação bialélica e trialélica, e os TA e fenótipos relacionados à ansiedade (CI e evitação de danos), em uma amostra de adolescentes ansiosos e não-ansiosos e seus familiares. Metodologia: Um total de 510 indivíduos participou do estudo. Os participantes foram 225 adolescentes (129 casos e 96 controles para TA) e seus familiares biológicos (194 mães, 63 pais e 22 irmãos). Foi realizado o diagnóstico psiquiátrico através de entrevista clínica e do Schedule for Affective Disorders and Schizophrenia for School-Age Children – Present and Lifetime Version Diagnostic Interview (K-SADS-PL). A escala Temperament and Character Inventory (TCI) e uma adaptação da escala Retrospective Self-report Scale of Behavioral Inhibition foram usadas para mensurar a evitação de danos e o comportamento inibido, respectivamente. As análises moleculares resultaram da extração de DNA da amostra de saliva dos adolescentes e seus familiares, seguida de amplificação do DNA por PCR e digestão enzimática com enzima MspI. Os genótipos foram agrupados pelo nível de expressividade: baixo (SS, LGS, LGLG); intermediário (LALG, LAS) e alto (LALA). A análise estatística foi realizada com o software PLINK e nível de significância α < 0.05. Resultados: Não foi encontrada associação entre o 5-HTTLPR, considerando a classificação bialélica e trialélica, e os transtornos de ansiedade, comportamento inibido e a evitação, tanto no caso-controle quanto no estudo de trios. Conclusões: Nossos resultados não sugerem a presença de associação entre o 5-HTTLPR e os transtornos de ansiedade e seus fenótipos relacionados (comportamento inibido e evitação de danos) em adolescentes. Diante de resultados controversos descritos na literatura, estudos de meta-análises podem ser necessários para auxiliar no esclarecimento sobre essa questão. / Introduction: Anxiety disorders (AD) are prevalent in childhood and adolescence and results in significant impairments. It usually persists into adulthood. Anxiety traits such as behavioral inhibition and harm avoidance may also be considered. The serotonin neurotransmitter plays an important role in the development and the plasticity of the brain. The serotonin transporter gene (5-HTT) is considered a strong candidate and the Serotonin Transporter Gene-linked Polymorphic Region (5-HTTLPR) functional and, therefore, relevant in studies concerning the association between genetic and psychiatric disorders. Objectives: To investigate the association between 5-HTTLPR (biallelic and triallelic classification) and AD and anxiety related phenotypes (behavioral inhibition and harm avoidance) in a sample of adolescents and their families. Methodology: A total of 510 subjects participated in this study. Participants were 225 adolescents (129 anxiety cases and 96 community controls) and their biological families (194 mothers, 66 fathers and 22 siblings). We assessed psychiatric diagnosis throughout a clinical interview and using the Schedule for Affective Disorders and Schizophrenia for School-Age Children – Present and Lifetime Version Diagnostic Interview (K-SADS-PL). The Temperament and Character Inventory (TCI) and an adaptation of the Resnick Behavioral Inhibition Scale were used in order to measure harm avoidance and behavioral inhibition, respectively. The molecular analysis resulted in the extraction of DNA from saliva sample of the adolescents and their families, followed by DNA amplification by PCR and enzymatic digestion with the MspI. The genotypes were grouped by level of expression: low (SS, LGS, LGLG); intermediated (LALG, LAS) and high (LALA). Statistical analysis was performed with the software PLINK, with the significance level of α < 0.05. Results: No association was found between 5-HTTLPR, considering the biallelic or triallelic analysis, and anxiety disorders, behavioral inhibition and harm avoidance in both case-control and trios studies. Conclusion: Our results do not support a major role of 5-HTTLPR in anxiety disorders and anxiety-related phenotypes (behavioral inhibition and harm avoidance) in adolescents. In the face of mixed results, further investigations and meta-analytic studies are needed in order to clarify this research question.

A depressão pós-parto (DPP) é um importante problema de saúde pública podendo provocar uma ruptura do vínculo entre a mãe e o bebê e até estar associada com respostas trágicas, como suicídio materno e infanticídio. A DPP é multifatorial e o seu surgimento pode ser favorecido por componentes hormonais, genéticos e ambientais. O ciclo gravídico-puerperal é considerado um período de risco, pois algumas mulheres possuem uma sensibilidade particular as alterações hormonais. O risco de DPP é aumentado em mulheres que possuem histórico de depressão na família, logo, um componente genético determina maior suscetibilidade. Segundo o DMS-IV, existe uma relação entre a sintomatologia depressiva e as alterações na concentração cerebral de neurotransmissores, com destaque para serotonina. O transportador de serotonina (SERT) controla a intensidade e duração da re-captação da serotonina nas sinapses serotonérgicas. Diversos trabalhos associam os polimorfismos do SERT com transtornos mentais, como unipolar, bipolar, depressão e esquizofrenia. Nosso objetivo foi analisar as concentrações salivares de cortisol (CORT), as freqüências alélicas e genotípicas dos polimorfismos 5-HTTVNTR e 5- HTTLPR no gene SLC6A4 entre mulheres que desenvolveram ou não DPP. A amostra foi constituída por 128 mulheres brancas da cidade de Pelotas/RS, triadas em ambulatórios do SUS. A avaliação diagnóstica foi realizada através de entrevista psiquiátrica e diagnóstica usando como instrumento o Beck Depression Inventory entre 30 a 45 dias após o nascimento das crianças. A coleta de material biológico (leucócitos e saliva) foi realizada no turno da manhã, respeitando o período de duas horas de jejum. A região promotora do gene contendo o polimorfismo 5-HTTLPR (inserção/deleção) e a região do segundo íntron contendo o polimorfismo 5- HTTVNTR (repetições em tandem) foram amplificadas através da reação em cadeia da polimerase. A dosagem do CORT foi realizada a partir da saliva por técnica de ELISA utilizando kit específico. A mediana e o intervalo interquartil das concentrações salivares do CORT entre os portadores dos diferentes genótipos foram comparados entre os grupos estudados usando o teste de Kruskal-Wallis e Mann-Whitney. A comparação das freqüências alélicas e genotípicas dos polimorfismos estudados entre as mulheres que apresentarem ou não DPP foram feitas pelo teste do Qui-quadrado com correção de Yates (p£ 0,05). A análise da distribuição das freqüências genotípicas dos polimorfismos 5-HTTLPR e 5- HTTVNTR do SERT permitiu verificar que a população está sob Equilíbrio Hardy– Weinberg. Quando os polimorfismos foram analisados isoladamente, não foi observada associação entre os polimorfismos 5-HTTLPR (p=0,48) e 5-HTTVNTR (p=0,77) e o diagnóstico para DPP. Porém, a análise combinada dos haplótipos dos polimorfismos 5-HTTLPR e 5-HTTVNTR demonstraram que mulheres portadoras do diplótipo L-12/L-12 apresentaram escores menores de sintomas depressivos (mediana: 0,5; intervalo inter-quartil: 0,00-4,00, p=0,04) quando comparadas com mulheres portadoras de outros diplótipos (mediana: 4,0; intervalo inter-quartil: 1,00- 10,00). O polimorfismo 5-HTTVNTR foi associado com as concentrações salivares de CORT (p=0,03), já o polimorfismo 5-HTTLPR não foi associado (p=0,41). Nossos achados são inovadores, visto que até a presente data a associação dos genótipos 5-HTTLPR e 5-HTTVNTR com DPP e concentrações salivares de CORT ainda não haviam sido investigados. / The postpartum depression (PPD) is an important public health problem that may cause a rupture of the bond between the mother and the baby and may even be associated with tragic responses such as maternal suicide and infanticide. The DPP is multifactorial and its appearance can be favored by hormonal components, genetic and environmental factors. The pregnancy and childbirth is considered a risk period, because some women have a particular sensitivity to hormonal changes. The rate of DPP is increased in women who have a record of depression in the family, so a genetic component determines higher susceptibility. According to the DSM-IV, there is a relationship between depressive symptoms and brain concentration changes of neurotransmitters, particularly serotonin. The serotonin transporter (SERT) controls the intensity and duration of re-uptake of serotonin in serotonergic synapses. Several studies linking polymorphisms of SERT with mental disorders such as unipolar, bipolar, depression, and schizophrenia. Our objective was to analyze the concentrations of salivary cortisol (CORT), the allele and genotype frequencies of polymorphisms 5-HTTLPR and 5-HTTVNTR SLC6A4 gene in women who developed or not DPP. The sample consisted of 128 white women from Pelotas, RS, sorted out from public health clinics. The diagnostic evaluation was conducted through interviews and psychiatric diagnostic instrument as using the Beck Depression Inventory among 30 to 45 days after the birth of children. The collection of biological materials (leukocytes and saliva) was performed in the morning, observing the twohour period of fasting. The promoter region of the gene containing the 5-HTTLPR polymorphism (insertion/deletion) and the region containing the second intron polymorphism 5-HTTVNTR (tandem repeats) were amplified by polymerase chain reaction. The dose of CORT was performed from the saliva by ELISA using the specific kit. The median and interquartile interval of salivary concentrations of CORT among patients of different genotypes were compared between groups using the Kruskal-Wallis and Mann-Whitney. The comparison of allele and genotype frequencies of polymorphisms among women who developed or not DPP were made by chi-square test with Yates correction (p <0.05). The analysis of the distribution of genotype frequencies of polymorphisms 5-HTTLPR and 5-HTTVNTR SERT showed that the population is under Hardy-Weinberg Equilibrium. When the polymorphisms were analyzed alone, no association was observed between the 5-HTTLPR (p=0.48) and 5-HTTVNTR (p=0.77) polymorphisms and the PPD diagnosis. But, the information from these analyses combined with information regarding the haplotypes of the 5-HTTLPR and 5-HTTVNTR polymorphisms demonstrated that women carriers of diplotype L-12/L-12 have a lower depression symptoms score (median: 0.5; interquartile range: 0.00-4.00; p=0.04) than women with other diplotypes (median: 4.0; inter-quartile range: 1.00-10.00). The 5-HTTVNTR polymorphism was associated with the salivary concentrations of CORT (p=0.03), whereas the 5-HTTLPR polymorphism was not associated (p=0.41). Our findings are innovative since the association of 5- HTTLPR genotypes and 5-HTTVNTR with DPP and salivary concentrations of CORT had not been investigated before.

Introdução: Os transtornos de ansiedade (TA) são prevalentes na infância e na adolescência e geram prejuízos significativos, podendo persistir na vida adulta. Os traços de personalidade e temperamento associados à ansiedade, como o comportamento inibido (CI) e a evitação de danos, também devem ser considerados. O neurotransmissor serotonina possui um papel crítico no desenvolvimento e na plasticidade do encéfalo. O gene do seu transportador (5-HTT) é um forte candidato para estudos de associação genéticos e psiquiátricos. O polimorfismo na região promotora do gene do 5-HTT (5-HTTLPR) é funcional e, portanto, de relevância para estudos de associação na psiquiatria. Objetivos: Investigar a associação entre o 5-HTTLPR, através da sua classificação bialélica e trialélica, e os TA e fenótipos relacionados à ansiedade (CI e evitação de danos), em uma amostra de adolescentes ansiosos e não-ansiosos e seus familiares. Metodologia: Um total de 510 indivíduos participou do estudo. Os participantes foram 225 adolescentes (129 casos e 96 controles para TA) e seus familiares biológicos (194 mães, 63 pais e 22 irmãos). Foi realizado o diagnóstico psiquiátrico através de entrevista clínica e do Schedule for Affective Disorders and Schizophrenia for School-Age Children – Present and Lifetime Version Diagnostic Interview (K-SADS-PL). A escala Temperament and Character Inventory (TCI) e uma adaptação da escala Retrospective Self-report Scale of Behavioral Inhibition foram usadas para mensurar a evitação de danos e o comportamento inibido, respectivamente. As análises moleculares resultaram da extração de DNA da amostra de saliva dos adolescentes e seus familiares, seguida de amplificação do DNA por PCR e digestão enzimática com enzima MspI. Os genótipos foram agrupados pelo nível de expressividade: baixo (SS, LGS, LGLG); intermediário (LALG, LAS) e alto (LALA). A análise estatística foi realizada com o software PLINK e nível de significância α < 0.05. Resultados: Não foi encontrada associação entre o 5-HTTLPR, considerando a classificação bialélica e trialélica, e os transtornos de ansiedade, comportamento inibido e a evitação, tanto no caso-controle quanto no estudo de trios. Conclusões: Nossos resultados não sugerem a presença de associação entre o 5-HTTLPR e os transtornos de ansiedade e seus fenótipos relacionados (comportamento inibido e evitação de danos) em adolescentes. Diante de resultados controversos descritos na literatura, estudos de meta-análises podem ser necessários para auxiliar no esclarecimento sobre essa questão. / Introduction: Anxiety disorders (AD) are prevalent in childhood and adolescence and results in significant impairments. It usually persists into adulthood. Anxiety traits such as behavioral inhibition and harm avoidance may also be considered. The serotonin neurotransmitter plays an important role in the development and the plasticity of the brain. The serotonin transporter gene (5-HTT) is considered a strong candidate and the Serotonin Transporter Gene-linked Polymorphic Region (5-HTTLPR) functional and, therefore, relevant in studies concerning the association between genetic and psychiatric disorders. Objectives: To investigate the association between 5-HTTLPR (biallelic and triallelic classification) and AD and anxiety related phenotypes (behavioral inhibition and harm avoidance) in a sample of adolescents and their families. Methodology: A total of 510 subjects participated in this study. Participants were 225 adolescents (129 anxiety cases and 96 community controls) and their biological families (194 mothers, 66 fathers and 22 siblings). We assessed psychiatric diagnosis throughout a clinical interview and using the Schedule for Affective Disorders and Schizophrenia for School-Age Children – Present and Lifetime Version Diagnostic Interview (K-SADS-PL). The Temperament and Character Inventory (TCI) and an adaptation of the Resnick Behavioral Inhibition Scale were used in order to measure harm avoidance and behavioral inhibition, respectively. The molecular analysis resulted in the extraction of DNA from saliva sample of the adolescents and their families, followed by DNA amplification by PCR and enzymatic digestion with the MspI. The genotypes were grouped by level of expression: low (SS, LGS, LGLG); intermediated (LALG, LAS) and high (LALA). Statistical analysis was performed with the software PLINK, with the significance level of α < 0.05. Results: No association was found between 5-HTTLPR, considering the biallelic or triallelic analysis, and anxiety disorders, behavioral inhibition and harm avoidance in both case-control and trios studies. Conclusion: Our results do not support a major role of 5-HTTLPR in anxiety disorders and anxiety-related phenotypes (behavioral inhibition and harm avoidance) in adolescents. In the face of mixed results, further investigations and meta-analytic studies are needed in order to clarify this research question.

Autism Spectrum Disorder (ASD) is a diverse disorder, and the heterogenous range of possible presentations hinders our understanding of its aetiology. Recently there has been a surge of genome wide association studies for ASD, while historically psychological theories were relied on to explain the emergence of ASD. These fields continue to provide insights into ASD, but they tend to operate in parallel – genetic studies often lack comprehensive psychological phenotyping and theoretical backing, and psychological studies tend to lack genetic explanations. I propose that moving forward, genotype-phenotype studies should have a strong foundation in both fields and should focus on genes and theories with realworld implications for ASD diagnostics and/or interventions. This approach can be supported by focusing on established, well supported psychological theories, and selected ASD candidate genes that could be implicated in these theories, and ideally the genetic mechanism implicated should be one that can be targeted by existing medications. I therefore selected two prominent psychological theories, the Social Motivation Theory for ASD (Panksepp, 1979) and the ToM Theory for ASD (Baron-Cohen et al., 1985), and selected an ASD candidate gene that was likely implicated in each of these theories, namely the mu-opioid receptor gene (OPRM1) and the serotonin transporter promoter length polymorphism (5-HTTLPR) respectively. For the first study of this protocol, I assessed the possible relationships between social motivation, OPRM1, and the ASD phenotype. For the second study, I assessed possible relationships between ToM deficits, 5-HTTLPR, and the ASD phenotype. These two studies shared a sample of 153 male children 4-16 years old; 51 boys per group (i.e. non-verbal ASD; verbal ASD; neurotypical). All ASD children completed ADOS2 assessment for ASD phenotyping. For the Social Motivation Study, attachment was used as a proxy to assess level of social motivation in all participants, and 76 of the 102 children with ASD provided DNA for OPRM1 genotyping. Comparisons across all three groups showed that the ASD samples had significantly lower social motivation than the neurotypical sample, with the non-verbal ASD group displaying the most severely reduced level of social motivation. Reduced social motivation was associated with ASD-related deficits in the non-verbal ASD sample but not the verbal ASD sample. Finally, I was unable to statistically assess the role of OPRM1 as hypothesized, due to an unprecedentedly high rate of the OPRM1 G allele, which indicated atypical mu-opioid processes. This clearly implicated OPRM1 in ASD and is the first study to show this so convincingly. Overall, this study's findings led me to suggest that Panksepp's (1979) theory could be updated to include a threshold effect such that more severely reduced social motivation in ASD is associated with little-to-no language acquisition, while those with less severely reduced social motivation are able to develop language and this protects against associations between social motivation and ASD-related deficits in childhood. For the Theory of Mind Study, the verbal ASD sample and neurotypical sample completed a developmental ToM Battery (i.e. University of Cape Town Theory of Mind Battery) and WASI assessment to establish verbal intelligence quotient (VIQ) scores, and 70 of the children with ASD were successfully genotyped for 5-HTTLPR. This study found that verbal male children with ASD tended to be one developmental stage behind age-matched neurotypical peers on ToM tasks. ToM deficits were associated with greater impairment in overall ASD severity and in symptoms from the social communication and interaction domain. For the non-verbal ASD sample, the 5-HTTLPR short allele, which is implicated in atypical serotonergic transmission, was associated with greater impairment overall and in the restricted and repetitive behaviours and interests symptom domain. No associations between 5-HTTLPR and ToM, or with ASD-related symptoms, was found for the verbal ASD group. This again suggested that language acquisition is an important consideration in genotypephenotype studies in male children with ASD.

Early life stress has been found to be a strong predictor of depression severity, with genetic risk factors such as the serotonin transporter promotor (5-HTTLPR) and brain derived neurotrophic factor (BDNF) polymorphisms moderating depression development. Our investigation aims to extend these findings to examine the impact of depression severity, genetic risk factors, and childhood maltreatment on neuronal connectivity changes using tract based spatial statistics (TBSS) and probabilistic tractography. Fifty-five medication-free patients with major depressive disorder (MDD) [x̅ age: 36.4, M/F: 22/33] and 18 controls [x̅ age: 33.2, M/F: 8/10] underwent diffusion tensor imaging scanning, genotyping and completed the Childhood Trauma Questionairre. Corrected TBSS findings revealed trends toward significantly reduced FA in the right anterior internal capsule [p=0.051], fornix [p=0.085] and right anterior corona radiata [p=0.084] in the MDD group. Probabilistic tractography analysis examined fractional anisotropy (FA) in the cingulum bundle, uncinate fasciculus and superior longitudinal fasciculus. Individuals scoring high in depression severity and who experienced severe childhood physical neglect (PN) and emotional neglect (EN) had higher FA in the uncinate [PN: p=0.003, EN: p=0.029] and superior longitudinal fasciculus [PN: p=0.0748], with BDNF and 5-HTTLPR moderating these associations. BDNF polymorphisms also exhibited a stronger impact on uncinate FA in individuals with high depression severity, with val-BDNF exhibiting higher FA than met carriers [p=0.021]. In conclusion, MDD patients exhibit widespread decreases in FA across many neural regions. Furthermore, the impact that depression severity has on FA is considerably influenced by early life neglect. / Thesis / Master of Science (MSc)