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The association between chronotypes and season of birth (SOB) remains an inconclusive issue due, in some extension, to the lack of investigations of mediation mechanisms. We evaluated the association of photoperiod at birth (PAB) with chronotypes and sleep duration in Brazil (n = 810), and the mediating effect of meteorological factors, sex, age and rs4753426 polymorphism in the melatonin receptor MTNR1B. Longer PAB was associated with a delayed mid-sleep phase with a suppressive effect of maximum environmental temperature. No significant interactions were identified for the other variables. These findings suggest that photoperiod and environmental temperature modulate chronotype development at early stages.

Background Evidences suggest that alterations in circadian rhythms trigger the development of mental disorders. Eveningness, sleep behavior, and circadian genes polymorphisms have been associated with depression and anxiety symptomatology. However, the mechanism underlying these interactions is not well understood. We investigated the contribution of diurnal preference, sleep habits, and PER3 VNTR polymorphism (rs57875989) to depression and anxiety symptoms in a Northeast sample from the Brazilian population. Methods Eight hundred and four young adults completed the Morningness-Eveningness (MEQ), Munich Chronotype (MCTQ), Center for Epidemiologic Studies – Depression (CES-D), and Beck Anxiety Inventory (BAI) questionnaires. All participants were genotyped and linear regression was performed to test the interactions between the genetic /behavioral variants and depression/ anxiety symptoms. Results Eveningness and sleep behaviors (bedtime, wake-up time, sleep duration, and midpoint of sleep) were correlated with depression symptomatology, specifically in somatic factors of the CES-D questionnaire. No correlation was found between diurnal preference/sleep habits with anxiety symptoms for both BAI total score and its factors. However, women with PER34/4 genotype showed less interpesonal affect in depression symptomatology and more anxiety symptoms in four factors of the BAI questionnaire. Limitations Mainly because this study was based on self-report questionnaires and was limited to undergraduate students aging 18 to 30 years old. Conclusion These results reinforce a role for sleep and diurnal preference in depression, and PER3 VNTR polymorphism in anxiety symptomatology, particularly in women.

Recent studies have shown that transcriptomes from different tissues present circadian oscillations. Therefore, the endogenous variation of total RNA should be considered as a potential bias in circadian studies of gene expression. However, normalization strategies generally include the equalization of total RNA concentration between samples prior to cDNA synthesis. Moreover, endogenous housekeeping genes (HKGs) frequently used for data normalization may exhibit circadian variation and distort experimental results if not detected or considered. In this study, we controlled experimental conditions from the amount of initial brain tissue samples through extraction steps, cDNA synthesis, and quantitative real time PCR (qPCR) to demonstrate a circadian oscillation of total RNA concentration. We also identified that the normalization of the RNA’s yield affected the rhythmic profiles of different genes, including Per1-2 and Bmal1. Five widely used HKGs (Actb, Eif2a, Gapdh, Hprt1, and B2m) also presented rhythmic variations not detected by geNorm algorithm. In addition, the analysis of exogenous microRNAs (Cel-miR-54 and Cel-miR-39) spiked during RNA extraction suggests that the yield was affected by total RNA concentration, which may impact circadian studies of small RNAs. The results indicate that the approach of tissue normalization without total RNA equalization prior to cDNA synthesis can avoid bias from endogenous broad variations in transcript levels. Also, the circadian analysis of 2−Cycle threshold (Ct) data, without HKGs, may be an alternative for chronobiological studies under controlled experimental conditions.

Objective To evaluate gene polymorphisms and their association with susceptibility to dengue. Methods A retrospective case-control study was performed with 262 subjects, comprising 78 dengue fever (DF) patients, 49 dengue hemorrhagic fever (DHF) patients and 135 healthy controls. Genotypic and allelic profiles were identified using polymerase chain reaction based in real time and amplification-refractory mutation system. Results We observed a protective association of IL-10 (−819 C/T) C allele (P = 0.028, OR = 0.56, CI = 0.34–0.91) against DHF, while the C/T (P = 0.047, OR = 2.10, CI = 1.01–4.38) and T/T (P = 0.008, OR = 3.82, CI = 1.38–10.59) genotypes were associated with DHF and DF, respectively. The dominant model TNFA −308 GA + AA (P = 0.043, OR = 0.45, CI = 0.20–1.00) genotypes were found to have protective effect against dengue infection. A protective association among the IFNG (+874 A/T) A/T genotype against DF (P = 0.02, OR = 0.46, CI = 0.24–0.89) and DHF (P = 0.034, OR = 0.43, CI = 0.19–0.95) was observed. When the studied single-nucleotide polymorphism was analyzed in combination, the combination GTA (P = 0.022, OR = 2.95, CI = 1.18–7.41) was statistically significantly associated with susceptibility to DF and the combination GCT (P = 0.035, OR = 0.28, CI = 0.08–0.90) with protection against the development of DHF. Conclusions This research identifies the association of the IFNG (+874 A/T), TNFA (−308G/A), IL-10 (−819 C/T) genotypes as a factor for protection, susceptibility and severity to dengue.

Background Seasonal variations in suicides have been reported worldwide, however, there may be a different seasonal pattern in suicide attempts. The aim of this study was to perform a systematic review on seasonality of suicide attempts considering potential interfering variables, and a statistical analysis for seasonality with the collected data. Method Observational epidemiological studies about seasonality in suicide attempts were searched in PubMed, Web of Science, LILACS and Cochrane Library databases with terms attempted suicide, attempt and season. Monthly or seasonal data available were evaluated by rhythmic analysis softwares. Results Twenty-nine articles from 16 different countries were included in the final review. It was observed different patterns of seasonality, however, suicide attempts in spring and summer were the most frequent seasons reported. Eight studies indicated differences in sex and three in the method used for suicide attempts. Three articles did not find a seasonal pattern in suicide attempts. Cosinor analysis identified an overall pattern of seasonal variation with a suggested peak in spring, considering articles individually or grouped and independent of sex and method used. A restricted analysis with self-poisoning in hospital samples demonstrated the same profile. Limitations Grouping diverse populations and potential analytical bias due to lack of information are the main limitations. Conclusions The identification of a seasonal profile suggests the influence of an important environmental modulator that can reverberate to suicide prevention strategies. Further studies controlling interfering variables and investigating the biological substrate for this phenomenon would be helpful to confirm our conclusion.

Mesial Temporal Lobe Epilepsy (mTLE) characterized by progressive development of complex partial seizures originating from the hippocampus is the most prevalent and refractory type of epilepsy. One of the remarkable features of mTLE is the rhythmic pattern of occurrence of spontaneous seizures, implying a dependence on the endogenous clock system for seizure threshold. Conversely, circadian rhythms are affected by epilepsy too. Comprehending how the circadian system and seizures interact with each other is essential for understanding the pathophysiology of epilepsy as well as for developing innovative therapies that are efficacious for better seizure control. In this review, we confer how the temporal dysregulation of the circadian clock in the hippocampus combined with multiple uncoupled oscillators could lead to periodic seizure occurrences and comorbidities. Unraveling these associations with additional research would help in developing chronotherapy for mTLE, based on the chronobiology of spontaneous seizures. Notably, differential dosing of antiepileptic drugs over the circadian period and/or strategies that resynchronize biological rhythms may substantially improve the management of seizures in mTLE patients.

Background Melatonin modulates the master circadian clock through the activation of G-protein-coupled receptors MT1 and MT2. It is presumed, therefore, that genetic variations in melatonin receptors can affect both sleep and circadian phase. We investigated whether the −1193T > C (rs4753426) polymorphism in the promoter of MT2 receptor gene (MTNR1B) is associated with diurnal preference and sleep habits. This polymorphism was previously associated with sunshine duration, suggesting a role in circadian entrainment. Methods A total of 814 subjects who completed the Morningness–Eveningness and the Munich Chronotype questionnaires were genotyped for the selected polymorphism. Linear and multinomial regression were performed to test the interaction between gene variants and diurnal preference/sleep habits. Results The −1193C variant was associated with the extreme morningness phenotype in a codominant model (C/C vs. T/T), recessive model (C/C + C/T vs. T/T) and alleles (C vs. T). A negative correlation was found between −1193C alleles and social jetlag scores. The frequency of −1193T allele was higher in the group that stay in bed more than 8 h/night compared to the group that stay in bed less than 8 h/night on weekends. Conclusion To the best of our knowledge, these data provide the first insights into the role of MTNR1B gene in the regulation of sleep, biological rhythms, and entrainment in humans.

There is little evidence for the involvement of microRNAs (miRs) in the regulation of circadian rhythms, despite the potential relevance of these elements in the posttranscriptional regulation of the clock machinery. The present work aimed to identify miRs targeting circadian genes through a predictive analysis of conserved miRs in mammals. Besides 23 miRs previously associated with circadian rhythms, we found a number of interesting candidate genes, equally predicted by the 3 software programs used, including miR-9, miR-24, miR25, miR-26, miR-27, miR-29, miR-93, miR-211, miR-302, and miR-346. Moreover, several miRs are predicted to be regulated by circadian transcription factors, such as CLOCK/BMAL, DEC2, and REV-ERBalpha. Using real-time PCR we demonstrated that the selected candidate miR-27b showed a daily variation in human leukocytes. This study presents predicted feedback loops for mammalian molecular clock and the first description of an miR with in vivo daily variation in humans.

An association between Parkinson's disease (PD) and brain-derived neurotrophic factor (BDNF) was suggested by several studies, with contradictory results. BDNF is necessary for the survival of dopaminergic neurons in substantia nigra. Val66Met is a common polymorphism of the BDNF gene that affects cognitive and motor processes. The authors studied 104 Brazilian patients with PD and 96 control participants. The G/G genotype was significantly associated with depression and anxiety symptoms and development of PD. This is the first study that associates this genotype with PD.

Transtorno bipolar (TB) é uma doença comum que afeta aproximadamente 1% da população. Apresenta características crônicas e agudas graves, com índices de remissão de baixa e alta prevalência de comorbidades clínicas e psiquiátricas. O objetivo do presente artigo é sintetizar dados de vários artigos que investigaram polimorfismos genéticos associados com TB. Dentre os 129 artigos selecionados, identificaram-se 79 (85,87%) genes associados com TB. Essa análise identificou cinco genes que são os mais citados na literatura: CANAC1C, DAOA, TPH2, ANK3 e DISC1. Dos 92 genes identificados nesses artigos, 33 (35,87%) não mostraram associação com TB. Essa análise mostrou que, apesar dos avanços recentes com relação ao papel do polimorfismo genético na predisposição para TB, mais pesquisas ainda são necessárias para elucidar sua influência sobre esse transtorno. Bipolar disorder (BD) is a common disorder that affects approximately 1% of the population. It is associated with both chronic and acute severe features, such as low remission rates and a high prevalence of clinical and psychiatric comorbidities. The aim of the present article is to synthesize data from various articles that investigated genetic polymorphisms associated with BD. The 129 articles selected identified 79 (85.87%) genes associated with BD. This analysis identified the five genes that are the most cited in the literature: CANAC1C, DAOA, TPH2, ANK3 and DISC1. Of the 92 genes identified in these articles, 33 (35.87%) showed no association with BD. This analysis showed that, despite recent advances with respect to the role of genetic polymorphism in predisposition to BD, further research is still required to elucidate its influence on this disorder.

We are reporting here the results of differential gene expression experiments comparing two siblings, a 21-yr-old male and a 19-yr-old female, with the same alpha-thalassemia genotype (-alpha(3.7)/(--SEA)) and quite different levels of Hb H in the peripheral blood (18.7 and 5%, respectively). By using mRNA differential-display reverse-transcription-PCR and suppression subtractive hybridization, two main transcripts were selected in both procedures and validated by qRT-PCR, one corresponding to the phosphatidylinositol phosphate 4-kinase type II-alpha (PIP4KIIA) gene and the other to the beta-globin gene, both over expressed in the patient with the higher percentage of Hb H. Type II PIP kinases produce phosphatidylinositol 4,5 biphosphate, a critical and pleiotropic regulatory molecule involved in diverse cellular activities, including gene expression. Our results suggest that PIP4KIIA may be one of the factors related to the regulation of the beta-globin gene expression and the different levels of Hb H in alpha-thalassemic patients.

Juvenile myoclonic epilepsy (JME) accounts for 26% of generalized idiopathic epileptic syndromes. The highest levels of thrombin activity are closely involved in the development of neurological diseases, including epilepsy. The prothrombin c.20210G>A (rs1799963) variation, which alters prothrombin mRNA stability, is associated with high plasma prothrombin levels. Objective : The present study was designed to investigate whether the SNP rs1799963 is a risk factor for JME in the northeastern Brazilian population. Results : The polymorphism was genotyped in 207 controls and 123 patients using polymerase chain reaction-restriction fragment length polymorphism method. No significant differences were observed in the genotype and allele frequencies of this polymorphism between cases and controls. Conclusion : These results present no evidence for an association of rs1799963 with JME. Further studies including other types of epilepsy are required to investigate the involvement of prothrombin gene in the genetic susceptibility to chronic seizure. Epilepsia mioclônica juvenil (EMJ) representa 26% das síndromes epilépticas idiopáticas generalizadas. Níveis elevados de atividade da trombina estão intimamente envolvidos no desenvolvimento de distúrbios neurológicos, incluindo epilepsia. A variante c.20210G>A (rs1799963) do gene de protrombina, que altera a estabilidade do RNAm, está associada com altos níveis de protrombina no plasma. Objetivo: Investigar se o SNP rs1799963 é um fator de risco para EMJ em uma amostra da população do nordeste brasileiro. Resultados : O polimorfismo foi genotipado em 123 pacientes e 207 controles usando a reação de polimerase em cadeia com restrição de polimorfismo. Não observamos diferença significativa nas frequências alélicas e genotípicas deste polimorfismo, entre as populações de pacientes e controle. Conclusão : Estes resultados não demonstram evidências para uma associação do polimorfismo rs1799963 com EMJ. Estudos posteriores, incluindo outros tipos de epilepsia, são necessários para investigar o envolvimento do gene protrombina na susceptibilidade genética a crises crônicas.

The genetic mechanisms underlying the continued expression of the gamma-globin genes during the adult stage in deletional hereditary persistence of fetal hemoglobin (HPFH) and deltabeta-thalassemias are not completely understood. Herein, we investigated the possible involvement of transcription factors, using the suppression subtractive hybridization (SSH) method as an initial screen to identify differentially expressed transcripts in reticulocytes from a normal and a HPFH-2 subject. Some of the detectable transcripts may participate in globin gene regulation. Quantitative real-time PCR (qRT-PCR) experiments confirmed the downregulation of ZHX2, a transcriptional repressor, in two HPFH-2 subjects and in a carrier of the Sicilian deltabeta-thalassemia trait. The chromatin remodeling factors ARID1B and TSPYL1 had a very similar pattern of expression with an incremental increase in HPFH and decreased expression in deltabeta-thalassemia. These differences suggest a mechanism to explain the heterocellular and pancellular distribution of fetal hemoglobin in deltabeta-thalassemia and deletional HPFH, respectively. Interestingly, alpha-globin mRNA levels were decreased, similar to beta-globin in all reticulocyte samples analyzed.

The aim of the present study was to analyze if the genetic polymorphisms might predict suicide attempts in mental disorder patients. The literature review and meta-analysis were conducted using the PubMed/Medline, Web of science and Scopus database using the terms: “5-HTT or SLC6A4 or 5-SERT and suicide, suicidal ideation or suicidal behavior or suicidal attempt”. Thirty articles were analyzed. We found 17 articles that showed association and 13 articles that showed no association between LPR serotonin transporter polymorphism and suicide. A higher study of suicide identified the serotonin transporter polymorphism in patients with schizophrenia, mental disorder, major depression and bipolar disorder. There is an association between the serotonin-transporter-linked polymorphic region and suicidal behavior. The mental disorders with greater relationship with the suicide were the bipolar disorder, major depression and schizophrenia. The L allele had higher risk for suicide.

Circadian rhythms are controlled by a molecular mechanism that is organized in transcriptional and translational feedback loops of gene expression. Recent studies have been demonstrating the involvement of microRNAs (miRs) in post-transcriptional/translational control of circadian rhythms. In the present study we aimed to analyze the daily variations of miR-16 and miR-181a expression in human leukocytes. These miRs were independently associated with hematopoiesis and circadian rhythms in previous studies using experimental models. Peripheral blood from 6 subjects was sampled in a 24 hour period for expression analysis using quantitative real-time PCR (RT-qPCR). Initially, we evaluated the expression stability of RNU6-2, RNU1A-1, RNU5A-1, SNORD-25, SCARNA-17 and SNORA-73A as candidate genes for normalization of RT-qPCR data. The combination of the four most stable genes (SNORA-73A/SCARNA-17/SNORD-25/RNU6-2) was indicated to provide a better normalization of miRs expressions. The results show a daily variation of miR-181a and miR-16 expression in human leukocytes, suggesting a potential participation of these genes in the modulation of the circadian rhythms present in blood cells.

Sleep disturbance is common in several epilepsy types, such as juvenile myoclonic epilepsy (JME). Genetic background could increase susceptibility to seizure and sleep abnormalities. From this perspective, a susceptibility gene for sleep disturbance or chronotype could contribute to the genetic susceptibility threshold for epilepsy and vice versa. Accordingly, we investigated whether functional clock gene polymorphisms (PER2 111C>G, CLOCK 3111T>C, and PER3 VNTR) might influence the risk for JME. All these polymorphisms have recently been reported to be associated with sleep disturbance, diurnal variation, and neurological diseases. The polymorphisms were genotyped in 97 patients and 212 controls using polymerase chain reaction or restriction fragment length polymorphism methods. No significant differences were observed in the genotypic and allelic frequencies of these polymorphisms between cases and controls even when analyses were restricted to patients that presented a diurnal preferential seizure occurrence. We also tested for interactions between polymorphisms by multifactor dimensionality reduction analysis. None of the combined genotypes differed significantly between the groups. These results present no evidence for an association of these polymorphisms with JME. Further studies including other types of epilepsy and/or other functional polymorphisms are required to investigate the possible relationship between clock genes and the genetic susceptibility to chronic seizure.

This study aimed to characterize the profiles of suicide attempts that were attended to in the Hospital of Alagoas in the year 2010. Four hundred sixty-one charts and service bulletins were analyzed. Patients attempting suicide were predominately female. There were significant difference for suicide attempts (SAs) among men and women in the age of 10 to 19 years and 60 to 69 years. Women have tried more suicide aged between 10 and 19 years and men between 60 and 69 years. The ingestion of drugs was the most frequent method for women; and poisoning, use of sharp objects and hanging for men. The results of this study may contribute to elaboration, planning and implementation of preventive measures to reduce cases of SAs. Este estudo teve como objetivo caracterizar o perfil das tentativas de suicídio (TS) que foram atendidos num Hospital de Alagoas, no ano de 2010. Quatrocentos e sessenta e um boletins foram analisados. Houve diferença significativa para as tentativas de suicídio entre homens e mulheres nas faixas etárias de 10 a 19 anos e de 60 a 69 anos. As mulheres cometem mais suicídio na faixa etária entre 10 e 19 anos e os homens entre 60 e 69 anos. A ingestão de drogas foi o método mais frequente para as mulheres; o envenenamento, uso de objetos pontiagudos e enforcamento para homens. Os resultados deste estudo podem contribuir para a elaboração, planejamento e implementação de medidas preventivas em casos de TS.

Real-time quantitative RT-PCR (qPCR) is one of the most powerful techniques for analyzing miRNA expression because of its sensitivity and specificity. However, in this type of analysis, a suitable normalizer is required to ensure that gene expression is unaffected by the experimental condition. To the best of our knowledge, there are no reported studies that performed a detailed identification and validation of suitable reference genes for miRNA qPCR during the epileptogenic process. Here, using a pilocarpine (PILO) model of mesial temporal lobe epilepsy (MTLE), we investigated five potential reference genes, performing a stability expression analysis using geNorm and NormFinder softwares. As a validation strategy, we used each one of the candidate reference genes to measure PILO-induced changes in microRNA-146a levels, a gene whose expression pattern variation in the PILO injected model is known. Our results indicated U6SnRNA and SnoRNA as the most stable candidate reference genes. By geNorm analysis, the normalization factor should preferably contain at least two of the best candidate reference genes (snoRNA and U6SnRNA). In fact, when normalized using the best combination of reference genes, microRNA-146a transcripts were found to be significantly increased in chronic stage, which is consistent with the pattern reported in different models. Conversely, when reference genes were individually employed for normalization, we failed to detect up-regulation of the microRNA-146a gene in the hippocampus of epileptic rats. The data presented here support that the combination of snoRNA and U6SnRNA was the minimum necessary for an accurate normalization of gene expression at the different stages of epileptogenesis that we tested.

Background: Juvenile myoclonic epilepsy (JME) is an idiopathic generalized epilepsy syndrome with genetic basis and accounts for 10% of all forms of epilepsy. Despite the existence of rare mutations responsible for some familial forms inherited in a Mendelian pattern, the genetics of JME is complex and probably involves multiple genes. Because of widespread distribution in the central nervous system (CNS) and their ability to produce postsynaptic inhibition, GABA (A) receptor subunits (GABRs) encoding genes represent high ranking candidates for epilepsy susceptibility. Aim: This case/control study was designed to investigate whether the rs211037 of the GABRG2 gene is a risk factor for JME in the Brazilian population. Materials and Methods: The polymorphism was genotyped in 98 patients and 130 controls using polymerase chain reaction-restriction fragment length polymorphism method. Descriptive and statistical analyses were performed using SNP stat software. Results: Genotype proportions and allele frequencies for the rs211037 polymorphism of the GABARG2 gene did not differ significantly between the groups, even when the odds ratio was adjusted for clinical variables. Conclusion: These results present no evidence for an association of rs211037 with JME. Further studies are required to investigate the involvement of the GABRG2 gene in the genetic susceptibility to this epileptic syndrome.

It is well recognized that the reference gene in a RT-qPCR should be properly validated to ensure that gene expression is unaffected by the experimental condition. We investigated eight potential reference genes in two different pilocarpine PILO-models of mesial temporal lobe epilepsy (MTLE) performing a stability expression analysis using geNorm, NormFinder and BestKepeer softwares. Then, as a validation strategy, we conducted a relative expression analysis of the Gfap gene. Our results indicate that in the systemic PILO-model Actb, Gapdh, Rplp1, Tubb2a and Polr1a mRNAs were highly stable in hippocampus of rats from all experimental and control groups, whereas Gusb revealed to be the most variable one. In fact, we observed that using Gusb for normalization, the relative mRNA levels of the Gfap gene differed from those obtained with stable genes. On the contrary, in the intrahippocampal PILO-model, all softwares included Gusb as a stable gene, whereas B2m was indicated as the worst candidate gene. The results obtained for the other reference genes were comparable to those observed for the systemic Pilo-model. The validation of these data by the analysis of the relative expression of Gfap showed that the upregulation of the Gfap gene in the hippocampus of rats sacrificed 24 hours after status epilepticus (SE) was undetected only when B2m was used as the normalizer. These findings emphasize that a gene that is stable in one pathology model may not be stable in a different experimental condition related to the same pathology and therefore, the choice of reference genes depends on study design.

Using a suppression subtractive hybridization method, we have previously identified genes differentially expressed in erythroid cells heterozygous for large deletions in beta-like globin cluster. Herein, we investigated the expression of four newly detected red cell-related genes in erythroid differentiation. ARID1B and TSPYL1, genes with chromatin remodeling properties, presented similar patterns of expression with an upregulation after erythropoietin (EPO) addition, similar to previous data found in reticulocytes. ZHX2, a transcriptional repressor, was downregulated, and a redoxin-related gene, SH3BGRL2, had higher levels of expression on differentiation. These are the first investigations of these newly described genes in erythroid differentiation and demonstrate that the expression of these genes is affected by EPO stimulation. These genes may participate in globin regulation and may be important in the normal physiology of erythrocytes.

Orientador: Fernando Ferreira Costa Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas Resumo: Persistência Hereditária de Hemoglobina Fetal (PHHF) consiste num grupo heterogêneo de alterações hereditárias, sem manifestações clínicas significativas, onde ocorrem falhas na mudança perinatal normal de hemoglobina fetal para hemoglobina adulta, resultando em altos níveis de Hb F durante a vida adulta. Nos tipos delecionais ocorre normalmente aumento de ambas as cadeias, ? A e ? G, até 30%, estando associadas a deleções de seqüências de DNA dentro do grupo de genes ß. As (dß)º - talassemias também se originam a partir de deleções, em muitos casos bastante similares às que originam as formas delecionais de PHHF, entretanto com níveis de Hb F aumentados em menor proporção (5-20%), sendo acompanhadas nos heterozigotos por hipocromia e microcitose nas hemácias. Três hipóteses principais foram propostas para explicar a relação destas deleções com a ausência de supressão normal dos genes ? na fase adulta: competição entre promotores pelo LCR (Locus Control Region); justaposição de elementos acentuadores, normalmente encontrados a 3' do cluster, nas proximidades dos genes ?; remoção de elementos silenciadores, localizados entre ? A e d. Neste trabalho, realizamos estudos funcionais com uma região intergênica, aqui denominada R/PYR, potencialmente envolvida com o silenciamento de ? e a troca na síntese das cadeias de globinas. Os resultados obtidos com transfecção estável em células MEL apontam uma possível participação deste elemento na regulação dos genes de globina. Também investigamos o possível envolvimento de fatores de transcrição, construindo Bibliotecas de cDNA Subtrativas Supressivas para identificar transcritos diferencialmente expressos em reticulócitos de um sujeito com PHHF-2. A análise dos perfis diferenciais de expressão gênica em reticulócitos de PHHF-2 e normais mostram alterações em larga escala e que podem estar associadas com o fenótipo de PHHF. Estas modificações podem ser decorrentes de perturbações nas interações cromossômicas e/ou nos níveis de RNAs regulatórios não codificantes e ajudarem a melhor compreender a disposição e organização dos genes transcricionalmente ativos no genoma, e de que maneira são regulados. A presença de fatores de transcrição e remodeladores de cromatina, como ARID1B, TSPYL1 e ZHX2, dentre outros, com expressão alterada indicam fortemente que outros mecanismos genéticos estão envolvidos com o aumento da síntese de gama globina no estágio adulto, além dos modelos previamente descritos na literatura. Além disto, são candidatos potenciais a reguladores de globina que ainda não haviam sido descritos, e podem ajudar a compreender as diferenças fenotípicas encontradas entre dß-talassemia e PHHF delecional. Identificamos ainda outros genes com expressão diferencial, além de seqüências sem similaridade a genes categorizados, como ORFs, ESTs e seqüências genômicas, que podem ter papel importante na fisiologia normal das células vermelhas ou nas hemoglobinopatias hereditárias. Além disto, a observação de que os níveis de RNAm de alfa globina estão diminuídos em PHHF-2 e dß-Talassemia Siciliana sugere um mecanismo de regulação pós-transcripcional compensatório para as cadeias de globina ou de regulação gênica por interação física direta entre os cromossomos 11 e 16. Ao nosso conhecimento, esta é a primeira descrição demonstrando as implicações na expressão gênica em larga escala em células contendo alterações no DNA como as deleções apresentada Abstract: The genetic mechanisms underlying the continued expression of the ?-globin genes during the adult stage in deletional hereditary persistence of fetal hemoglobin (HPFH) and dß-thalassemias are not completely understood. For deletional HPFH, three main hypotheses were proposed to explain the relationship between these deletions and the non-suppression of ? -genes in the adult; 1- the removal of competitive regions that interact with the LCR; 2- the juxtaposition of enhancer elements located downstream from the breakpoint region; and 3- the removal of gene silencer elements. Herein, we studied a region, called R/PYR, that lies 1-3 Kb upstream from the d gene. This region has been implicated in ? globin repression and globin gene switching. Stable transfections in MEL cells suggest a possible involvement of R/PYR in globin regulation. We also investigated the possible involvement of transcription factors, using the SSH method to identify differentially expressed transcripts in reticulocytes from a normal and a HPFH-2 subject. Some of the detectable transcripts may participate in globin gene regulation. Quantitative RT-PCR experiments confirmed the downregulation of ZHX2, a transcriptional repressor, in two HPFH-2 subjects and in a carrier of the Sicilian dß-thalassemia trait. The chromatin remodeling factors ARID1B and TSPYL1 had a very similar pattern of expression with an incremental increase in HPFH and decreased expression in dß-thalassemia. These differences suggest a mechanism to explain the heterocellular and pancellular distribution of F cells in dß-thalassemia and deletional HPFH, respectively. Interestingly, a-globin mRNA levels were decreased, similar to ß-globin in all reticulocyte samples analyzed, which may be the result of a common regulatory process affecting a-like and ß-like globin synthesis. Part of the altered gene expression detected within the subtracted libraries may be an indirect effect due to the increased concentration of HbF in the cells. However, genetic alterations similar to those presented in this work may be implicated in the perturbation of chromosome interactions and/or disruption of the expression of regulatory non-coding transcripts with functional consequences for cellular gene expression. Additionally, other genes may have modifications of their expression, but were not detected in our experiments. To our knowledge, this is the first description of wide-ranging gene expression alterations in cells containing deletional HPFH and thalassemia. Finally, the dissection of the differential expression data presented in this study may help elucidate important pathways of erythroid differentiation and maintenance of red cells in peripheral circulation, as well as aid in the understanding of genetic mechanisms involved in erythrocyte pathologies such as sickle cell disease and thalassemias Doutorado Biologia Estrutural, Celular, Molecular e do Desenvolvimento Doutor em Fisiopatologia Médica

We developed a semi-automated approach to detect large deletions in the β-globin gene cluster, based on the quantitative differences in the amplifications of samples by a fluorescent PCR. With this strategy, we were able to detect the presence of HPFH-2 in an African-Brazilian subject, confirmed by sequencing analysis. Differently from other PCR strategies, GAP-PCR for example, it has the potential to identify new deletions. Am. J. Hematol. 72:225–227, 2003. © 2003 Wiley-Liss, Inc.

Heterogeneous amounts of Hb H have been detected in peripheral blood of alpha-thalassemic patients even when they carry the same mutation association. This suggests the involvement of other modulating factors besides the thalassemia determinants. To address this issue, we investigated differential gene expression in reticulocytes from two Brazilian siblings of mixed ethnical origin (Chinese and African) in whom Hb H disease is caused by the -a3.7/--SEA genotype. One is a 21-year-old male, and the other a 19-year-old female. Their hemoglobin H levels are 18.7 and 5.0%, respectively. By using the mRNA differential-display reverse-transcription polymerase chain reaction (DDRT-PCR) and suppression subtractive hybridization (SSH) techniques, we identified two main transcripts in both procedures, one corresponding to the phosphatidylinositol-4-phosphate-5-kinase type II-alpha (PIP5KIIA) gene and the other corresponding to the beta-globin gene, both overexpressed in the patient with the higher percentage of Hb H. This result was validated by quantitative RT-PCR using two endogenous genes (GAPDH and BAC). PIP5KIIA expression, in arbitrary units (AU), was 0.108701 in the patient with the higher Hb H, 0.031646 in the other and 0.01561 in the normal control, while beta-globin gene expression was 1.13882, 0.71080 and 0.37631 AU, respectively. Reticulocyte RNA samples obtained from three beta-thalassemia intermedia patients showed a very low level of PIP5KIIA expression. Type II PIP kinases produce phosphatidylinositol 4.5 biphosphate (PI4.5P2) by phosphorylating phosphatidylinositol-5-phosphate. PI4.5P2 is a pleiotropic regulatory molecule involved in diverse cellular activities, including modulation of actin cytoskeleton, protein localization and gene expression. Our results suggest that PIP5KIIA may be one of the factors related to the beta-globin gene expression and to the different levels of Hb H in alpha-thalassemic patients. Its exact mechanism of action, however, remains to be determined.

Hereditary Persistence of Fetal Hemoglobin is a rare, heterogeneous and benign group of hereditary disorders with an abnormal switch from fetal to adult hemoglobin, resulting in high levels of Hb F in the adult stage. A total of six deletions related to HPFH have been described, associated with increased levels of both gamma chains. Three main hypotheses have been proposed to explain the relationship between these deletions and the non-suppression of gamma genes: the removal of competitive regions that interact with the LCR; the juxtaposition of enhancer elements; and the removal of silencers. Despite evidence to support these hypotheses, however, they are not conclusive. Recently, Xiang and cols (Abstract #1215, 2004 ASH Meeting; Blood, Volume 104, issue 11, November 16, 2004) developed a YAC construct with the whole beta-globin locus containing a deletion of approximately 83.5 Kb responsible for the HPFH-2. Unexpectedly, the gamma gene was completely silenced in the adult transgenic mice. These data suggest that other mechanisms could be involved in the increased levels of HbF in these conditions. The authors speculate that other regions upstream from the cluster may harbor this activity. We, herein, investigate the possible involvement of transcription factors, using the subtractive hybridization method to identify differentially expressed transcripts in reticulocytes from a normal subject and a HPFH-2 subject. We have identified 56 and 106 unique genes in the normal and HPFH-2 cDNA libraries, respectively. Some of these are transcription (zinc fingers and homeobox proteins) and chromatin remodeling (NAP and SWI like proteins) factors that could participate in globin gene regulation. These genes are located in cis or in trans to the deletion and their altered gene expression has been confirmed by Quantitative Real-time PCR in other two HPFH subjects. The data may present new clues about globin gene regulation, the increased expression of gamma gene in deletional HPFH and the dynamic organization of genes and chromosomes in cells.

Os padrões temporais de atividade no tuco-tuco de Anillaco (_Ctenomys_ aff. _knighti_), um roedor subterrâneo, foram estudados extensivamente em laboratório e arenas semi-naturais. No entanto, essas condições controladas não reproduzem completamente o que os animais fazem na natureza. O presente estudo amplia nosso entendimento dos padrões de atividade dos tuco-tucos e descreve sua atividade diária em condições de vida livre. Nós coletamos dados de 21 tuco-tucos usando acelerômetros e sensores de luz ao longo de 2019 e 2020, em todas as estações do ano. Além disso, usamos modelos ocultos de Markov (HMMs) para classificar os dados do acelerômetro em três estados comportamentais distintos que correspondem, aproximadamente, a repouso e atividades de média e alta intensidade. Os registros do sensor de luz confirmam uma mudança anual no padrão temporal diário de emergência da superfície, a qual já havia sido descrita para animais em arenas semi-naturais. Os registros de acelerômetro confirmam que os tuco-tucos são diurnos em campo, sem alteração no padrão temporal diário ao longo do ano. Em contrapartida, em julho, os tuco-tucos apresentaram uma redução significativa nos níveis de atividade diária. Em termos de mudanças de estado comportamental, os tuco-tucos apresentaram mudanças significativas no tempo alocado em diferentes estados ao longo do ano. Em julho, houve uma redução no tempo diário gasto em atividades de alta intensidade. No entanto, observamos que os tuco-tucos não repousam mais em julho; em vez disso, eles alocam mais tempo em atividades de média intensidade. Esses resultados sugerem que as mudanças anuais nos níveis de atividade diária se devem a mudanças no tempo gasto em diferentes estados comportamentais. Em geral, confirmamos que os tuco-tucos são diurnos e apresentam mudanças anuais em seus níveis gerais de atividade, o que provavelmente é reflexo de uma mudança comportamental anual. Temporal patterns of activity of the Anillaco tuco-tuco (Ctenomys aff. knighti), a neotropical subterranean rodent, have been extensively studied in laboratory and semi-natural enclosures. However, the laboratory-controlled settings do not fully reproduce what animals do in nature. The present study extends our understanding of tuco-tucos activity patterns and describes their daily activity in free-living conditions. We collected data from 21 tuco-tucos using animal-borne accelerometers and light loggers during 2019 and 2020, covering all seasons. Additionally, we used hidden Markov models (HMMs) to classify the accelerometer data into three distinct behavioral states that roughly correspond to rest, medium, and high-intensity activity. Lightlogger data confirms the annual change in the daily temporal pattern of surface emergence already described for animals in semi-natural enclosures. Our accelerometer data confirm that tuco-tucos are diurnal in the field, with no change in the daily temporal pattern throughout the year. Nevertheless, in July, tuco-tucos displayed a significant reduction in daily activity levels. In terms of behavioral state changes, tuco-tucos displayed significant changes in the time allocated in different states throughout the year. In July, there was a reduction in the daily time spent in high-intensity activity. However, we observed that tuco-tucos do not rest more in July; instead, they allocate more time in medium-intensity activities. These results suggest that the annual changes in daily activity levels are due to changes in the time spent in different behavioral states. All in all, we confirm that tuco-tucos are diurnal and exhibit annual changes in their general activity levels, which is probably a reflection of an annual behavioral change.