Orientador: Wellerson Rodrigo Scarano / Resumo: A fertilidade masculina e a espermatogênese estão diretamente ligadas à capacidade das células de Sertoli em produzir fatores associados ao desenvolvimento das células germinativas. As células de Sertoli expressam receptores para FSH e testosterona e são os principais reguladores da espermatogênese. Aproximadamente 60-70% dos casos de infertilidade masculina são considerados idiopáticos, devido aos mecanismos moleculares envolvidos na espermatogênese ainda serem desconhecidos. Estudos recentes relatam que os microRNAs (miRNAs), são capazes de modular a função testicular durante a espermatogênese e sua expressão alterada pode estar envolvida na infertilidade masculina. miRNAs podem desempenhar papel importante na resposta aos xenobióticos que têm todas as consequências adversas para a saúde. Um grupo importante de compostos orgânicos com potencial tóxico são as dioxinas, como o 2,3,7,8-tetraclorodibenzo-p-dioxina (TCDD). Modelos experimentais de exposição ao TCDD, em camundongos, demonstraram que sua exposição provoca baixa contagem de espermatozóides e atraso na puberdade. Neste estudo, analisamos o efeito do TCDD nas células de Sertoli humanas in vitro após 72h a uma dose de 10nM. Nossos resultados mostraram que as enzimas antioxidantes catalase, superóxido dismutase e glutationa peroxidase diminuíram sua atividade e confirmaram o estresse oxidativo causado pelo TCDD nesse tipo celular. 78 miRNAs apresentaram expressão alterada, com regulação positiva de 73 e regulação negat... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Male fertility and spermatogenesis are directly linked to the ability of Sertoli cells to produce factors associated with the development of germ cells. Sertoli cells express receptors for FSH and testosterone, and are the major regulators of spermatogenesis. Approximately 60-70% of male infertility cases are considered idiopathic, due to the molecular mechanisms involved in spermatogenesis are still unknown. Recent studies report that microRNAs (miRNAs) are capable of modulating spermatogenesis in testicular function and its altered expression may be involved in male infertility. miRNAs may play a role in response to xenobiotics that have all the adverse consequences for health. An important group of organic compounds that are potentially toxic are the dioxins such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Experimental models of exposure to TCDD in mice showed that its exposure causes low sperm count and delayed puberty. In this study, we analyzed the effect of TCDD on human Sertoli cells after a exposure of 72h in vitro at a dose of 10nM. Our results showed that the antioxidant enzymes catalase, superoxide dismutase and glutathione peroxidase decreased their activity and confirmed the oxidative stress caused by TCDD in this cell type. 78 miRNAs showed altered expression with upregulation of 73 miRNAs and downregulation of 5 miRNAs compared to the control group. Regarding the gene expression profile, 51 genes showed deregulated, of which 46 genes with upregulation and d... (Complete abstract click electronic access below) / Doutor

Orientador: Danillo {UNESP] Pinhal / Resumo: A tilápia do Nilo (Oreochromis niloticus) possui importância econômica na piscicultura mundial. Com a finalidade de atender à demanda do mercado consumidor, foram desenvolvidas várias linhagens com maior viabilidade econômica, dentre elas a Chitralada, que possui rápido crescimento, e a Red Stirling, com filé de cor rosado, mais apreciado pelo consumidor. Com o objetivo de combinar estas características, foi desenvolvido um híbrido, que apresentou heterose. A pesquisa genética em peixes mostrou a interferência direta e indireta de vários genes no desempenho animal, principalmente os genes relacionados ao eixo GH/IGF e a miostatina (MSTN). Além disso, uma classe de RNAs não-codificadores, os microRNAs (miRNAs), possuem papel fundamental na regulação de vários pontos em vias biológicas conhecidas, principalmente na modulação de genes codificadores de proteínas relacionadas ao crescimento. Portanto, o objetivo deste trabalho foi avaliar a expressão gênica dos genes relacionados ao eixo GH/IGF e MSTN; determinar os miRNAs envolvidos e seus alvos; e avaliar o conjunto de proteínas relacionando-as aos respectivos fenótipos. Amostras biológicas foram coletadas das linhagens Chitralada e Red Stirling e do híbrido (7/8 Chitralada). Para a análise de expressão gênica por RT-qPCR, foram coletadas amostras de cérebro, fígado e músculo branco. Para a análise de miRNAs por RNA-seq, foram utilizados pools das amostras de músculo branco, assim como para a análise por ESI-q-TOF e shotgun. Os ... (Resumo completo, clicar acesso eletrônico abaixo) / Doutor

Filarial infections (lymphatic filariasis and onchocerciasis) are amongst the major neglected tropical diseases, and together account for more than 120 million infections in tropical and subtropical regions. The gold-standard technique for the diagnosis of filariases relies on the detection of microfilariae (mf) either in blood smears (lymphatic filariasis) or in skin biopsies (onchocerciasis). The secretion of extracellular RNAs (exRNAs) by parasitic nematodes has opened new avenues for the development of novel biomarkers for helminthiases, including filariasis. However, rather little is known about the origin and regulation of these RNAs inside the nematodes. One outstanding question is whether the secretion of small RNAs is distinct across the developmental stages of parasitic nematodes. Similarly, it is not clear whether the secretion of miRNAs is affected by treatment with anthelminthic chemotherapy or their potential as biomarkers for infection. Litomosoides sigmodontis is a murine filarial nematode closely related to filarial nematodes of medical and veterinary importance, including Onchocerca spp. and Brugia spp. L. sigmodontis has been extensively used to decipher multiple aspects of filarial biology, including parasite development, vaccine, and host-pathogen interactions. Therefore, we decided to use this model to address fundamental questions regarding the secretion of small RNAs and their biomarker potential. Our in vitro studies demonstrate that some extracellular miRNAs are enriched in a sexand stage-specific manner in the Excretion/Secretion (ES) products from early larval and adult stages from the rodent filarial nematode Litomosoides sigmodontis. Moreover, our data demonstrates that the gravid adult female worms secrete a plethora of miRNAs enriched in the secretome of this developmental stage when compared to adult males or mf. Further characterization studies show that the miRNAs are likely to be secreted in association with extracellular vesicles (EVs), as previously reported for other parasitic nematodes, including the human pathogen Brugia malayi. Interestingly, Ivermectin, which is typically used to treat filarial infections, does not have consistent effects on the secretion of miRNAs by gravid adult female worms in vitro, requiring further in vivo experiments to determine the effect of IVM on detection of extracellular parasite-derived miRNAs. In vivo experiments, using murine models of infection with L. sigmodontis (gerbils and BALB/c mice), as well as human samples from patients infected with Onchocerca volvulus and cattle infected with Onchocerca ochengi, demonstrated the presence of filarial-derived miRNAs, including female-specific miRNA markers, in biofluids from infected hosts. Further statistical analysis showed that two parasite-derived miRNAs, miR-71 and miR-100d, can significantly discriminate infected animals from naïve controls with high sensitivity/specificity (> 80%/100%). The results presented in this PhD thesis provide an initial framework to understand the secretion of small RNAs throughout nematode development, the potential interactions between anthelminthic chemotherapy and small RNA trafficking and secretion, as well as the use of parasite-derived miRNAs for the development of a new generation of biomarkers for filarial infections.

The aim of these studies was to identify circulating miRNAs that can be used as biomarkers in patients with paracetamol-induced liver injury. Whether the miRNAs discovered in humans could be back-translated to zebrafish with the aim of developing a liver toxicity model to replace rodent use was also investigated. First, the miRNA signature of DILI induced by paracetamol was defined. Plasma miRNAs were quantified in paracetamol overdose patients. A signature of 16 miRNAs was discovered that best separated patients with liver injury from those without liver injury. This signature was tested in a second cohort and resulted in the detection of paracetamol-induced liver injury with high specificity and sensitivity. At first presentation to hospital miR-122-5p was the most sensitive single miRNA and superior to ALT activity in predicting liver injury. In order to further qualify miR-122-5p, three detailed studies relevant to possible clinical scenarios were performed. The effect of acute alcohol ingestion (commonly co-ingested with paracetamol overdose) on circulating concentrations of miR-122-5p in healthy volunteers was investigated. Alcohol ingestion induced a small, non-clinically relevant, increase in miR-122-5p. The effect of chronic kidney disease (CKD) and haemodialysis (HD) on circulating miR-122-5p concentrations was explored because kidney dysfunction has been associated with a reduction in the concentration of circulating miRNAs. HD patients had lower concentrations of miR- 122-5p compared to healthy volunteers and CKD patients. To facilitate miRNA measurement outwith hospitals, miR-122-5p was measured in a blood drop from a finger prick. miR-122-5p was readily measurable in finger prick samples and concentrations were significantly higher in the blood drop from DILI patients compared with healthy volunteers. To complement miR-122-5p as a marker of toxicity, circulating paracetamol metabolites were measured in plasma samples from paracetamol overdose patients. A higher percentage of circulating metabolites formed by cytochrome P450 enzymes were present in patients with liver injury and these metabolites were superior to both ALT and paracetamol concentration with regard to early patient stratification. To reduce need for rodent studies, miRNAs were back-translated into zebrafish. In order to study circulating miR-122-5p in adult zebrafish, a bloodletting method by collecting blood retro-orbitally was developed. After studying different dosing regimens of paracetamol in adult and larvae zebrafish the model was determined to be too variable with regard to liver injury. A new drug, triptolide, originating from traditional Chinese medicine and responsible for DILI in China, was tested as an alternative model for drug-induced liver injury in zebrafish larvae. miRNA-122-5p decreased in zebrafish larvae after triptolide treatment and triptolide-induced liver injury could be tracked by fluorescent microscopy. Selective plane illumination microscopy was able to track the decrease in liver volume during triptolide exposure. In order to identify the toxic pathways involved in triptolide-induced liver injury, RNA-sequencing was performed. This identified KEGG pathways including ribosome, spliceosome and notch signalling as pathways affected by triptolide. In summary, miRNAs can be used as highly sensitive biomarkers to detect acute liver injury in patients and zebrafish. Zebrafish may represent an alternative model species to study DILI, further work is needed.

All mammalian cells encode microRNAs (miRNAs), which are small non-coding RNAs (~ 22 nucleotides) that control numerous physiological processes via regulation of gene expression. A number of viruses, in particular herpesviruses, also encode miRNAs. Gammaherpesviruses such as Epstein-Barr virus (EBV) and Kaposi’s sarcoma associated herpesvirus (KSHV) are associated with lymphoproliferative disorders and some types of cancer in humans. Gammaherpesvirus-encoded miRNAs are predicted to contribute to pathogenesis and virus life cycle by suppressing host and viral target genes. However, the exact functions of these miRNAs during virus infection in the natural host are largely unknown. Strict species specificity has limited research on the human gammaherpesviruses mainly to in vitro studies. Murine gammaherpesvirus 68 (MHV-68) encodes at least 15 miRNAs and provides a unique tractable small animal model to investigate in vivo gammaherpesvirus pathogenic features that are difficult to assess in humans. Following intranasal infection of lab mice, the virus undergoes primary lytic infection in the lung epithelial cells and then spreads to the spleen establishing latent infection in splenic B lymphocytes, macrophages, and dendritic cells. The peak of the latent viral load occurs in the spleen at 14 dpi and then it decreases over time, but the virus is not completely eliminated and the latent viral genomes remain in the host cells for lifetime and can reactivate to produce infectious virus under certain conditions. The aims of my project were to: (1) establish and develop quantitative reverse transcription polymerase chain reaction (qRT-PCR) assays for quantification of the MHV-68 miRNAs, (2) determine the miRNAs expression profiles during the two stages of virus infection (lytic and latent infection), (3) investigate the kinetics of the miRNAs expression during latency in vivo, (4) construct an MHV-68 miRNA mutant virus lacking 9 miRNAs (designated MHV-68.ΔmiRNAs), and (5) carry out thorough phenotypic characterisation of this mutant virus in order to determine the possible functions MHV-68 miRNAs in the context of natural host infection. It was found that the MHV-68 miRNAs expression pattern varied during different stages of infection, suggesting a differential regulation of the expression of these miRNAs depending on the phase of infection. In order to investigate the kinetics of miRNAs expression during latency in vivo, BALB/c mice were infected intranasally with MHV- 68 virus and spleens were harvested at days 10, 14, 21, and 32 post infection. The levels of miRNAs expression were determined by qRT-PCR in the splenocytes from infected mice. Interestingly, in contrast to the lytic MHV-68 protein coding genes, the expression of the miRNAs increased over time after 21 dpi, suggesting that the MHV-68-encoded miRNAs may play more fundamental roles during later stages of latent infection. In order to determine the potential roles of the MHV-68 miRNAs in virus pathogenesis, a miRNA mutant virus lacking the expression of 9 miRNAs, named MHV- 68.ΔmiRNAs, was constructed. The miRNA mutant virus replicated with the same kinetics as wild type virus in vitro and in vivo demonstrating that the deleted MHV-68 miRNAs are dispensable for virus lytic replication. To examine the roles of the miRNAs during virus latency, the MHV-68.ΔmiRNAs virus was characterised throughout a 49- day course of infection. Although the level of ex vivo reactivation of the MHV-68.ΔmiRNAs virus was comparable to that of the WT virus during the establishment of latency and as late as 28 dpi, the reactivation of the MHV-68.ΔmiRNAs virus was approximately 18-times higher than that of the WT virus at 49 dpi despite the similar levels of the genomic viral DNA loads at the same time-point. This suggests that the MHV-68 miRNAs suppress virus reactivation and promote maintenance of long-term latency. Moreover, the lytic viral gene expression levels were higher in splenocytes from the MHV-68.ΔmiRNAs-infected mice than the basal expression levels in the splenocytes from WT MHV-68-infected mice, suggesting that the MHV-68 miRNAs may suppress viral lytic gene expression during long-term latency in vivo and thus help the virus lay low.

Anemia falciforme (AF) é considerada a doença monogênica mais prevalente no Brasil, e resulta de uma mutação pontual no gene da beta-globina que leva à produção de uma molécula de hemoglobina anormal (HbS). A HbS polimeriza quando submetida a baixas tensões de oxigênio, precipitando e causando a deformação dos eritrócitos pois torna rígida sua membrana plasmática que apresentará uma série de alterações e danos. Além disso, a falcemização dos eritrócitos ocasiona anemia severa, lesão de isquemia/reperfusão, superprodução de espécies reativas de oxigênio, inflamação e vaso-oclusão (VO). A VO manifesta-se clinicamente como crises de dor, ou crises vaso-oclusivas (CVOs) que pode levar ao bloqueio de vasos e capilares e ao comprometimento de órgãos. Estes pacientes possuem alta susceptibilidade a infecções, principalmente na infância. Os mecanismos envolvidos no desenvolvimento da VO, no entanto, não estão totalmente elucidados. Estudos acerca deste tema têm sugerido que a VO seria o resultado da interação entre eritrócitos falcêmicos, leucócitos, plaquetas, células endoteliais e substâncias presentes no plasma dos indivíduos afetados. Tais observações como que AF seria o resultado de uma resposta inflamatória exacerbada que estes indivíduos desenvolvem, levaram à formulação da hipótese de que a anemia falciforme se comporta como uma condição inflamatória crônica e sugerindo que uma modulação do sistema imune poderá ser útil para a regulação da sintomatologia clínica. Salientando a carência de dados na literatura referentes à correlação de polimorfismos descritos para genes do sistema imune e a patofisiologia da AF, nosso estudo tem como objetivo principal: Analisar a correlação para polimorfismos descritos para genes do sistema imune e correlacionar-lho com a patofisiologia da AF. Sabemos que o HLA-G é uma molécula HLA não-clássica, que mostrou ser expressa em sítios de inflamação e nas doenças inflamatórias. Na região promotora além de ser altamente polimórfica, encontramos a região UTR 3' que parece desempenhar um papel importante na regulação da expressão do HLA-G. Então, dentre dos polimorfismos avaliados específicamente temos os dos genes HLA-G (14pb) e MiRNA (+3142). Nossos resultados indicam que os polimorfismos do HLA-G de 14pb e +3142 em 93 pacientes com AF, 21 pacientes apresentaram uma infecção pelo VHC e 16 pacientes com AF (22,2%) eram homozigotos para o genótipo +3142C e nenhum deles era positivo para HCV. Nenhum dos resultados obtidos indicou qualquer tipo de imunodeficiência mas pelo contrário, sugerem a existência de uma tendência inflamatória crônica na clínica da AF. Assim fica evidente a importância do polimorfismo +3142 sobre a susceptibilidade a infecções entre os pacientes com SCD.

xvii, 125 p. : ill. (some col.) A print copy of this thesis is available through the UO Libraries. Search the library catalog for the location and call number. / MicroRNAs are small regulatory RNAs that control various developmental and physiological processes in animals and plants. To study the involvement of microRNAs in skeletal development, I manipulated the expression of miR-140, which is strongly expressed in the developing skeleton, and miR-196, which is located among the body patterning Hox cluster genes. I found that miR-140 regulates zebrafish palate formation by interfering with neural crest cell migration through the inhibition of the expression of the platelet derived growth factor receptor alpha ( pdgfra ) gene. I also found that miR196 regulates zebrafish pectoral fin initiation by regulating the expression of the retinoic acid receptor alpha b ( rarab ) gene and that miR-196 is involved in the patterning of zebrafish pharyngeal arches and vertebrae. These results illuminate previously unknown regulatory mechanisms of skeletal development. I also reviewed current knowledge concerning microRNAs in skeletal development and evolution and discussed potential relationships between microRNAs and skeletal disease. This dissertation includes previously published and unpublished coauthored material. / Committee in charge: Judith Eisen, Chairperson, Biology; John Postlethwait, Advisor, Biology; Charles Kimmel, Member, Biology; William Cresko, Member, Biology; J. Andrew Berglund, Outside Member, Chemistry

QUIDUTE, Ana Rosa Pinto. Expressão dos genes GNAS e BTG2 e de um painel de microRNAs em somatotrofinomas esporádicos com e sem mutação no gene GNAS. 2013. 138 f. Tese (Doutorado em Farmacologia) - Universidade Federal do Ceará, Fortaleza, 2013. / Submitted by denise santos (denise.santos@ufc.br) on 2014-03-11T13:16:20Z No. of bitstreams: 1 2013_tese_arpquidute.pdf: 3847087 bytes, checksum: e9928440696d538b7170d304639ee18e (MD5) / Approved for entry into archive by denise santos(denise.santos@ufc.br) on 2014-03-11T13:17:36Z (GMT) No. of bitstreams: 1 2013_tese_arpquidute.pdf: 3847087 bytes, checksum: e9928440696d538b7170d304639ee18e (MD5) / Made available in DSpace on 2014-03-11T13:17:36Z (GMT). No. of bitstreams: 1 2013_tese_arpquidute.pdf: 3847087 bytes, checksum: e9928440696d538b7170d304639ee18e (MD5) Previous issue date: 2013 / Introduction: Mutations in GNAS and AIP genes are present in 35% and 3%, respectively, of the sporadic somatotropinomas. Recently, increased biological importance of microRNAs (miRNAs) has been observed in pituitary tumorigenesis. However, the molecular mechanisms involved in the pathogenesis of 60% of these tumors remain to be elucidated. Objectives: To identify the prevalence of mutations in GNAS and AIP genes in a series of sporadic somatotropinomas. Compare clinical, bioquimical parametrer at diagnosis as age, tumor size and theirs aggressiveness, pre-operative growth hormone (GH), prolactin (PRL) and insulin-like growth factor-I (IGF-1) levels and treatment responsiveness between somatotropinomas with (gsp+) and without (gsp-) GNAS mutation.To analyze the expression of GNAS and BTG2 genes and a panel of miRNAs between somatotrofinomas and normal pituitaries (NP) and the association between the expression of these genes and miRNAs with aggressiveness, as well as disease control with surgery or control with all adjuvant therapeutic approaches. Material and Methods: 26 patients with acromegaly. GH basal ≤2.5μg/L or nadir after OGTT ≤1μg/L and normal IGF-I matched for age and sex were used as diagnosis and for cure criteria after transsphenoidal surgery (TS). As control after somatostatin analogues (SA), we adopted the normalization of IGF-I matched for age and sex. Tumor size was evaluated by MRI/CT and the degree of invasiveness by Hardy score (I to IV).Tumor samples (26) were obtained during TS, processed for histopathology and stored at -70°C for molecular studies. NP (07) were obtained during autopsy. Total DNA and RNA were extracted by TRIzol®. Codons 201 and 227 of the GNAS gene and the whole AIP gene were sequenced. Relative expression of BTG2 and GNAS genes and miRNAs let-7a, miR-16a, miR-21, miR-141, miR-143, miR-15a, miR-145, miR-23a, miR-23b, and miR-24-2 was measured by qPCR (TaqMan probes) using 2-ΔΔCt method. Results: Frequencies of GNAS and AIP mutations were 35% and 3.8%, respectively. There was no difference between the mean age (39.0 ± 11.5 vs 43.6 ± 9.0 years, p=0.32), basal GH (62.4±128.1 vs 39.9 ± 48.3 μg/L; p=0.39), IGF-I (435.5 ± 230.8 vs. 556.9 ± 238.3; p=0.32) and PRL (25.7 ± 29.8 vs. 30.9 ± 32.8 ng/L, p=0.69) in plasma concentration, and tumor aggressiveness (p=1.00) between (gsp+) and (gsp-) groups. We observed that 80% (04/05) of gsp+ whereas 33% (02/06) of the gsp- achieved control (p=0.07) after SA therapy adjuvant to TS. When SA, dopamine agonists and/or external radiotherapy were associated 100% (05/05) of gsp+ group and 44% (04/09) of gsp- group (p=0.08) showed disease control.There was no difference in GNAS expression between somatotropinomas and NP (1.07 ± 0.55 vs 0.98 ± 0.28, p=0.97) as well as between somatotropinomasgsp+ and gsp- (1.04 ± 0.59 vs 1.10 ± 0.55, p=0.97, respectively). Hardy I/II tumors showed higher GNAS expression than Hardy III/IV (p=0.02), but there was no association between GNAS expression and disease control with surgery alone or associated with other adjuvant therapies. We observed hypoexpression of BTG2 and miR-16a and miR-141 in somatotropinomas compared with NP (-6.6 fold, p=0.002; -10.0 fold, p=0.01; and -50.0 fold, p=0.0003, respectively) with no difference between gsp+ and gsp- somatotropinomas. There was miR-21 overexpression in somatotropinomas compared with NP (20.2 ± 18.5 vs 2.5 ± 3.6; 10.2 fold, p=0.02), with no difference between gsp+ and gsp- somatotropinomas. However, miR-145 and miR-23b were more hipoexpressed in gsp+ compared to gsp- (-4.8fold, p=0.03 and-2.7 fold, p=0.02). There was no association between the expression of BTG2 and a panel of miRNAs with aggressiveness or disease control. Conclusion: In this series of assumed sporadic somatotopinomas, the frequencies of mutations in GNAS (35%) and AIP (3.8%) were similar to the literature. There were no differences in clinical and biochemical characteristics, aggressiveness, response to therapy, and GNAS expression in patients with gsp+ and gsp- somatotropinomas. Hypoexpression of BTG2, a tumor suppressor gene related to p53 and Rb signaling pathways, low expression of tumor suppressor miRNAs and high expression of oncomirs in somatotropinomas suggest a role in the somatotrophic tumorigenesis. / Introdução: Mutações nos genes GNAS e AIP estão presentes em 35% e 3%, respectivamente, dos somatotrofinomas esporádicos. Recentemente, observa-se importância biológica crescente dos microRNAs (miRNAs) na tumorigênese hipofisária. Entretanto, mecanismos moleculares envolvidos na patogênese de 60% desses tumores permanecem não elucidados. Objetivos: Identificar a prevalência de mutações nos genes GNAS e AIP em um grupo de somatotrofinomas esporádicos. Comparar parâmetros clínicos e bioquímicos ao diagnóstico como idade, tamanho tumoral e agressividade (escore Hardy), hormônio do crescimento (GH), prolactina (PRL) e Fator de Crescimento Insulin-Like I (IGF-1) e resposta as terapêuticas entre os grupos com (gsp+) e sem (gsp-) mutação no GNAS. Analisar a expressão dos genes GNAS e BTG2 e miRNAs entre somatotrofinomas e hipófises normais (HN) e a associação entre a expressão com agressividade, a resposta à cirurgia e a todas as terapêuticas adjuvantes disponíveis. Material e Métodos: 26 pacientes com diagnóstico de acromegalia. Tamanho tumoral foi avaliado por RNM/CT e o grau de invasibilidade pelo escore de Hardy (I a IV). GH basal ≤2.5μg/L ou nadir de GH após o GTT≤1μg/L e IGF-1 normal para idade e sexo foram utilizados como critério de cura após cirurgia transesfenoidal (CTE). Como controle com o análogo da somatostatina (AS), adotamos a normalização dos níveis de IGF-1 para idade e sexo. As amostras tumorais (n=26) foram obtidas durante a CTE, realizado histopatológico e armazenadas a -70 °C, para estudos moleculares. HN (07) foram obtidas durante autópsias. RNA e DNA total foram extraídos pelo TRIzol®. Os códons 201 e 227 do gene GNAS e o AIP completo foram sequenciados. Expressão relativa dos genes GNAS e BTG2 e dos miRNAs let-7a, miR-16a, miR-21, miR-141, miR-143, miR-15a, miR-145, miR-23a, miR-23b e miR-24-2 foi avaliada por qPCR (sondas TaqMan), pelo método 2-ΔΔCt. Resultados: A frequência de mutações no GNAS foi de 35% e no AIP 3,8%. Não houve diferença entre as médias de idade (39,0±11,5 vs 43,6±9,0 anos; p=0,32), nas concentrações plasmáticas basais de GH (62,4±128,1 vs 39,9±48,3µg/L; p=0,39), IGF-1 (435,5±230,8 vs 556,9± 238,3 %ULNR; p=0,32), PRL (25,7±29,8 vs 30,9±32,8 ng/L; p=0,69) e agressividade tumoral entre os gsp+ e gsp-(p=1,00). Ao analisar o uso do AS como terapêutica adjuvante à CTE, observamos que 04/05 (80%) dos indivíduos com somatotrofinoma gsp+ obtiveram controle da doença, enquanto que no grupo gsp- 02/06 (33%) obtiveram controle (p=0,08). Quando associamos ao AS, os agonistas dopaminérgicos e/ou radioterapia externa, observamos que 05/05 (100%) dos pacientes gsp+ tiveram critério de controle da doença, contra (04/09) 44% no grupo gsp- (p=0,09). Não houve diferença na expressão de GNAS entre os somatotrofinomas e as HN (1,07±0,55 vs 0,98±0,28; p=0,97), e entre os gsp+ e gsp- (1,04±0,59 vs 1,10±0,55; p=0,97, respectivamente). Os tumores Hardy I / II apresentaram maior expressão do GNAS do que os tumores classificados como III / IV (p=0,02). Não houve associação entre a expressão do GNAS e o controle da doença com cirurgia isolada ou com o uso de todas as terapêuticas adjuvantes. Observamos hipoexpressão do BTG2 e dos miR-16a e miR-141 em somatotrofinomas quando foram comparados com as HN (p=0,002, fold=-6,63; p=0,01, fold=-10,00; p=0,0003, fold=-50,00, respectivamente) sem diferenças entre os gsp+ e gsp-. Houve hiperexpressão do miR-21 (p=0,02;fold=10,18) em somatotrofinomas (20,16±18,48) quando comparado com as HN (2,52 ±3,56), sem diferença entre os gsp + e gsp-. Não houve diferença na expressão entre os grupos gsp+ e gsp- para os miRNAs let-7a, miR-21, miR-143, miR-15a, miR-23a e miR-24-2. Entretanto, miR-145 e miR-23b foram mais hipoexpressos no grupo gsp+ quando comparados ao gsp- (p=0,03, fold=-4,83 e p=0,02, fold=-2,77, respectivamente). Não houve associação entre a expressão do BTG2 e o painel de miRNAs com agressividade e com o controle da doença. Conclusão: Na presente série de somatotrofinomas, assumidos como esporádicos, a frequência de mutações nos genes GNAS (35%) e AIP (3,8%) foram semelhantes aos relatados na literatura. Não houve diferenças nas características clínicas e bioquímicas, agressividade, resposta às terapêuticas, e na expressão diferencial do GNAS entre os pacientes com tumores gsp+ e gsp-. Hipoexpressão de BTG2 (gene supressor tumoral relacionado às vias de sinalização do p53 e do Rb), baixa expressão de miRNAs (supressores tumorais) e alta expressão de oncomirs em somatotrofinomas sugerem um papel desses na tumorigênese somatotrófica.

Submitted by Nuzia Santos (nuzia@cpqrr.fiocruz.br) on 2015-06-18T17:41:26Z No. of bitstreams: 1 Fábio Ribeiro Queiroz - Caracterização da maquinaria de processamento de miRNAs e piRNAs em Biomphalaria glabrata e o efeito da infecção por Schistosoma mansoni no processo - 2015.pdf: 3625694 bytes, checksum: fa248ee7f88a52b2c4f2165fa5d9b5e4 (MD5) / Approved for entry into archive by Nuzia Santos (nuzia@cpqrr.fiocruz.br) on 2015-06-18T17:41:36Z (GMT) No. of bitstreams: 1 Fábio Ribeiro Queiroz - Caracterização da maquinaria de processamento de miRNAs e piRNAs em Biomphalaria glabrata e o efeito da infecção por Schistosoma mansoni no processo - 2015.pdf: 3625694 bytes, checksum: fa248ee7f88a52b2c4f2165fa5d9b5e4 (MD5) / Approved for entry into archive by Nuzia Santos (nuzia@cpqrr.fiocruz.br) on 2015-06-18T17:41:47Z (GMT) No. of bitstreams: 1 Fábio Ribeiro Queiroz - Caracterização da maquinaria de processamento de miRNAs e piRNAs em Biomphalaria glabrata e o efeito da infecção por Schistosoma mansoni no processo - 2015.pdf: 3625694 bytes, checksum: fa248ee7f88a52b2c4f2165fa5d9b5e4 (MD5) / Made available in DSpace on 2015-06-18T17:41:47Z (GMT). No. of bitstreams: 1 Fábio Ribeiro Queiroz - Caracterização da maquinaria de processamento de miRNAs e piRNAs em Biomphalaria glabrata e o efeito da infecção por Schistosoma mansoni no processo - 2015.pdf: 3625694 bytes, checksum: fa248ee7f88a52b2c4f2165fa5d9b5e4 (MD5) Previous issue date: 2015 / Fundação Oswaldo Cruz. Centro de Pesquisa René Rachou / A Organização Mundial de Saúde estima que cerca de 240 milhões de pessoas, em 78 países, necessitam de tratamento para esquistossomose, uma doença crônica causada por trematódeos do gênero Schistosoma. No Brasil, o Schistosoma mansoni é o único representante deste gênero, cuja passagem pelo hospedeiro invertebrado, caramujos do gênero Biomphalaria, é obrigatória antes de infectar um hospedeiro mamífero, entre eles o homem. A interação parasito-hospedeiro invertebrado é complexa, podendo o grau de susceptibilidade do caramujo à infecção variar desde extremamente permissivo até completamente resistente. Com o genoma e transcriptoma de B. glabrata disponíveis, o estudo de genes relacionados à regulação da expressão gênica, principalmente aqueles responsáveis pela maquinaria de processamento de miRNAs e piRNAs poderão auxiliar no entendimento da biologia do vetor B. glabrata bem como sua relação como o parasito S. mansoni. Alguns aspectos da interação parasito-hospedeiro invertebrado continuam pouco explorados, incluindo a participação dos pequenos RNAs não codificadores de proteínas como miRNAs, siRNAs e piRNAs. Utilizando ferramentas de bioinformática e PCR quantitativa, buscamos identificar e caracterizar a maquinaria de processamento de miRNAs e piRNAs em B. glabrata. Nosso trabalho demonstra que a maquinaria necessária para o processamento destas moléculas está ativa em B. glabrata com expressão gênica diferencial dos genes Argonauta, Drosha, Piwi, Exportina 5 e Tudor em diferentes estágios de desenvolvimento do animal bem como durante a infecção pelo S. mansoni. As análises de bioinformática utilizadas nesse trabalho mostraram a alta conservação dos genes envolvidos na maquinaria de miRNAs e piRNAs utilizando análise e distribuição de domínios conservados, análise de resíduos do sítio catalítico bem como análise filogenética. Estes dados sugerem que a maquinaria de silenciamento mediada por miRNAs e piRNAs interferem na biologia do caramujo durante todo o seu ciclo de vida, além de contribuir, de maneira ainda indefinida, na relação B. glabrata/S. mansoni. Estudos mais detalhados necessitarão ser realizados para confirmar a participação das preditas proteínas da via de miRNAs e piRNAs na relação parasito/hospedeiro, principalmente sua participação efetiva em seus genes alvos, uma vez que o parasito S. mansoni não possui expressa a via de piRNAs em seu genoma / The World Health Organization (WHO) estimates that about 240 million people in 78 countries require treatment for schistosomiasis, a chronic disease caused by trematodes of the genus Schistosoma. In Brazil, the Schistosoma mansoni is the only representative specie, whose passage through the invertebrate host, snails of the genus Biomphalaria, is mandatory before infecting a mammalian host, including humans. The interaction between the invertebrate host and the parasite is complex and the degree of susceptibility of the snail to infection ranges from extremely permissive to completely resistant. With the genome and transcriptome of B. glabrata available, the study of genes related to the regulation of gene expression, particularly those responsible for miRNAs and piRNAs processing machinery may assist in vector biology understanding B. glabrata well as its relation to the parasite Schistosoma mansoni. Some aspects of this interaction are still poorly explored, including the participation of small RNAs not protein-coding as miRNAs, siRNAs and piRNAs. Using bioinformatics tools and quantitative PCR, we seek to identify and characterize the processing machinery of miRNAs and piRNAs in B. glabrata. Our work shows that the protein machinery required for processing these molecules are active at the level of gene transcription in B. glabrata with a differential expression of the genes Argonauta, Drosha, Piwi, Exportin 5 and Tudor in different developmental stages and also during infection with S. mansoni. The bioinformatics analysis used in this study showed the high conservation of genes involved in miRNAs and piRNAs machinery using analysis and distribution of conserved domains, the catalytic site residue analysis and phylogenetic analysis. These data suggest that the silencing machinery mediated by miRNAs and piRNAs interfere in the snail biology throughout its life cycle, and contribute, even indefinitely, in relation B. glabrata/S. mansoni. More detailed studies need to be performed to confirm the participation of the predicted proteins of miRNAs and piRNAs pathway in the parasite/host relationship, mainly their effective participation in their target genes, since the parasite S. mansoni has not expressed the piRNAs pathway in its genome.

O adenocarcinoma ductal pancreático (ADP) é uma doença altamente letal e agressiva. Alteração no perfil de acetilação das histonas envolvendo desacetilases de histonas (HDAC), assim como modificações da expressão de miRNAs podem levar ao desenvolvimento tumoral. Neste estudo, foi avaliada a expressão das HDAC1, HDAC2, HDAC3 e HDAC7 em ADP e amostras de tecido pancreático não tumoral (TN) usando análises experimentais e de banco de dados. Os níveis de expressão foram correlacionados com características clínico-patológicas dos pacientes e foi realizada uma investigação in silico de miRNAs reguladores de efeito das HDACs. Os níveis de expressão das HDACs foram avaliadas por qRT-PCR a partir de 25 amostras de ADP e 23 amostras de TN e a análise da expressão diferencial (ED) e correlação entre HDACs e miRNAs em ADP foi realizada utilizando perfis de expressão de seis microarranjos do Gene Expression Omnibus. Potenciais relações miRNA-HDACs foram coletadas em bases de dados de interação de miRNAs. Um valor de P<0,05 foi considerado estatisticamente significativo. Encontramos expressão reduzida em ADP comparado com TN para todas as HDACs analisadas, com P<0,05 para HDAC1, 2 e 3. Entretanto, os fold-changes foram muito baixos e provavelmente sem relevância biológica, e a expressão da HDAC2 e HDAC7 foi correlacionada com a idade ao diagnóstico. Nenhuma outra correlação entre a expressão das HDACs e características clínico-patológicas foi identificada. Análises de ED sugeriram significativa superexpressão das HDAC1, 2 e 7 e subexpressão da HDAC3, contudo todas apresentaram fold-changes pequenos. As análises dos bancos de dados identificaram 728 miRNAs como reguladores das HDACs. Interseções entre os conjuntos de miRNAs (GSE41369 e GSE43796) e aqueles recuperados da análise de expressão diferencial indicaram cinco miRNAs que influenciam a HDAC1 (miR-188-5p, miR-539, miR-708, miR -4269 e miR-3616-3p) e três que influenciam a HDAC2 (miR-4307, miR-944 e miR-195). A expressão das HDACs provavelmente não é um biomarcador de prognóstico robusto para o ADP, uma vez que a expressão diferencial entre os grupos é sutil. Ainda, este e estudos anteriores indicam nenhuma ou pouca associação entre a expressão HDACs e características clínico-patológicas relacionadas com o prognóstico. Finalmente, miRNAs provavelmente não estão exercendo um papel central na regulação da HDACs no ADP. / Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal and aggressive disease. The disruption of histone acetylation through histones deacetylases (HDACs) and expression regulation by miRNAs can lead to tumor development. In this study we assessed HDAC1, HDAC2, HDAC3 and HDAC7 expression in PDAC and non-tumoral tissue (NT) samples using experimental and databases analysis, correlated their expression levels with clinical and pathological features in patients and performed in silico investigation of HDACs regulation by miRNAs. Expression levels of HDACs were measured by qRT-PCR from 25 PDAC and 23 NT. An analysis of differential expression (DE) and correlation of HDACs and miRNAs in PDAC was performed using six Gene Expression Omnibus microarray datasets. Potential miRNA-HDACs relationships were collected from miRNA interaction databases. A P<0.05 was considered statistically significant. We found reduced expression in PDAC compared with NT for HDAC1, HDAC2 and HDAC3, with P<0.05. Expression levels of HDAC7 did not significantly differ between groups. However, fold-changes were very small and probably not biologically relevant. Only HDAC2 and HDAC7 were associated with age at diagnosis and no other associations between HDAC expression and clinical features were identified. DE analysis suggested significant up-regulation of HDAC1, HDAC2 and HDAC7, and down-regulation of HDAC3, albeit all of them associated with small fold changes. Databases analysis identified 728 miRNAs that could be HDACs regulators. Intersections among the set of miRNAs found in differential expression analysis of GSE41369 and GSE43796 and those retrieved from target prediction identified five miRNAs targeting HDAC1 (miR-188-5p, miR-539, miR-708, miR-4269 and miR-3616-3p) and three targeting HDAC2 (miR-4307, miR-944 and miR-195). HDACs expression is likely not a robust prognostic biomarker in PDAC since differential expression between groups is subtle. Also, this and previous studies indicate no or only very few associations between HDACs expression and clinicopathological features related to prognosis. Finally, miRNAs are probably not exerting a central role in HDAC regulation in PDAC.