Abstract Hereditary hemochromatosis (HH) is a common autosomal recessive disorder estimated to affect one out of every 250–400 Caucasian individuals. It is a disorder of iron metabolism, in which excessive iron accumulation in the body may induce serious clinical manifestations (e.g. liver cirrhosis, hepatocellular carcinoma, diabetes, and cardiomyopathy). HH is caused by mutations in the HFE gene, and HFE genotyping thus enables early diagnosis of the disease and detection of the individuals at risk for HH. HFE mutations have also been proposed to predispose to certain other diseases, such as various hematological malignancies and cardiomyopathy. The present evaluation of the clinical utility and outcome of HFE genotyping in search for HH was based on data obtained from 137 subjects referred for HFE mutation analysis during the years 1999–2001. The C282Y and H63D mutations were determined for each subject. HFE genotyping was also used to examine the association between HFE mutations with various hematological disorders and idiopathic dilated cardiomyopathy (IDCM). The C282Y and H63D mutations were determined from 232 patients with various hematological disorders and the C282Y, H63D, and S65C mutations from 91 patients with IDCM and 102 control subjects. High frequencies of C282Y homozygotes (16.8%) and C282Y/H63D compound heterozygotes (5.1%) were found among the subjects referred for HFE genotyping, and the rate of positive findings for HH increased steadily over the years 1999–2001. The frequencies of HFE mutations did not differ significantly in patients with various hematological disorders and IDCM compared to controls. At the end of the follow-up period, left ventricular end-diastolic diameter (LVEDD) was significantly higher in IDCM patients carrying the C282Y mutation than in those without this mutation (p = 0.037). The present study supports active testing for the HFE gene mutations C282Y and H63D in public health care. Serum transferrin saturation is considered the most useful test for selecting subjects for such analysis. Although increasing numbers of HH cases are recognized by physicians, it may still be an underdiagnosed disease. HFE mutations do not seem to significantly increase the risk for various hematological disorders or IDCM. The C282Y mutation may, nevertheless, mediate the progression of IDCM by modifying LV dilation and remodeling.

QUARESMA, Fabrízia da Silva. Concentração letal média (CL50-96h) e efeitos subletais da amônia não ionizada sobre parâmetros hematológicos de alevinos de tambacu (Colossoma macropomum fêmea x Piaractus mesopotamicus macho). 2016. 53 f. : Dissertação (mestrado) - Universidade Federal do Ceara, Centro de Ciências Agrárias, Departamento de Engenharia de Pesca, Fortaleza-CE, 2016 / Submitted by Nádja Goes (nmoraissoares@gmail.com) on 2016-07-22T14:52:37Z No. of bitstreams: 1 2016_dis_fsquaresma.pdf: 1262731 bytes, checksum: 837440ddfa1a47f8014fdf9a4a826ce7 (MD5) / Approved for entry into archive by Nádja Goes (nmoraissoares@gmail.com) on 2016-07-22T14:52:53Z (GMT) No. of bitstreams: 1 2016_dis_fsquaresma.pdf: 1262731 bytes, checksum: 837440ddfa1a47f8014fdf9a4a826ce7 (MD5) / Made available in DSpace on 2016-07-22T14:52:53Z (GMT). No. of bitstreams: 1 2016_dis_fsquaresma.pdf: 1262731 bytes, checksum: 837440ddfa1a47f8014fdf9a4a826ce7 (MD5) Previous issue date: 2016 / Ammonia is a toxic compound to aquatic organisms and at high concentrations in the water can cause various changes in the animal. Therefore, it becomes important to determine the tolerance of farmed aquatic animals for that substance. Thus, the aim of this study was to determine the median lethal concentration (LC50-96h) of un-ionized ammonia (NH3) in fingerlings tambacu (Colossoma macropomum x Piaractus mesopotamicus) in acute toxicity test and evaluate the effects of concentrations sublethal through the analysis of hematological parameters. In the acute toxicity test, the fingerl ings were exposed to ammonia at concentrations of: 0,09 (control); 0,54; 1,23; 2,52; 3 ,44 and 3,66 mg L-1NH3, which were obtained from the application of NH4Cl. The test lasted 96 hours and mortalities were recorded over that period. The LC50 was determin ed by statistical method Trimmed Spearman Karber . In sublethal exposure to ammonia test concentrations used were : 0,04 (control), 0,19 and 0,35mg L-1NH3.For analysis of hematological parameters, blood samples were collected before the addition of NH4Cl(timezero) and after 96 hours of exposure.The hematological parameters analyzed were: hematocrit ( Ht), hemoglobin(Hb), red blood cell count(RBC) mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH) and mean corpuscular hemoglobin concentration(MCHC). Data hematological parameters were subjected to analysis of variance (ANOVA) and when there was a significant difference between treatments , the averages were compared in pairs through the Tukey's HSD test (α=0,05).LC50-96h of theun - ionized ammonia (NH3) to fingerlings the hybrid tambacu was 1,63m g L-1. After 96h exposure to 0,35 mg L-1NH3, fish showed significant reductions in hematocrit and the MCV and significant increase in MCHC. Already Hb,RBC and MCH did not differ significantly between treatments and time 0. The exposure to fingerlins tambacu for short period to 0,19 mg L -1 of un-ionized ammonia (NH3) does not cause significant changes in hematological parameters of these fish. / A amônia é um composto tóxico para os organismos aquáticos e em elevadas concentrações na água pode causar diversas alterações no animal. Dessa forma, torna-se importante determinar o limite de tolerância dos animais aquáticos cultivados à essa substância. Sendo assim, o objetivo do trabalho foi determinar a concentração letal média (CL50-96h) da amônia não ionizada (NH3) em alevinos de tambacu (Colossoma macropomum x Piaractus mesopotamicus), em teste de toxicidade aguda e avaliar os efeitos de concentrações subletais através da análise de parâmetros hematólogicos. No teste de toxicidade aguda, os alevinos foram expostos à amônia, nas concentrações de: 0,09 (controle); 0,54; 1,23; 2,52; 3,44 e 3,66 mg L-1 NH3, que foram obtidas a partir da aplicação de NH4Cl. O teste teve duração de 96h e as mortalidades foram registradas ao longo desse período. A CL50 foi determinada pelo método estatístico Trimmed Spearman Karber. No teste de exposição subletal à amônia, utilizou-se as concentrações: 0,04 (controle), 0,19 e 0,35 mg L-1 NH3. Para análise dos parâmetros hematológicos, foram realizadas coletas de sangue antes da adição de NH4Cl (tempo zero) e após 96h de exposição. Os parâmetros hematológicos analisados foram: hematócrito (Ht), taxa de hemoglobina (Hb), número de eritrócitos (RBC), volume corpuscular médio (VCM), hemoglobina corpuscular média (HCM) e concentração de hemoglobina corpuscular média (CHCM). Os dados dos parâmetros hematológicos foram submetidos à análise de variância (ANOVA) e quando houve diferença significativa entre os tratamentos, as médias foram comparadas duas a duas através do teste de Tukey (α=0,05). A CL50-96h da amônia não ionizada (NH3) para alevinos do híbrido tambacu foi 1,63 mg L-1. Após exposição de 96h a 0,35 mg L-1 NH3, os peixes apresentaram reduções significativas no Ht e VCM e aumento significativo na CHCM. Já Hb, RBC e HCM não diferiram significativamente entre os tratamentos e o tempo 0. A exposição de alevinos de tambacu por curto período de tempo a 0,19 mg L-1 de amônia não ionizada (NH3) não causa alterações significativas nos parâmetros hematológicos desses peixes.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / A amÃnia à um composto tÃxico para os organismos aquÃticos e em elevadas concentraÃÃes na Ãgua pode causar diversas alteraÃÃes no animal. Dessa forma, torna-se importante determinar o limite de tolerÃncia dos animais aquÃticos cultivados à essa substÃncia. Sendo assim, o objetivo do trabalho foi determinar a concentraÃÃo letal mÃdia (CL50-96h) da amÃnia nÃo ionizada (NH3) em alevinos de tambacu (Colossoma macropomum x Piaractus mesopotamicus), em teste de toxicidade aguda e avaliar os efeitos de concentraÃÃes subletais atravÃs da anÃlise de parÃmetros hematÃlogicos. No teste de toxicidade aguda, os alevinos foram expostos à amÃnia, nas concentraÃÃes de: 0,09 (controle); 0,54; 1,23; 2,52; 3,44 e 3,66 mg L-1 NH3, que foram obtidas a partir da aplicaÃÃo de NH4Cl. O teste teve duraÃÃo de 96h e as mortalidades foram registradas ao longo desse perÃodo. A CL50 foi determinada pelo mÃtodo estatÃstico Trimmed Spearman Karber. No teste de exposiÃÃo subletal à amÃnia, utilizou-se as concentraÃÃes: 0,04 (controle), 0,19 e 0,35 mg L-1 NH3. Para anÃlise dos parÃmetros hematolÃgicos, foram realizadas coletas de sangue antes da adiÃÃo de NH4Cl (tempo zero) e apÃs 96h de exposiÃÃo. Os parÃmetros hematolÃgicos analisados foram: hematÃcrito (Ht), taxa de hemoglobina (Hb), nÃmero de eritrÃcitos (RBC), volume corpuscular mÃdio (VCM), hemoglobina corpuscular mÃdia (HCM) e concentraÃÃo de hemoglobina corpuscular mÃdia (CHCM). Os dados dos parÃmetros hematolÃgicos foram submetidos à anÃlise de variÃncia (ANOVA) e quando houve diferenÃa significativa entre os tratamentos, as mÃdias foram comparadas duas a duas atravÃs do teste de Tukey (α=0,05). A CL50-96h da amÃnia nÃo ionizada (NH3) para alevinos do hÃbrido tambacu foi 1,63 mg L-1. ApÃs exposiÃÃo de 96h a 0,35 mg L-1 NH3, os peixes apresentaram reduÃÃes significativas no Ht e VCM e aumento significativo na CHCM. Jà Hb, RBC e HCM nÃo diferiram significativamente entre os tratamentos e o tempo 0. A exposiÃÃo de alevinos de tambacu por curto perÃodo de tempo a 0,19 mg L-1 de amÃnia nÃo ionizada (NH3) nÃo causa alteraÃÃes significativas nos parÃmetros hematolÃgicos desses peixes. / Ammonia is a toxic compound to aquatic organisms and at high concentrations in the water can cause various changes in the animal. Therefore, it becomes important to determine the tolerance of farmed aquatic animals for that substance. Thus, the aim of this study was to determine the median lethal concentration (LC50-96h) of un-ionized ammonia (NH3) in fingerlings tambacu (Colossoma macropomum x Piaractus mesopotamicus) in acute toxicity test and evaluate the effects of concentrations sublethal through the analysis of hematological parameters. In the acute toxicity test, the fingerl ings were exposed to ammonia at concentrations of: 0,09 (control); 0,54; 1,23; 2,52; 3 ,44 and 3,66 mg L-1NH3, which were obtained from the application of NH4Cl. The test lasted 96 hours and mortalities were recorded over that period. The LC50 was determin ed by statistical method Trimmed Spearman Karber . In sublethal exposure to ammonia test concentrations used were : 0,04 (control), 0,19 and 0,35mg L-1NH3.For analysis of hematological parameters, blood samples were collected before the addition of NH4Cl(timezero) and after 96 hours of exposure.The hematological parameters analyzed were: hematocrit ( Ht), hemoglobin(Hb), red blood cell count(RBC) mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH) and mean corpuscular hemoglobin concentration(MCHC). Data hematological parameters were subjected to analysis of variance (ANOVA) and when there was a significant difference between treatments , the averages were compared in pairs through the Tukey's HSD test (α=0,05).LC50-96h of theun - ionized ammonia (NH3) to fingerlings the hybrid tambacu was 1,63m g L-1. After 96h exposure to 0,35 mg L-1NH3, fish showed significant reductions in hematocrit and the MCV and significant increase in MCHC. Already Hb,RBC and MCH did not differ significantly between treatments and time 0. The exposure to fingerlins tambacu for short period to 0,19 mg L -1 of un-ionized ammonia (NH3) does not cause significant changes in hematological parameters of these fish.

Les protéines de la famille TET catalysent l'oxydation des 5-méthylcytosines (5mC) en 5-hydroxyméthylcytosines (5hmC) et jouent ainsi un rôle dans la régulation épigénétique de la transcription et dans le processus de déméthylation de l'ADN. Les interactions entre la méthylation de l'ADN et les autres marques épigénétiques sont encore mal connues.Des mutations inactivatrices du gène TET2 ont été décrites dans les hémopathies myéloïdes et lymphoïdes et l'inactivation conditionnelle (cKO) de ce gène évaluée chez la souris a permis d'identifier de multiples anomalies de l'hématopoïèse, ainsi que le développement tardif d'hémopathies myéloïdes. Cette latence importante suggère la nécessité d'évènements oncogéniques coopératifs pour la transformation hématopoïétique. Chez l'Homme, les mutations de TET2 sont observées en association avec de nombreuses autres mutations, et en particulier avec des mutations du gène DNMT3A impliqué dans la méthylation de novo des cytosines de l'ADN, et avec des mutations du gène EZH2 responsable de methylation de la lysine 27 de l'histone H3. Nous avons testé fonctionnellement ces associations en utilisant des modèles murins.L'utilisation d'un modèle de transplantation de moelle osseuse nous a permis d'identifier une coopération de l'inactivation de Tet2 et du mutant DNMT3AR882H dans la transformation des lignées myéloïdes et lymphoïde T, correspondant aux hémopathies humaines porteuses de ces mutations. Dans la transformation lymphoïde, nos données indiquent que la dérégulation de la méthylation entraine une surexpression du gène NOTCH1 et de l'activité de la voie de signalisation correspondante.L'analyse de souris invalidées de manière conditionnelle pour Tet2 et Ezh2 a montré que les souris correspondantes meurent d'aplasie médullaire, dont l'origine est imputée à la disparition de cellules souches hématopoïétiques capables de reconstituer l'hématopoïèse à long terme (LT-HSC). Ezh2 et Tet2 ont donc des rôles primordiaux dans le maintien de l'autorenouvellement des cellules souches hématopoïétiques, dont les mécanismes moléculaires, génétiques et épigénétiques restent à définir. / TET family proteins catalyzing the conversion of 5-methylcytosines (5mC) into 5-hydroxymethylcytosines (5hmC) are crucial for epigenetic regulation of transcription and for DNA demethylation. Interactions between DNA methylation and other epigenetic marks are not fully understood.TET2 inactivating mutations have been identified in both myeloid and lymphoid malignancies. The conditional inactivation (cKO) of this gene in mice highlights pleiotropic hematopoietic abnormalities as well as myeloid transformation at late stages. This latency suggest cooperativity between Tet2 and other oncogenic events during transformation. Human TET2 mutations are frequently found associated with other mutations, and more particularly with mutations in DNMT3A, involved in de novo methylation of cytosines and with mutations in EZH2, responsible for lysine 27 of histone H3 methylation. We decided to functionally assess these mutation associations in mice.Bone marrow transplantation of Tet2 inactivated and DNMT3AR882H mutated cells allowed us to identify myeloid and T-cell transformations, corresponding to human hematological disorders harboring these mutations. Our results on T-cell transformations clearly demonstrate that the deregulation of methylation leads to NOTCH1 overexpression and activation of the corresponding signaling pathway.Analyses of Tet2 and Ezh2 inactivated mice show that these Ezh2 Tet2 mice succumb to bone marrow exhaustion, attributed to long term hematopoietic stem cells (LT-HSC) disappearance. Ezh2 and Tet2 show major roles in LT-HSC maintenance whose molecular, genetic and epigenetic mechanism remains to be investigated.