Submitted by GABRIELA RONCADA HADDAD null (gabriela.haddad@yahoo.com) on 2017-09-10T22:08:03Z No. of bitstreams: 1 Tese Doutorado 27 jul 2017 - Gabriela.pdf: 1172239 bytes, checksum: d6ae9b8e2d02ba5890bf385b02539433 (MD5) / Approved for entry into archive by Monique Sasaki (sayumi_sasaki@hotmail.com) on 2017-09-12T16:46:39Z (GMT) No. of bitstreams: 1 haddad_gr_dr_bot.pdf: 1172239 bytes, checksum: d6ae9b8e2d02ba5890bf385b02539433 (MD5) / Made available in DSpace on 2017-09-12T16:46:39Z (GMT). No. of bitstreams: 1 haddad_gr_dr_bot.pdf: 1172239 bytes, checksum: d6ae9b8e2d02ba5890bf385b02539433 (MD5) Previous issue date: 2017-08-21 / Introdução: A influência do ácido retinoico (AR) sobre o coração é bastante relevante, ocorrendo durante a embriogênese, diferenciação e desenvolvimento cardíaco. Estudos experimentais mostram que o AR pode induzir hipertrofia excêntrica com melhora da função cardíaca em coração de ratos sadios, e também reduzir a hipertrofia, modulando o sistema renina angiotensina aldosterona (SRAA), em animais hipertensos. Pouco se sabe sobre os efeitos do AR em coração de humanos. Pacientes portadores de acne fazem uso de um tipo de AR que é o 13- cis-AR, também chamado de isotretinoína e por isso possibilitam o estudo do papel do AR em humanos. Estudo prévio mostrou que com 2 meses de uso do 13-cis-AR houve remodelação cardíaca. Entretanto, não se sabe sobre os mecanismos ou se essas alterações são reversíveis. Objetivos: Portanto, os objetivos desse trabalho foram de comparar a avaliação morfofuncional cardíaca e variáveis do SRAA entre pacientes em uso de isotretinoína com um grupo controle. Adicionalmente, avaliar se as alterações são reversíveis em pacientes em uso de isotretinoína. Casuística e Métodos: Foram estudados 35 adolescentes e adultos jovens, com idade entre 14 e 23 anos, do sexo masculino, sendo 20 deles em uso de isotretinoína oral, na dose de 0,5 mg/kg/dia a 0,75 mg/kg/dia, acompanhados no ambulatório de dermatologia do Hospital das Clínicas da Faculdade de Medicina de Botucatu (FMBUNESP), aos 6 meses de tratamento. Os outros 15 pacientes foram convidados na comunidade ou apresentavam acne leve com indicação apenas de tratamento tópico. Foram realizados avaliação morfofuncional e doppler tissular por meio de ecocardiografia transtorácica, dosagens bioquímicas de rotina e dosagens de componentes do SRAA renina, angiotensina I, angiotensina II, aldosterona, angiotensina 1-7 e alamandina. Essas variáveis foram comparadas nos grupos controle e isotretinoína pelo teste t de student ou Mann Whitnney. Os pacientes que receberam isotretinoína foram estudados antes do início do tratamento, com 6 meses de tratamento e 2 meses após o término do tratamento. Esses momentos foram comparados por meio do teste de Anova de 1 via de medidas repetidas. O nível de significância adotado foi de 5%. Resultados: Os pacientes do grupo controle e isotretinoína apresentaram a mesma idade, índice de massa corcoral, pressão arterial e frequência cardíaca. Dosagens bioquímicas habitualmente solicitadas durante o tratamento como enzimas hepáticas, função renal e triglicérides também foram semelhantes entre os grupos. Os dados morfológicos mostraram aumento do diâmetro diastólico do ventrículo esquerdo (DDVE) acompanhado de aumento do débito cardíaco e do fluxo transmitral avaliado por E/E’. Houve aumento do volume do átrio esquerdo (AE), no limite da significância e tendência ao aumento da massa do ventrículo esquerdo (VE) e com espessura relativa da parede semelhante entre os grupos. Sobre o SRAA houve redução da angiotensina II e da renina. Na avaliação ecocardiográfica apenas dos pacientes em uso de isotretinoína observou-se que houve redução do AE e do índice de massa do VE (IMVE) após 2 meses do término do tratamento. Embora não significativo, o comportamento de E/E’ também foi de redução após o tratamento. Discussão: O 13-cis-AR promove remodelação cardíaca, provavelmente induzida por hipervolemia, levando a um padrão de hipertrofia excêntrica com melhora da função. Essas alterações provavelmente levaram a menor ativação do SRAA visto pela redução da renina e angiotensina II. Esse perfil de remodelação e de bloqueio do SRAA é semelhante ao que ocorre no exercício físico. Nesse estudo foi possível apenas avaliar o grupo isotretinoína, quanto às variáveis ecocardiográficas antes, durante e após o tratamento. Observa-se que o término do estimulo com 13-cis-AR reduz algumas variáveis como átrio esquerdo e massa do VE. Portanto, em corações normais de adultos jovens, o AR atenuou o efeito de SRAA e promoveu remodelação cardíaca do tipo excêntrica, com melhora da função, compatível com sobrecarga volêmica e com caráter transitório. / Background: The influence of retinoic acid (RA) in the heart is very relevant, occurring during the embryogenesis, differentiation and cardiac development. Experimental studies shown that RA induces excentric hypertrophy, with improvement of cardiac function in heart of healthy rats. In addition, it was observed that RA regulates renin angiotensin aldosterone system (RAAS), a regulatory system involved in blood pressure, volume homeostasis and cardiac hypertrophy. There is a lack of information about the role of RA on cardiac remodeling in adult humans. Otherwise there are patients with Acne that uses 13- cis-RA, also called isotretinoin, and this population allow the investigation of cardiac remodeling and RA treatment. In fact, previously study shown that the use of 13-cis-RA, for acne, for 2 months induced cardiac remodeling, however, no one knows if these changes are persistent and reversible. Objectives: the objectives of these study is to compare the cardiac morphofunctional evaluation and RASS variables in patients using isotretinoin and in control group. Additionally, evaluate if these changes are reversible in isotretinoin group. Casuistic and Methods: Study1: 35 young men, between 14 to 23 years, 20 in use of 13-cis-RA, in the dose of 0,5 mg/kg/day to 0,75 mg/kg/day, from dermatology clinic of São Paulo State University (FMB-UNESP), at 6 months of treatment. The others 15 patients had mild acne only with topic treatment. Morphofunctional evaluation, tissular Doppler, Biochemical evaluation, dosage of RAAS components were performed. Results were compared in isotretinoin and control group by t student or Mann Whitnney tests. Study 2: Only the isotretinoin group were evaluated before beginning of treatment (initial moment - M0), after 6 months of treatment (final moment - M1) and two months after finishing the medication (M2). This results were compared by One way pared Anova. The statistically significant level was set at 5%. Results: control and isotretinoin group presents similar age, body mass index, blood pressure and heart rate. Biochemical evaluation was also similar. The present study showed that young patients receiving 13-cis-RA for Acne treatment presented left ventricle and atrium chamber enlargement and increase in cardiac output and in mitral flows. There was a trend toward higher ventricular mass with preserved relative wall thickness. RAAS showed decreased in angiotensin II and renin. Considering only patients that received isotretinoin, it was observed that cardiac atrium size and flows returned to baseline 2 months after the end of treatment and cardiac structures such as ventricle mass and thickness reduced. Discussion: 13-cis-RA promotes cardiac remodeling, probably induced by hypervolemia, taking to a pattern of eccentric hypertrophy, with improvement of function. It is possible that hypervolemia or a direct effect of 13-cis-RA, reduces renin and angiotensin II. The remodeling phenotype described is compatible with cardiac remodeling induced by physical activity, marked by hypervolemia, excentric hypertrophy and increased cardiac output. In the isotretinoin group, the end of treatment reduces left atrium size and left ventricle mass. Therefore, in normal hearts of young adults, RA reduces the effect of RAAS and promotes eccentric cardiac remodeling, with improvement of function, compatible with volume overload and with transitory character.

Submitted by Irene Nascimento (irene.kessia@ufpe.br) on 2017-03-14T16:58:48Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Dissertação Pollianne Barbosa versão final.pdf: 2566531 bytes, checksum: e5e800736b597cae6798fe49bcab9b9b (MD5) / Made available in DSpace on 2017-03-14T16:58:48Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Dissertação Pollianne Barbosa versão final.pdf: 2566531 bytes, checksum: e5e800736b597cae6798fe49bcab9b9b (MD5) Previous issue date: 2015-10-21 / Quando se trata da caracterização de fármacos ou produtos farmacêuticos deve-se ter em mente que as metodologias empregadas forneçam o maior número possível de informações e sejam complementares, essa caracterização se torna ainda mais importante quando são alteradas as propriedades físico-químicas do fármaco, sendo necessário um monitoramento. Nos últimos anos os pesquisadores têm estudado os fármacos com outro enfoque, tornando o que antes era considerando um efeito colateral como uma ação principal, ou ainda descobrindo novos usos com diferentes vias de administração; é o caso do ácido retinoico (AR), objeto de estudo desse trabalho. Com o objetivo de avaliar a aplicação das ferramentas analíticas na caracterização físico químicas do ácido retinoico, o presente trabalho utilizou DSC, FTIR e PDRX para a análise da formação de cocristais e dispersões sólidas entre o ácido retinoico (AR) e os adjuvantes tecnológicos ácido cítrico (AC), ácido sórbico (AS) e nicotinamida (NI). No estudo de estabilidade foi utilizada a termogravimetria, com o modelo cinético de Osawa. Os resultados das análises revelaram a formação de dispersão sólida cristalina em acido retinoico – acido sórbico e acido retinoico – nicotinamida e de cocristal em acido retinoico – cítrico; a técnica de FTIR demonstrou a provável formação de pontes de hidrogênio em ARAC LIO e a PDRX demonstrou a ocorrência em todas as amostras do sistema cristalino monoclínico, apesar de apresentarem grupos espaciais, volumes de célula unitária e parâmetros de rede distintos. As análises por termogravimetria demonstraram que ARAC LIO mostrou-se a mais instável das amostras e que AR apresentou cinética de degradação de ordem superior influenciada pela razão de aquecimento. Dessa forma foi possível concluir que as ferramentas analíticas utilizadas foram elucidativas, rápidas e de grande importância na pesquisa do AR e adjuvantes tecnológicos, utilizando a chamada “química verde”, mais ecológica e econômica que as convencionais. / When it comes to the characterization of drugs or pharmaceutical products must be borne in mind that the methodologies employed to provide the largest possible amount of information and are complementary, this characterization becomes even more important when the physicochemical properties of the drug are changed and require a monitoring. In recent years researchers have studied the drugs with another approach, such as making a major action than was previously considering a side effect, or finding new uses with different routes of administration; in the case of retinoic acid (AR), object of study of this work. In order to evaluate the application of analytical tools in the physicochemical characterization of drugs, this study used DSC, FTIR and PDRX to analyze the formation of cocrystals and solid dispersions between the drug retinoic acid (AR) and processing aids citric acid ( AC), sorbic acid (AS) and nicotinamide (NI). The stability prediction was used thermogravimetry with the kinetic model Osawa. The results of the analysis revealed the formation of crystalline solid dispersion in ARAS LIO and ARNI LIO and cocrystal in ARAC LIO; FTIR technique demonstrated the probable formation of hydrogen bridges and ARAC LIO and PDRX demonstrated to occur in all of the samples monoclinic crystal system, space group though present, and unit cell volumes different lattice parameters. Analysis by thermogravimetry showed that ARAC LIO proved to be more unstable and AR samples showed higher order degradation kinetics influenced by the ratio of heating. Thus it was concluded that the analytical tools used were enlightening, fast and very important in AR research and processing aids, using the so-called "green chemistry", more ecological and economical.

Orientador: Francisco Eduardo Martinez / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-18T07:43:37Z (GMT). No. of bitstreams: 1 Fontanelli_BeatrizAparecidaFioruci_M.pdf: 2160007 bytes, checksum: 5e358e9b41de70f7fd7c801fae0e0378 (MD5) Previous issue date: 2011 / Resumo: A exposição ao etanol altera a concentração do retinol e do all-trans-ácido retinóico (atAR) em vários tecidos. Os retinóides, retinol e atAR, são importantes para a diferenciação e manutenção das células epiteliais da próstata. O atAR se liga aos receptores de ácido retinóico (RARa, ß e y) e a interação receptor/ligante com a sequência responsiva ao retinóide no DNA, levam à transcrição de genes alvos. Assim, o atAR exerce efeitos no crescimento celular, diferenciação e apoptose, sendo essencial no desenvolvimento e diferenciação de órgãos e tecidos. Nosso objetivo foi analisar o retinol, o ácido retinóico e seus receptores, bem como, o índice de proliferação celular e de apoptose no lobo dorsolateral da próstata de ratos adultos UCh. Os animais foram divididos em quatro grupos experimentais (n=10/grupo): UChA (ingestão voluntária de etanol a 10% (v/v); UChACo (controle - ausência de etanol); UChB (ingestão voluntária de etanol a 10% (v/v) e UChBCo (controle - ausência de etanol). Após 150 dias de experimentação, os animais foram eutanasiados por decapitação e o sangue do tronco e os lobos dorsolaterais das próstatas foram coletados e processados: (1) para análises da concentração do retinol e do atAR no plasma e na próstata por meio de HPLC; (2) e análises de microscopia de luz para a proliferação celular (Ki-67), apoptose (Tunel) e para os receptores de ácido retinóico, por meio dos anticorpos anti-RARa, -ß e -y. O consumo crônico de etanol diminuiu a concentração do retinol no plasma dos grupos UChB (consumo alto de etanol) e UChA (consumo baixo de etanol). A concentração do retinol foi ainda menor no plasma do grupo UChB comparado ao UChA. No entanto, a concentração do retinol no tecido prostático não teve diferença significativa entre os grupos. O atAR aumentou significativamente somente no plasma do grupo UChB. Na próstata, a concentração do atAR aumentou no grupo UChB, enquanto que no UChA não houve diferença estatística. O RAR? na próstata dorsal e lateral dos ratos UCh não foi alterada em função do consumo de etanol. Já os RARß e -? apresentaram aumento do sinal na próstata dorsal do grupo UChB. Não houve diferença no índice de proliferação celular e de apoptose nas próstatas dorsais e laterais dos grupos experimentais. Conclui-se que o etanol altera a concentração do retinol e do atAR no plasma. Essa alteração é diretamente proporcional à quantidade de etanol consumida. Já na próstata, o retinol não é alterado pelo etanol. O consumo alto de etanol altera a concentração do atAR na próstata dorsolateral e a expressão dos RAR ß e y na próstata dorsal. A alteração da expressão dos RAR pode aumentar a sensibilidade da próstata à ação do atAR. O etanol não altera a proliferação celular e a apoptose na próstata dorsal e lateral / Abstract: Ethanol exposure alters the concentration of retinol and all-trans retinoic acid (atAR) in several tissues. Retinoids (retinol and atAR) are essential for the differentiation and homeostasis of the prostate epithelial cells. atAR binds to retinoic acid receptors (RAR a, ß and ?) and the interaction receptor/ligand with the sequence responsive to retinoid into DNA lead to transactivation of target genes. Thus, atAR directly produces their effects on cell growth, differentiation and apoptosis. This study aimed to analyze the retinol and all-trans-retinoic acid concentrations and its atAR receptors as well as the cell proliferation and apoptosis index upon the dorsolateral prostate lobe of adult UCh rats. All animals were divided into four experimental groups (n = 10/group): UChA (10% ethanol (v / v) voluntary intake); UChACo (without ethanol consumption); UChB (10% ethanol (v / v) voluntary intake) and UChBCo (without ethanol consumption). After 150 days of experimentation, animals were sacrificed followed by decapitation and trunk blood and dorsolateral prostate lobes collected. Samples of plasma and prostate by concentration analysis of the retinol and atAR were processed for HPLC. The cell proliferation and apoptosis immunoreactivities were assessed by Ki-67 and Tunel, respectively, and nuclear receptors by anti-RAR a,-ß and-y. Chronic ethanol consumption reduced the concentration of plasma retinol in UChB (high ethanol intake) and UChA groups (low ethanol intake). The retinol concentration in plasma was even lower in UChB compared to UChA group. However, the retinol concentration in prostate tissue was not significantly different between the groups. Concentration of atAR increased in plasma of UChB group, and was 96% higher in the UChA group. The prostate, atAR increased in the UChB group, while in UChA group no statistical difference. There was no statistical difference in proliferation cell and apoptosis in the dorsal and lateral prostate lobes between the groups. The expression of RAR a in the dorsal and lateral prostate of UCh rats was not altered as a function of ethanol consumption. Already RAR ß and-y showed increased signal in the dorsal prostate UChB group. We conclude that ethanol alters the concentration of retinol and atAR in plasma. This change is directly proportional to the amount of ethanol consumed. In the prostate, retinol is not altered by ethanol. The high ethanol intake alters the concentration of atAR in dorsolateral prostate and the expression of RARß and RARy in the dorsal prostate. Alteration in expression of RAR can increase sensitivity to the action of the atAR in prostate. Ethanol does not alter cell proliferation and apoptosis in the dorsolateral prostate / Mestrado / Anatomia / Mestre em Biologia Celular e Estrutural

Made available in DSpace on 2014-08-20T13:32:47Z (GMT). No. of bitstreams: 1 dissertacao_eduarda_schultze.pdf: 1903452 bytes, checksum: 1fd6d0e0478c9c31687ae8d7882532ea (MD5) Previous issue date: 2013-02-26 / Retinoid derivatives and analogs have been widely studied as antitumor agents due to their effects on cell proliferation and differentiation. Tretinoin (TT), also known as retinoic acid is a retinoid derivative that has been used as an adjuvant in the treatment of acute promyelocytic leukemia with excellent rates of remission. This compound has antiproliferative activity in various tumor types. However, non small cell lung cancer in general exhibit strong resistance to the effects of TT, which may be related to the deficiency in the cellular up-take of TT in that cell type. A strategy to enhance the antiproliferative activity of TT is to increase the cellular internalization of the compound through carriers such as liposomes or other vesicles or nanospheres or nanocapsules. Here we evaluated TT lipid-core nanocapsules (TT-LCNC) for their power to inhibit growth, induce apoptosis and interfere with the cell cycle of lung adenocarcinoma, A549 cell line, which is resistant to treatment with TT. The results showed that TT-LCNC was able to overcome the cellular resistance to treatment with TT, reducing cell viability and inducing apoptosis, upregulation of P21 and cell cycle arrest in G1 phase. / Derivados e análogos retinóides têm sido largamente estudados como agentes antitumorais devido a seus efeitos sobre a proliferação e diferenciação celular. Tretinoína (TT), também conhecida como ácido retinóico é um derivado retinóide que tem sido usado como adjuvante no tratamento de leucemia promielocítica aguda com excelentes índices de remissão da doença. Este composto exerce atividade antiproliferativa em diversos tipos de tumores. Entretanto, células de adenocarcinoma de pulmão humano em geral exibem uma forte resistência aos efeitos da tretinoína, a qual pode estar relacionada com a deficiência na internalização celular de tretinoína nesse tipo de célula. Uma estratégia para aumentar a atividade antiproliferativa de tretinoína é aumentar a captação celular do composto através de carreadores como lipossomas ou outras vesículas como nanocápsulas ou nanoesferas. Neste trabalho nanocápsulas de núcleo lipídico contendo tretinoína (TT-LCNC) foram avaliadas quanto ao seu potencial de inibir o crescimento, induzir a apoptose e interferir com o ciclo celular de células de adenocarcinoma de pulmão, linhagem A549, resistentes ao tratamento com TT livre. Os resultados demonstraram que TT-LCNC foi capaz de superar a resistência celular ao tratamento com TT, reduzindo a viabilidade celular e induzindo apoptose, superexpressão de P21 e parada do ciclo celular em G1.

Human immunodeficiency virus type-1 (HIV-1) is the etiologic agent of acquired immune deficiency syndrome (AIDS). In most cases HIV-1 infection in humans, leads to AIDS, which is characterized by opportunistic infections leading to death. The role various infectable cell types play in the course of infection is unclear. However, it is becoming increasingly more evident that cells of the monocyte/macrophage lineage are very important at several stages of disease. They are involved in the transmission, establishment and dissemination of infection as well as the AIDS related complication of dementia and pulmonary dysfunction. The regulation of virus expression in monocyte/macrophages while maintaining normal cell function would be of great benefit. Retinoic acid (RA) is a bioactive metabolite of vitamin A, an essential nutrient, and acts as a transcriptional regulator of many genes. RA is also a potent modulator of myeloid cell differentiation and function; it is currently used clinically. Clinical data indicate that serum vitamin A levels are inversely correlated with various aspects of HIV-1 induced disease. Furthermore, work done by several groups has demonstrated that RA directly modulates HIV-1 replication in cells of the myeloid lineage. RA is capable of either stimulating or repressing HIV-1 replication depending on the cell type used. This dichotomy appears to depend upon the differentiation state of the cells. Changes in differentiation states are associated with the altered expression of many cellular proteins including transcriptional regulators. Experiments indicate that the TATA box of HIV-1 is required for full levels of gene expression. I hypothesized that RA was modulating replication at the level of LTR-directed gene expression, and that the differentiation state of the cell influences the RA modulation. This thesis demonstrates that the RA effect is at the level of gene expression mapping to a promoter proximal element for both HIV-1 and simian immunodeficiency virus (SIVmac.) The ability of RA to stimulate or repress expression depends upon the differentiation state of the cells. Using U937 promonocyte cells, I demonstrate that RA increases SIVmac and HIV-1 transcription. When THP-1 monocytes or primary macrophages are used, I demonstrate that RA induces repression of HIV-1 and SIVmac. This RA modulation of expression is associated with altered complexes binding to the promoter proximal regions of HIV-1 and SIVmac. There has been a great deal of interest in CD8+ T cell derived factors which modulate HIV-1 replication. Work done by Levy and colleagues over a decade ago demonstrated that factors secreted by CD8+ T cells could block HIV-1 replication. Others have shown that the β-chemokines, released by activated CD8+ T cells, can block the entry of HIV-1 into macrophages. Center and colleagues identified a lymphocyte chemoattractant factor as IL-16. IL-16 is released by activated CD8+ T cells and it's receptor is CD4. IL-16 induces the migration of CD4+ T lymphocytes, and has been shown to activate many signaling pathways in CD4+ T lymphocytes. Kurth et al. demonstrated that IL-16 blocked the replication of HIV-1 in CD8+ depleted PBMC. In these experiments, it was not determined whether IL-16 was blocking viral entry (preventing viral binding to CD4) or whether IL-16 had inhibitory effects on subsequent steps in the virus life cycle. While IL-16 and HIV-1 share CD4 as their receptor, IL-16 binding was mapped to a separate epitope on CD4 from the HIV-1 binding site. Therefore I began experiments to determine how IL-16 regulates HIV-1 expression in T cells. I hypothesized that the IL-16 signaling pathway is involved in repressing HIV-1 gene expression. Experiments presented here demonstrate that IL-16 represses LTR-directed gene expression in T cell lines in a CD4 dependent manner. The IL-16 mediated repression is dependent on a DNA binding site contained within the viral core enhancer region. The data are also consistent with IL-16 inducing a repressor which binds within or adjacent to the HIV-1 core enhancer region.

microRNAs (miRNAs), ~21-24 nucleotide-long RNAs that post-transcriptionally regulate gene expression, have rapidly become one of the most extensively studied mechanisms of the past decade. Since their discovery as temporal regulators of post-embryonic development in C. elegans, miRNAs have been functionally implicated in almost every cellular process investigated to date. miRNAs are integral to the complex biological processes of embryonic development and aging. In this research, we sought to determine whether misregulation of miRNAs could be responsible for eliciting adverse effects during these two distinct developmental stages. First, to uncover the potential role of miRNAs in teratogenicity, we investigated whether miRNAs were involved in regulation of retinoic acid (RA) induced vertebrate axis defects. Global miRNA expression profiling revealed that RA exposure suppressed the expression of miR-19 family members during zebrafish somitogenesis. Bioinformatics analyses predict that miR-19 targets cyp26a1, a key RA detoxifying enzyme, and a physiological reporter assay confirmed that cyp26a1 is a bona fide target of miR-19. Transient knockdown of miR-19 phenocopied RA-induced body axis defects. In gain-of-function studies, exogenous miR-19 rescued the axis defects caused by RA exposure. Our findings indicate that the teratogenic effects of RA exposure result, in part, from repression of miR-19 and the subsequent misregulation of cyp26a1. This highlights a previously unidentified role of miR-19 in facilitating vertebrate axis development. Next, to explore whether age-related changes in miRNAs trigger deficits in regeneration capacity, we performed mRNA and small RNA sequencing on regenerating and non-regenerating caudal fin tissue from aged, adult and juvenile zebrafish. An unbiased approach identified cbx7 as the most abundant transcript with significantly increased expression in regenerative-competent adult and juvenile tissue and decreased expression in regenerative-compromised aged tissue. While cbx7 is a known regulator of aging, this is the first report of its role in tissue regeneration. A computational approach was used to discover mRNAs expressed during regeneration, which are potential targets of the significantly expressed miRNAs in regenerating tissue. miR-21 was one of the most abundant and significantly increased miRNAs in regenerating tissue and exhibited an aberrant age-dependent expression profile. Bioinformatics predicts miR-21 to target the 3' UTR of cbx7 and a reporter assay confirmed that miR-21 targets cbx7 in vivo. Transient knockdown of miR-21 inhibited tissue regeneration, suggesting a role for miRNA mediated regulation of cbx7 during regeneration. These findings reveal a novel, age-dependent regenerative function of cbx7 and emphasize the importance of miR-21 as a master regulator of vertebrate regenerative responses. This research, when combined, underscores the negative consequences misregulation of miRNAs has on embryonic development and aging. / Graduation date: 2013 / Access restricted to the OSU Community at author's request from Dec. 5, 2012 - Dec. 5, 2013

Interactions between the retinoic acid receptor alpha (RARalpha) and coregulators play a key role in coordinating gene transcription and myeloid differentiation. In acute promyelocytic leukemia (APL), RARalpha is fused with the promyelocytic leukemia (PML) gene, resulting in the expression of the fusion protein PML/RARalpha. Here, I report that topoisomerase II beta (topoIIbeta) associates with and negatively modulates PML/RARalpha and RARalpha transcriptional activity, and increased levels and association of topoIIbeta cause resistance to retinoic acid (RA) in APL cell lines. Knock down of topoIIbeta was able to overcome resistance by permitting RA-induced differentiation and increased RA-gene expression. Overexpression of topoIIbeta, in clones from an RA-sensitive cell line, conferred resistance by a reduction in RA-induced expression of target genes and differentiation. Chromatin immunoprecipitation assays indicate that topoIIbeta is bound to an RA-response element, and inhibition of topoIIbeta causes hyper-acetylation of histone 3 at lysine 9 and activation of transcription. These results identify a novel mechanism of resistance in APL and provide further insights to the role of topoIIbeta in gene regulation and differentiation. / Studies to determine the mechanism by which topoIIbeta protein is regulated found that levels of protein kinase C delta (PKCdelta) correlated with topoIIbeta protein expression. Moreover, activation of PKCdelta, by RA or PMA, led to an increase of topoIIbeta protein levels. Most notably, in NB4-MR2 cells, we observed increased phosphorylation levels of threonine 505 on PKCdelta, a marker of activation. Inhibition of PKCdelta was able to overcome the topoIIbeta repressive effects on RA-target genes. In addition, the combination of RA and PKCdelta inhibition led to increased expression of the granulocytic marker, CD11c, in NB4 and NB4-MR2 cells. These results suggest that PKCdelta regulates topoIIbeta expression, and a constitutively active PKCdelta in the NB4-MR2 cell line leads to overexpression of topoIIbeta. / In conclusion, these studies demonstrate that topoIIbeta associates with RARalpha, binds to RAREs and plays a critical role in RA dependent transcriptional regulation and granulocytic differentiation. In addition, I show that topoIIbeta overexpression leads to RA resistance and provide evidence that topoIIbeta protein levels are regulated via a mechanism involving the PKCdelta pathway. This work has contributed to an enhanced understanding of the role of topoIIbeta in gene regulation and brings novel perspectives in the treatment of RA-resistance in APL.

Diss. (sammanfattning) Umeå : Univ., 2007. / Härtill 3 uppsatser.

Fibroblast growth factor (Fgf) and Retinoic acid (RA) are known to be involved in patterning the posterior embryo. Work has shown that Fgf can convert anterior tissue into posterior fates and that embryos deficient in Fgf signaling lack posterior trunk and tail structures. Likewise, studies performed on RA have shown that overexpression of RA posteriorizes anterior tissue, while disrupting RA signaling yields a loss of posterior fates. While it appears these signals are necessary for posterior development, the role Fgf and RA play in development of the hindbrain is still enigmatic. A detailed study of the requirements for Fgf and RA in the early vertebrate hindbrain are lacking, namely due to a deficiency in gene markers for the presumptive hindbrain at early developmental stages. In this study, we make use of recently isolated genes, which are expressed in the presumptive hindbrain region at early developmental stages, to explore Fgf and RA regulation of the early vertebrate hindprain. We employed both overexpression and loss of function approaches to explore the role of Fgf in early vertebrate development with an emphasis on the presumptive hindbrain region in zebrafish embryos. By loss of function analysis, we show that Fgf regulates genes expressed exclusively in the hindbrain region (meis3 and hoxbla) as well as genes whose expression domains encompass both the hindbrain and more caudal regions (nlz and hoxb1b), thus demonstrating a requirement for Fgf signaling throughout the anteroposterior axis of the hindbrain (rostral to caudal hindbrain) by mid-gastrula stages. To further characterize early gene regulation by Fgf, we utilized an in vitro system and found that Fgf is sufficient to induce nlz directly and hoxb1b indirectly, while it does not induce meis3 or hoxb1a. Furthermore, in vivo work demonstrates that Fgf soaked beads can induce nlz and hoxb1b adjacent to the bead and meis3at a distance. Given the regulation of these genes in vitro and in vivo by Fgf and their position along the rostrocaudal axis of the embryo, our results suggest an early acting Fgf resides in the caudal end of the embryo and signals at a distance to the hindbrain. We detect a similar regulation of hindbrain genes by RA at gastrula stages as well, suggesting that both factors are essential for early hindbrain development. Interestingly however, we find that the relationship between Fgf and RA is dynamic throughout development. Both signals are required at gastrula stages as disruption of either pathway alone disrupts hindbrain gene expression, but a simultaneous disruption of both pathways at later stages is required to disrupt the hindbrain. We suggest that Fgf and RA are present in limiting concentrations at gastrula stages, such that both factors are required for gene expression or that one factor is necessary for activation of the other. Our results also reveal a changing and dynamic relationship between Fgf and RA in the regulation of the zebrafish hindbrain, suggesting that at segmentation stages, Fgf and RA may no longer be limiting or that they are no longer interdependent. As we have demonstrated that an early Fgf signal is required for gastrula stage hindbrain development, we next questioned which Fgf performed this function. We have demonstrated that the early Fgf signal required for hindbrain development is not Fgf3 or Fgf8, two Fgfs known to be involved in signaling centers at the mid-hindbrain boundary (MHB) and rhombomere (r) 4. We further show that two recently identified Fgfs, Fgf4 and Fgf24 are also insufficient alone or in combination with other known Fgfs to regulate hindbrain gene expression. However, as Fgfs may act combinatorially, we do not rule out the possibility of their involvement in early hindbrain gene regulation. However, as time passes and additional Fgfs are isolated and cloned, the elusive Fgf signal required for early hindbrain development will likely be identified. Taken together, we propose that an early acting Fgf residing in the caudal end of the embryo regulates hindbrain genes together with RA at gastrula stages. We suggest that both Fgf and RA are required for gene expression at gastrula stages, but this requirements changes over time as Fgf and RA become redundant. We also demonstrate that the Fgf required for gastrula stage hindbrain development has yet to be identified.

No description available.