Type 2 diabetes is a metabolic disorder that results in β-cell dysfunction and ultimate destruction, and leads to impaired glucose homeostasis. High rates of proliferation and differentiation of pancreatic β-cells occurs mostly during neonatal development. However, research shows these mechanisms remain intact as β-cell proliferation has been observed during pregnancy and obesity. We have shown that overexpression of the β-cell transcription factor Nkx6.1 is sufficient to induce β-cell proliferation. Exploration of the transcriptional targets of Nkx6.1 has identified histone deacetylase 1 (HDAC1) as a down-stream target of Nkx6.1. Here we demonstrate that HDAC1 overexpression is sufficient to induce β-cell proliferation, enhance β-cell survival upon exposure to apoptotic stimuli and maintains glucose stimulated insulin secretion (GSIS). Our data suggests overexpression of HDAC1 leads to p15/INK4b suppression, a cell cycle inhibitor, potentially explaining the mechanism behind these observed effects. These data demonstrate that HDAC1 overexpression is sufficient to induce β-cell proliferation and enhance cell survival while maintaining glucose stimulated insulin secretion.

Epigenetic mechanisms are essential for normal cell development. Alteration in those normal processes leads to malignant cell transformation and with this to cancer development. Use of inhibitors that alter the epigenetics of DNA methylation and histone post translational modifications has lead to the exploration of the epigenetic mechanism involved in silencing of tumor suppressor genes in cancer, including acute myeloid leukemia (AML). Moreover, combinations of inhibitors that target various epigenetic enzymes have being recognized to be more effective in the re-activation of tumor suppressor genes than individual drug treatments. Here, we reported that p15, p21 and E-cadherin genes are more effectively re-expressed using a combination of DNA methyltransferase and histone methyltransferase inhibitors in AML cell lines. Re-expression of hypermethylated p15 and E-cadherin genes required reduced levels of promoter histone 3 lysine 9 (H3K9) methylation rather than inhibition of DNA methylation itself. Moreover, induction of p21 expression was associated with changes in promoter histone 3 lysine 9 methylation (H3K9Me) by achieving inhibition of the histone methyltransferase, SUV39H1, activity. Altogether, our results highlight the potential of combining epigenetic drugs in the re-activation of epigenetically silenced tumor suppressor genes and the need for evaluating histone methyltransferases as therapeutic targets for treatment of acute myeloid malignancies.

Epigenetic mechanisms are essential for normal cell development. Alteration in those normal processes leads to malignant cell transformation and with this to cancer development. Use of inhibitors that alter the epigenetics of DNA methylation and histone post translational modifications has lead to the exploration of the epigenetic mechanism involved in silencing of tumor suppressor genes in cancer, including acute myeloid leukemia (AML). Moreover, combinations of inhibitors that target various epigenetic enzymes have being recognized to be more effective in the re-activation of tumor suppressor genes than individual drug treatments. Here, we reported that p15, p21 and E-cadherin genes are more effectively re-expressed using a combination of DNA methyltransferase and histone methyltransferase inhibitors in AML cell lines. Re-expression of hypermethylated p15 and E-cadherin genes required reduced levels of promoter histone 3 lysine 9 (H3K9) methylation rather than inhibition of DNA methylation itself. Moreover, induction of p21 expression was associated with changes in promoter histone 3 lysine 9 methylation (H3K9Me) by achieving inhibition of the histone methyltransferase, SUV39H1, activity. Altogether, our results highlight the potential of combining epigenetic drugs in the re-activation of epigenetically silenced tumor suppressor genes and the need for evaluating histone methyltransferases as therapeutic targets for treatment of acute myeloid malignancies.

Introdução: A falta de previsibilidade no tratamento periodontal regenerativo de defeito de furca grau III, têm estimulado o estudo de alternativas para melhorar os resultados, através do emprego de diferentes técnicas e biomateriais. Um novo enxerto ósseo enriquecido com peptídeos - Matriz óssea bovina inorgânica/P15 (PepGen P15) - foi desenvolvido recentemente e, segundo a literatura, mostrou resultados significantes em relação à neoformação tecidual nos defeitos infra-ósseos testados. Assim, o presente estudo teve como objetivo verificar o potencial regenerativo da matriz óssea inorgânica/P15 particulada, no tratamento de defeitos de furca grau III em cães associado ou não ao uso de membrana de PTFE-e Material e Métodos: Foram utilizados seis cães, nos quais defeitos de furca grau III nos pré-molares inferiores foram confeccionados, sendo que no grupo teste foi utilizado a membrana de PTFE-e e a matriz óssea inorgânica/P15, no grupo controle foi utilizada somente a membrana e no grupo controle negativo, nos 2° prémolares, não foi colocado biomaterial. Os defeitos foram confeccionados e preenchidos com material de impressão (Impregum F) e após 21 dias foram debridados, as raízes aplainadas com cureta Gracey (Hu-friedy) e os dentes submetidos à profilaxia semanal com ultra-som e limpeza diária com digluconato de clorexidina a 0,12% durante 15 dias. Na segunda fase cirúrgica, foram realizadas marcações do nível ósseo nas raízes mesial e distal dos dentes P2, P3 e P4, colocação das membranas e do enxerto ósseo nos respectivos defeitos. Os dentes P3 e P4 foram aleatoriamente escolhidos para ser o grupo teste ou controle. Quatro semanas após a colocação das membranas, estas foram retiradas e, doze semanas após a remoção, os animais foram sacrificados. Os dentes e seus tecidos periodontais de proteção e suporte foram removidos, fixados em formalina tamponada a 10%, descalcificados em ácido tricloroacético a 10%, desidratados e seccionados no plano mésio-distal em cortes semi-seriados com 7m de espessura cada. Os cortes foram corados com hematoxilina e eosina (HE) ou tricrômico de Mallory (TM) sendo selecionados para a análise histomorfométrica 5 cortes representativos da porção central da bifurcação. Foram realizadas medidas da área total da bifurcação (AT), área de novos tecidos formados (ANT), área de epitélio (AE), área de tecido conjuntivo (ATC), área de novo osso (ANO) e medidas lineares da extensão representativa da altura do defeito (ED), extensão do novo tecido extensão (ENT), extensão do novo osso (EO), extensão da bifurcação (EB) e extensão do novo cemento (EC). Resultados: A análise histológica demonstrou características morfológicas similares entre os grupos avaliados. Adicionalmente, o grupo teste apresentou grânulos do enxerto ósseo, envoltos por uma matriz óssea imatura, aprisionado entre os tecidos neoformados. Os resultados das médias das medidas lineares (mm) e das medidas de área (mm2) foram respectivamente os seguintes para o grupo teste: 14,11 ± 1,74 (EF) e 17,62 ± 2,39 (ATF); 8,61 ± 3,24 (EC) e 14,66 ± 3,73 (ANT); 4,71 ± 0,54 (ED) e 0,90 ± 0,80 (AE); 3,78 ± 0,85 (ENT) e 5,36 ± 2,41 (ATC); 1,77 ± 1,54 (EO) e 6,52 ± 5,69 (ANO); 4,82 ± 2,98 (DO). As seguintes médias lineares e de área para o grupo controle respectivamente: 13,19 ± 2,03 (EF) e 15,11 ± 3,29 (ATF); 8,52 ± 3,54 (EC) e 11,88 ± 2,09 (ANT); 4,59 ± 0,65 (ED) e 0,95 ± 0,71 (AE); 3,54 ± 0,61 (ENT) e 5,19 ± 2,17 (ATC); 1,64 ± 1,06 (EO) e 4,17 ± 3,40 (ANO); 2,58 ± 1,71 (DO). E as seguintes médias lineares e de área para o grupo controle negativo respectivamente: 13,54 ± 1,41 (EF) e 14,99 ± 3,13 (ATF); 6,56 ± 2,11 (EC) e 11,13 ± 3,25 (ANT); 4,62 ± 0,47 (ED) e 0,95 ± 0,78 (AE); 3,59 ± 0,28 (ENT) e 5,89 ± 0,87 (ATC); 0,98 ± 0,48 (EO) e 2,88 ± 2,49 (ANO); 2,35 ± 2,00 (DO). A análise estatística dos dados, realizada através da aplicação do teste de Friedman Test (< 0,05), demonstrou haver diferenças estatisticamente significantes entre o grupo teste e controle negativo no parâmetro referente à ANO. Conclusão: Pode-se concluir que a matriz óssea inorgânica/P15 apresentou resultados semelhantes aos outros grupos em relação à regeneração periodontal do defeito. No entanto, quando a matriz óssea permaneceu no defeito, apresentou resultados satisfatórios através da formação óssea que circunscreveu as partículas. / Background: The aim of this study was to verify the regenerative potential of particulate ABM/Synthetic Peptide in class III furcation defects associated or not with ePTFE membranes. Material and Methods: Six dogs were used and class III furcation defects were produced in the lower pre-molars and filled with impression material. After 21 days, the membranes and the bone grafts were inserted and P3 and P4 were randomized to form the test and control groups. P2 was the negative control. The animals were sacrificed 3 months post-treatment. Results: Comparisons between groups by the Friedman Test (<0.05) showed statistically significant differences between the test and negative control groups, based on NBA parameter. In the negative control group, a new bone area (NBA) of 20% was observed. In the test group, a new bone area of 37% was observed. There was no statistically significant difference between the groups to the others parameters. Conclusions: The statistical analysis using Friedman Test (<0.05) showed statistically significant differences between the test and negative control groups, based on NBA parameter. Furthermore, when the Inorganic Bone Matrix/P15 remained inside the defects, it could be clearly observed that new bone formation not only circumscribed the particles but formed above the level of the particles.

<p>Pancreatic endocrine tumors (PETs) may cause typical syndromes of hormone excess, or appear clinically non-functioning without hormonal symptoms. PETs occur sporadically, in association with the multiple endocrine neoplasia type 1 (MEN1) syndrome, or rarely the von Hippel-Lindau syndrome. Molecular genetic investigations may reveal pathways important for tumor development, and be of clinical use.</p><p>The aim of this thesis was to investigate regulation of different genes involved in cell proliferation, and relate findings to signs of malignancy in PETs.</p><p>The MEN1 gene on chromosome 11q13 was mutated in three out of eleven sporadic malignant PETs. Two nonsense mutations, causing truncation of the protein, and one missense mutation were found.</p><p>Relation of allelic loss at 11q13 and 3p25 to malignant behavior was observed in sporadic PETs. Allelic loss at 18q21 was found in a subset of sporadic and MEN1-associated PETs, and mutation analysis of Smad4 excluded a tumor suppressor gene function.</p><p>In PETs with allelic loss on chromosome 3p25, mutation analysis of WNT7A and HDAC11 excluded function as tumor suppressor genes.</p><p>Menin, encoded by the MEN1 gene, was reported to regulate expression of the cyclin-dependent kinase inhibitors CDKN2C/p18, CDKN1B/p27, and CDKN2B/p15 in mouse pancreatic islet tumor models. Here, the mRNA expression of these genes was not related to MEN1 gene mutations in human PETs.</p><p>Cyclin-dependent kinase 4 (CDK4) and the protooncogene c-Myc were found to be overexpressed regardless of MEN1 gene mutational status of the PETs. The CDK4 gene was neither amplified nor mutated. Targeting of CDK4 may present an alternative to traditional chemotherapy of PETs in the future.</p>

Pancreatic endocrine tumors (PETs) may cause typical syndromes of hormone excess, or appear clinically non-functioning without hormonal symptoms. PETs occur sporadically, in association with the multiple endocrine neoplasia type 1 (MEN1) syndrome, or rarely the von Hippel-Lindau syndrome. Molecular genetic investigations may reveal pathways important for tumor development, and be of clinical use. The aim of this thesis was to investigate regulation of different genes involved in cell proliferation, and relate findings to signs of malignancy in PETs. The MEN1 gene on chromosome 11q13 was mutated in three out of eleven sporadic malignant PETs. Two nonsense mutations, causing truncation of the protein, and one missense mutation were found. Relation of allelic loss at 11q13 and 3p25 to malignant behavior was observed in sporadic PETs. Allelic loss at 18q21 was found in a subset of sporadic and MEN1-associated PETs, and mutation analysis of Smad4 excluded a tumor suppressor gene function. In PETs with allelic loss on chromosome 3p25, mutation analysis of WNT7A and HDAC11 excluded function as tumor suppressor genes. Menin, encoded by the MEN1 gene, was reported to regulate expression of the cyclin-dependent kinase inhibitors CDKN2C/p18, CDKN1B/p27, and CDKN2B/p15 in mouse pancreatic islet tumor models. Here, the mRNA expression of these genes was not related to MEN1 gene mutations in human PETs. Cyclin-dependent kinase 4 (CDK4) and the protooncogene c-Myc were found to be overexpressed regardless of MEN1 gene mutational status of the PETs. The CDK4 gene was neither amplified nor mutated. Targeting of CDK4 may present an alternative to traditional chemotherapy of PETs in the future.

Thesis (Ph. D.)--University of Alabama at Birmingham, 2007. / Title from first page of PDF file (viewed Feb. 18, 2009). Includes bibliographical references.

Introdução: A falta de previsibilidade no tratamento periodontal regenerativo de defeito de furca grau III, têm estimulado o estudo de alternativas para melhorar os resultados, através do emprego de diferentes técnicas e biomateriais. Um novo enxerto ósseo enriquecido com peptídeos - Matriz óssea bovina inorgânica/P15 (PepGen P15) - foi desenvolvido recentemente e, segundo a literatura, mostrou resultados significantes em relação à neoformação tecidual nos defeitos infra-ósseos testados. Assim, o presente estudo teve como objetivo verificar o potencial regenerativo da matriz óssea inorgânica/P15 particulada, no tratamento de defeitos de furca grau III em cães associado ou não ao uso de membrana de PTFE-e Material e Métodos: Foram utilizados seis cães, nos quais defeitos de furca grau III nos pré-molares inferiores foram confeccionados, sendo que no grupo teste foi utilizado a membrana de PTFE-e e a matriz óssea inorgânica/P15, no grupo controle foi utilizada somente a membrana e no grupo controle negativo, nos 2° prémolares, não foi colocado biomaterial. Os defeitos foram confeccionados e preenchidos com material de impressão (Impregum F) e após 21 dias foram debridados, as raízes aplainadas com cureta Gracey (Hu-friedy) e os dentes submetidos à profilaxia semanal com ultra-som e limpeza diária com digluconato de clorexidina a 0,12% durante 15 dias. Na segunda fase cirúrgica, foram realizadas marcações do nível ósseo nas raízes mesial e distal dos dentes P2, P3 e P4, colocação das membranas e do enxerto ósseo nos respectivos defeitos. Os dentes P3 e P4 foram aleatoriamente escolhidos para ser o grupo teste ou controle. Quatro semanas após a colocação das membranas, estas foram retiradas e, doze semanas após a remoção, os animais foram sacrificados. Os dentes e seus tecidos periodontais de proteção e suporte foram removidos, fixados em formalina tamponada a 10%, descalcificados em ácido tricloroacético a 10%, desidratados e seccionados no plano mésio-distal em cortes semi-seriados com 7m de espessura cada. Os cortes foram corados com hematoxilina e eosina (HE) ou tricrômico de Mallory (TM) sendo selecionados para a análise histomorfométrica 5 cortes representativos da porção central da bifurcação. Foram realizadas medidas da área total da bifurcação (AT), área de novos tecidos formados (ANT), área de epitélio (AE), área de tecido conjuntivo (ATC), área de novo osso (ANO) e medidas lineares da extensão representativa da altura do defeito (ED), extensão do novo tecido extensão (ENT), extensão do novo osso (EO), extensão da bifurcação (EB) e extensão do novo cemento (EC). Resultados: A análise histológica demonstrou características morfológicas similares entre os grupos avaliados. Adicionalmente, o grupo teste apresentou grânulos do enxerto ósseo, envoltos por uma matriz óssea imatura, aprisionado entre os tecidos neoformados. Os resultados das médias das medidas lineares (mm) e das medidas de área (mm2) foram respectivamente os seguintes para o grupo teste: 14,11 ± 1,74 (EF) e 17,62 ± 2,39 (ATF); 8,61 ± 3,24 (EC) e 14,66 ± 3,73 (ANT); 4,71 ± 0,54 (ED) e 0,90 ± 0,80 (AE); 3,78 ± 0,85 (ENT) e 5,36 ± 2,41 (ATC); 1,77 ± 1,54 (EO) e 6,52 ± 5,69 (ANO); 4,82 ± 2,98 (DO). As seguintes médias lineares e de área para o grupo controle respectivamente: 13,19 ± 2,03 (EF) e 15,11 ± 3,29 (ATF); 8,52 ± 3,54 (EC) e 11,88 ± 2,09 (ANT); 4,59 ± 0,65 (ED) e 0,95 ± 0,71 (AE); 3,54 ± 0,61 (ENT) e 5,19 ± 2,17 (ATC); 1,64 ± 1,06 (EO) e 4,17 ± 3,40 (ANO); 2,58 ± 1,71 (DO). E as seguintes médias lineares e de área para o grupo controle negativo respectivamente: 13,54 ± 1,41 (EF) e 14,99 ± 3,13 (ATF); 6,56 ± 2,11 (EC) e 11,13 ± 3,25 (ANT); 4,62 ± 0,47 (ED) e 0,95 ± 0,78 (AE); 3,59 ± 0,28 (ENT) e 5,89 ± 0,87 (ATC); 0,98 ± 0,48 (EO) e 2,88 ± 2,49 (ANO); 2,35 ± 2,00 (DO). A análise estatística dos dados, realizada através da aplicação do teste de Friedman Test (< 0,05), demonstrou haver diferenças estatisticamente significantes entre o grupo teste e controle negativo no parâmetro referente à ANO. Conclusão: Pode-se concluir que a matriz óssea inorgânica/P15 apresentou resultados semelhantes aos outros grupos em relação à regeneração periodontal do defeito. No entanto, quando a matriz óssea permaneceu no defeito, apresentou resultados satisfatórios através da formação óssea que circunscreveu as partículas. / Background: The aim of this study was to verify the regenerative potential of particulate ABM/Synthetic Peptide in class III furcation defects associated or not with ePTFE membranes. Material and Methods: Six dogs were used and class III furcation defects were produced in the lower pre-molars and filled with impression material. After 21 days, the membranes and the bone grafts were inserted and P3 and P4 were randomized to form the test and control groups. P2 was the negative control. The animals were sacrificed 3 months post-treatment. Results: Comparisons between groups by the Friedman Test (<0.05) showed statistically significant differences between the test and negative control groups, based on NBA parameter. In the negative control group, a new bone area (NBA) of 20% was observed. In the test group, a new bone area of 37% was observed. There was no statistically significant difference between the groups to the others parameters. Conclusions: The statistical analysis using Friedman Test (<0.05) showed statistically significant differences between the test and negative control groups, based on NBA parameter. Furthermore, when the Inorganic Bone Matrix/P15 remained inside the defects, it could be clearly observed that new bone formation not only circumscribed the particles but formed above the level of the particles.

Superheaters suffer large material loss during combustion of waste and biomass, causing a short life time for these expensive components. During combustion, corrosive ash particles are formed and erosion is caused by circulating bed material and sand particles, all contributing to the material loss. This study examines whether corrosion or erosion has the largest effect on this material loss by investigating two superheaters in loop seal during biomass and waste combustion of an 85 MW, Circulating Fluidized Bed (CFB) boiler in Händelö. The samples were investigated by SEM/EDX and XRD with regard to material loss and corrosion products. The superheaters have different thermal conditions since the material temperature in the first superheater that the steam passes is lower than in the one that comes after. In this report, a model to determine the tube temperature in steam boiler superheaters is also described due to the fact that the local tube temperature is of great importance of condensation of corrosive gases such as KCl and NaCl. Material loss was significantly greater on the cooler superheater compared with the warmer. The material temperatures on the outside of the tubes, were calculated to be about 574 °C for the cooler superheater and about 617°C for the warmer superheater. Overall, all analyzes showed low levels of corrosive substances, although there was a certain corrosion tendency, which indicates that material loss of the superheaters is caused by corrosion-assisted erosion. Lower material temperature of the superheater resulted in a higher degree of condensation of corrosive species such as alkali chlorides, which might have accelerated the erosion. The conclusion is that the dominant mechanism of material loss on the superheaters is erosion.

The Aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor and member of the basic helix-loop-helix Per/ARNT/Sim (bHLH/PAS) family of chemosensors and developmental regulators. As a member of the PAS domain family of transcription factors responsive to exogenous signals, the AhR exerts influence on many processes relating to cellular fate. The activation of AhR is widely associated with toxic endpoints related to dioxin exposure. However, the AhR also activates endogenous gene programs related to development, cellular growth, and differentiation. The AhR is able to bind a variety of ligands, leading to a wide range of biological outcomes. Recent reports have shown that the AhR can mediate tumor suppressive effects. As a ligand-activated transcription factor, the AhR has the potential to actuate a variety of transcriptional programs that are dependent on the AhR ligand. Our central hypothesis is that AhR ligands can be identified that are capable of initiating tumor suppressive functions of the AhR. We utilized complementary cell-based and in silico virtual screening approaches to identify potential AhR ligands. We developed homology models of the AhR ligand-binding domain (LBD) for virtual ligand screening (VLS) of small molecule libraries. This led to the identification of new AhR ligands 5,7- dihydroxyflavanone!and 5-hydroxy-7-methoxyflavone. Additional small molecule libraries were screened in parallel that led to identification of flutamide as a putative AhR ligand. Flutamide is clinically approved for the treatment of prostate cancer due to its ability to antagonize androgen receptor mediated transcription. We investigated the biological effects of flutamide in AhR positive cancer cells that do not express the androgen receptor and found that flutamide inhibited the growth of HepG2 cells. Suppression of AhR expression reversed the anti-proliferative effects of flutamide. We tested 15 structural analogs of flutamide, including the flutamide metabolite 2-hydroxyflutamide for activation of AhR transcriptional activity. Flutamide is unique in its ability to activate the AhR, and suppresses hepatoma cell growth. These data suggests that flutamide-induced AhR transcriptional activity is required to initiate the tumor suppressive effects. We examined changes in cell cycle checkpoint proteins after flutamide treatment and discovered increased expression of cell cycle inhibitory proteins p27[superscript Kip1] and p15[superscript INK]. We also found that transforming Growth Factor β1 (TGFβ1), which regulates both p27[superscript Kip1] and p15[superscript INK], is upregulated by flutamide. We demonstrate that TGFβ1 is upregulated by flutamide in an AhR-dependent manner and is required for suppression of proliferation by flutamide. We identify specific and unique transcriptional signatures of the AhR upon activation by flutamide, that are distinct from the potent AhR agonist 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD). In summary, we characterize flutamide as an AhR ligand and demonstrate its AhR-dependent tumor suppressive effects in hepatoma cells. We provide the first direct evidence that AhR regulates TGFβ signaling in a ligand dependent manner. We demonstrate that the AhR-induced downstream transcriptional signature and subsequent biological effects are specific to the AhR ligand. Our studies have broad impact for characterizing the AhR as a new therapeutic target in hepatocellular carcinoma. / Graduation date: 2013 / Access restricted to the OSU Community at author's request from March 28, 2013 - March 28, 2015