Thesis (Ph. D.)--West Virginia University, 2003. / Title from document title page. Document formatted into pages; contains viii, 123 p. : ill. (some col.). Includes abstract. Includes bibliographical references (p. 94-123).

Submitted by ROBERTO PAULO CORREIA DE ARAÚJO (ppgorgsistem@ufba.br) on 2015-07-17T20:13:43Z No. of bitstreams: 1 SILVA JUNIOR, Joaquim Custódio.pdf: 4910459 bytes, checksum: 7746a181edeca5d3d7a1104ec6e7fc66 (MD5) / Made available in DSpace on 2015-07-17T20:13:43Z (GMT). No. of bitstreams: 1 SILVA JUNIOR, Joaquim Custódio.pdf: 4910459 bytes, checksum: 7746a181edeca5d3d7a1104ec6e7fc66 (MD5) / FAPESB – Fundação de Amparo à Pesquisa do Estado da Bahia. / Introdução: A despeito de bom prognóstico na maioria dos casos, o manejo do câncer diferenciado de tireoide (CDT) ainda carece de avanços em situações de doença mais agressiva. O uso crescente da Biologia Molecular na Oncologia vem ganhando força como ferramenta útil no manejo de diversos tipos de câncer. Além da abordagem tradicional de busca de mutações relacionadas a tipos específicos de câncer, o estudo de características epigenéticas emerge como uma nova modalidade de análise e predição do comportamento dos tumores. HOPX é um gene homeobox com função de supressão tumoral, cuja hipermetilação e consequente redução de expressão gênica foi estudada em diversos tipos de câncer (pulmão, esôfago, estômago, pâncreas, cólon e útero). Em câncer colorretal, gástrico, pulmonar e de esôfago, a redução de expressão gênica de HOPX confere fenótipo mais agressivo e piora do prognóstico. HOPX possui papel controlador de diversas onco-proteínas (Cyr61, EMP1, EphA2, c-Fos, c-Jun, EGR1 e GLUT-3) e interage com fatores de transcrição tais como HDAC2/GATA4, SRF e EPC1. Objetivos: Analisar o perfil de expressão do gene homeobox HOPX em amostras de CDT, comparando com o tecido não-tumoral adjacente e com tecidos benignos, e buscar associação com aspectos clínico-patológicos. Metodologia: Estudo prospectivo envolvendo 32 pacientes submetidos à tireoidectomia total, dos quais 26 tiveram confirmação de diagnóstico de CDT e 6 com tumores benignos. Realizada dissecção de amostra de tecido tumoral (T) e tecido não-tumoral (NT) adjacente ao tumor. Extração de RNA das amostras pelo método Trizol® e conversão em cDNA. Análise de expressão gênica através da reação em cadeia da polimerase em tempo real utilizando aparelho 7500 Real Time PCR System (Applied Biosystems® ). O gene S8 foi utilizado como controle interno para normalização da expressão. Dados clínicos e patológicos foram obtidos através de entrevista com os pacientes e revisão de prontuários eletrônicos. Para cálculo da expressão gênica foi empregado o método ∆∆cT. A comparação da expressão gênica entre tecidos NT e T foi realizada utilizando testes de Wilcoxon. Para comparar os grupos benigno e maligno foi utilizado o teste ANOVA. As análises considerando os aspectos clínico-patológicos foram realizadas com o teste do Qui-Quadrado e o teste de Kruskal-Wallis. Resultados: A expressão de HOPX demonstrou marcada redução em amostras de CDT, quando comparado a neoplasias benignas de tireoide (p = 0,024). Verificou-se significativa redução (65,6%) da expressão gênica de HOPX nas amostras T quando comparadas com as NT (3,49 ± 8,3 vs. 10,14 ± 27,8) (p = 0,009). Em pacientes com CDT sem tireoidite associada, a redução da expressão de HOPX esteve associada a estádios mais iniciais de neoplasia (I e II, classificação TNM), em comparação com estádios mais avançados (III e IV) (p = 0,021). Redução de expressão de HOPX também esteve associada com idade menor que 45 anos (p = 0,048). Conclusão: Este é o primeiro estudo da literatura que evidencia redução da expressão de HOPX em câncer de tireoide, tanto em comparação com tecidos benignos, quanto comparado a tecido não-tumoral. Por outro lado, os achados de associação com estádios mais precoces e pacientes mais jovens sugerem uma possível especificidade dos mecanismos de controle da expressão gênica de HOPX em CDT, mediante influências microambientais e genéticas ainda não identificadas. Estudos adicionais permitirão confirmar estes achados em maior tamanho amostral, bem como definir o papel de HOPX como marcador de prognóstico clínico e risco de recorrência tumoral em câncer de tireoide. / Background: Despite good prognosis in majority of cases, differentiated thyroid cancer (DTC) management still lacks advances in more aggressive disease. Growing use of Molecular Biology in Oncology has emerged as useful tool in management of several types of cancer. In addition to the traditional approach of search tissue-specific related mutations, the study of epigenetic pattern constitutes a new way to analysis and prediction of tumor’s behavior. HOPX is a homeobox gene with tumor-suppressing function, whose hypermethylation and consequent reduction of gene expression was studied in several cancer types (lung, esophagus, stomach, pancreas, colorectal and uterus). In colorectal, gastric, lung and esophageal cancer, reduction of HOPX gene expression gives more aggressive phenotype and worse prognosis. HOPX acts controlling various oncoproteins (like Cyr61, EMP1, EphA2, c-Fos, c-Jun, EGR1 and GLUT-3) and interacts with transcription factors such as HDAC2/GATA4, SRF and EPC1. Objectives: Analysis of HOPX gene expression profile in DTC samples, comparing with the adjacent non-tumor tissue and benign thyroid samples, and looking for association with clinical-pathologic features. Methods: Prospective study enrolling 32 patients who undergone thyroidectomy, of which 26 has confirmed DTC diagnosis and 6 with benign tumors. Held sample dissection of tumor tissue (T) and nontumor tissue (NT) adjacent to the tumor. RNA extraction from samples with Trizol® method, and then converted in cDNA. Gene expression analysis with real time PCR method, using 7500 Real Time PCR System (Applied Biosystems® ). S8 ribosomal gene was used as housekeeping to normalize expression. Clinical and pathological data was obtained interviewing patients and reviewing electronic medical records. ∆∆cT method was used to calculate gene expression. Gene expression. comparison between T and NT tissues was done with Wilcoxon’s test. ANOVA test was used to compare malign and benign tumors. Clinicalpathologic features analysis were done with qui-square test and Kruskal-Wallis’s test. Results: HOPX expression markedly reduced in DTC samples, when compared to benign thyroid tissues (p = 0.024). There was a significant reduction (65.6%) of HOPX gene expression in tumor samples compared to non-tumoral (3.49 ± 8.3 vs. 10.14 ± 27.8) (p = 0.009). In DTC patients without thyroiditis, HOPX reduced expression was associated with earlier tumor stages (I and II, TNM staging system) compared with advanced stages (III and IV) (p = 0.021). HOPX reduced expression was also associated with age below 45 years (p = 0.048). Conclusion: This is the first study that evidences HOPX reduced expression in thyroid cancer, compared with benign thyroid tumors and non-tumoral tissues. However, finding of association with earlier stages and lower age suggests specific mechanisms controlling HOPX gene expression in DTC, mediated by microenvironmental and genetic influences, yet not identified. Additional studies may confirm these findings in larger samples, and establish the utility of HOPX use as prognostic and tumor recurrence marker in thyroid cancer.

polyhomeotic is a gene of the Polycomb-group required for proper segment determination in Drosophila. Genetic and molecular analysis has shown that ph has a repetetive structure. The DNA sequence presented here shows that ph consists of a direct tandem duplication with very high sequence conservation. Analysis of the sequence has revealed several conserved open reading frames and splice junctions, putative transcriptional promoter and terminator sequences, polyadenylation signals and translational start signals. In addition, the DNA sequence shows that ph contains a zinc finger sequence in each repeat. This suggests that ph may encode a DNA-binding protein. / Science, Faculty of / Zoology, Department of / Graduate

Orientadores: Sergio Roberto Peres Line, Marcelo Rocha Marques / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba / Made available in DSpace on 2018-08-20T05:35:08Z (GMT). No. of bitstreams: 1 Andrade_SimoneCaixetade_D.pdf: 127365203 bytes, checksum: 4736aaa809aadf03b1ad27e52dbb8b30 (MD5) Previous issue date: 2012 / Resumo: A dentição não é somente o maior componente do complexo crânio facial dos mamíferos, mas um modelo útil de desenvolvimento, combinado em um sistema de um único órgão, onde ocorrem fenômenos de organização espacial, simetria, aquisição de formas complexas e citodiferenciação órgão-específica. Durante os primeiros estágios do desenvolvimento do dente ocorre uma série de interações entre o epitélio oral e mesênquima, por meio de diferentes moléculas sinalizadoras ao longo do futuro processo alveolar. Bmp4 atua ativando Msx1 na região de incisivos e molares e restringe a ação de Barx1 apenas na região dos molares. A interação entre o sinal mesenquimal Bmp4 e o sinal epitelial Shh faz parte dos primeiros eventos da formação do germe dentário no estágio botão. As fases seguintes são os estágios capuz e sino. Mutações nos genes Msx1 e Barx1 impedem o desenvolvimento do germe dentário além do estágio botão. Para o gene Msx1 essa condição é permanente, enquanto para Barx1 é temporal e o crescimento do germe dentário é retomado após o estágio E14.5 em camundongos. O objetivo desse trabalho é investigar a interação genética entre Msx1 e Barx1 no desenvolvimento do primeiro molar inferior na fase sino (E16.5) e qual a influência da ausência do gene Msx1 na dimensão das estruturas vestigiais MS, R2 e do primeiro molar inferior nas fases botão (E13.5) e sino (E16.5). Esse estudo também se propôs a avaliar se genes Barx2, Barhl1 e Barhl2 estariam expressos em botões de molares inferiores (E13.5). Os resultados indicaram que na ausência completa de Barx1, um único alelo de Msx1 não é suficiente para promover a evolução do botão dentário a capuz em camundongos E16.5 Barx1;Msx1 e não houve expressão de Bmp4 nesses camundongos. A expressão nula dos genes Barx2, Barhl1 e Barhl2 em botões de molares inferiores em camundongos wild-type E13.5 indica que genes da família Bar não possuem nenhum papel no desenvolvimento de molares inferiores. Ainda, dados alométricos de Msx1-/- (E13.5 e E16.5) indicam que a mutação no gene Msx1 e consequente ausência de expressão de Smad1-5-8 e Shh não impede o crescimento no sentido proximal-distal. Conclui-se que existe interação entre Barx1 e Msx1. Barx1 é necessário para controlar os níveis de expressão de Bmp4. Barx2 possivelmente desempenha um papel importante na morfogênese de pré-molares e incisivos inferiores. O atraso no desenvolvimento dos molares inferiores de camundongos Msx1-/- é permanente ao estágio botão, contudo, os botões dentários destes camundongos continuam a exibir um crescimento no sentido proximal-distal no estágio E16.5 / Abstract: The dentition is not only the largest component of the craniofacial complex of mammals, but a useful model of development, combined in a single organ system, where phenomena of spatial organization, symmetry, complex shape acquisition and organ-specific cytodifferentiation occur. During the early stages of tooth development, series interactions take place between oral epithelial and mesenchyme by different signalling molecules along the future alveolar process. Bmp4 acts by activating Msx1 in the region of incisors and molars and it restricts the action of Barx1 only to the molars' region. The interaction between mesenchymal Bmp4 signal and Shh epithelial signal is part of the first events of tooth germ formation at the bud stage. The next stages are cap and bell. Mutations in Msx1 and Barx1 genes inhibit the development of the tooth germ beyond the bud stage. For the Msx1 gene, this condition is permanent, while for Barx1 is temporal and the growth of tooth germ is retaken after stage E14.5 in mice. This study investigates the genetic interaction between Msx1 and Barx1 in the first lower molar development at the bell stage (E16.5), the effect of the absence of Msx1 gene in the size of the vestigial structures MS, R2 and first lower molar bud (E13.5) and bell (E16.5). This study also aimed to assess whether Barx2, Barhl1 and Barhl2 genes were expressed in lower molars buds (E13.5). The results indicated that in the complete absence of Barx1, a single allele of Msx1 is not sufficient to promote the development of tooth bud to cap in E16.5 Barx1;Msx1 mice, and there was no expression of Bmp4 in these mice. The null expression of Barx2, Barhl1 and Barhl2 genes in lower molar buds in E13.5 wild-type mice indicates that genes of the Bar family do not exert any role in the development of lower molars. Still, allometric data on Msx1-/- (E13.5 and E16.5) indicates that Msx1 gene mutation and consequently the absence of Shh and SMAD1-5-8 expression do not prevent growth towards proximal-distal. It is concluded that there is interaction between Barx1 and Msx1. Barx1 is necessary to control the expression levels of Bmp4. Barx2 might play a role in the lower premolars and incisors morphogenesis. The delayed development of Msx1-/- lower molars is permanent at the bud stage, however, the tooth buds of these mice continue to exhibit growth towards proximal-distal at E16.5 stage / Doutorado / Histologia e Embriologia / Doutor em Biologia Buco-Dental

by Lam, Sau Hing. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1998. / Includes bibliographical references (leaves 141-158). / Abstract also in Chinese. / Content / Acknowledgements / Abbreviation / Project Objectives / Abstract / Chapter Chapter One --- Introduction / Chapter 1.1 --- The definition of homeobox genes / Chapter 1.2 --- Homeobox genes as a transcription factor / Chapter 1.3 --- Homeobox in Drosophila / Chapter 1.3.1 --- The development of Drosophila / Chapter 1.3.2 --- Maternal genes / Chapter 1.3.3 --- Segmentation genes / Chapter 1.3.4 --- Homeobox genes / Chapter 1.4 --- Family of Hox genes in the mammalian system / Chapter 1.5 --- Some possible chemical mechanism in the cascade system of the Hox genes in the vertebrate / Chapter 1.6 --- Hox (Antp-Class homeobox gene) in mammal / Chapter 1.6.1 --- Labial Like homeobox genes / Chapter 1.6.2 --- Proboscipedia Like homeobox genes / Chapter 1.7 --- Divergent homeobox genes / Chapter 1.7.1 --- Paired (prd) Class / Chapter 1.7.2 --- Even-Skipped (Eve) Class / Chapter 1.7.3 --- Distal-less (Dll) Class / Chapter 1.7.4 --- Muscle-Specific Homeobox (Msx) Class / Chapter 1.8 --- Orphan homeobox gene / Chapter 1.8.1 --- The characteristic of Hox 11 sequence in human and mouse / Chapter 1.8.2 --- Novel homeobox genes related to hox 11 gene family / Chapter 1.8.3 --- The mechanism of HOX 11 inducing gene regulation and signal transduction pathways / Chapter 1.8.4 --- HOX 11 in human / Chapter 1.8.5 --- Hox11 in mouse / Chapter 1.8.6 --- Hox11 L1 in mouse / Chapter 1.9 --- Homeobox gene involved in haematopoiesis / Chapter 1.10 --- Some translocations of homeobox genes in blood lineage / Chapter 1.11 --- The development of mouse / Chapter 1.11.1 --- Early organogenesis / Chapter 1.11.2 --- Nervous system development / Chapter 1.11.3 --- Somite development / Chapter 1.11.4 --- Eye development / Chapter 1.11.5 --- Neural crest cell migration / Chapter 1.11.6 --- Branchial arches development / Chapter Chapter Two --- Materials and methods / Chapter 2.1 --- Mouse Embryos / Chapter 2.2 --- RNA extraction / Chapter 2.3 --- Large plasmid preparation / Chapter 2.4 --- The synthesis of cDNAs using Reverse Transcription / Polymerase Chain Reaction (RT-PCR) and ligation into Bluescript® II KS / Chapter 2.4.1 --- The synthesis of RT-PCR products / Chapter 2.4.2 --- The formation of blunt ends of cDNA / Chapter 2.4.3 --- The ligation of cDNA with plasmid vectors / Chapter 2.4.4 --- Transformation / Chapter 2.4.5 --- The miniprep plasmid purification / Chapter 2.5 --- T7 sequencing / Chapter 2.6 --- Double stranded DNA cycle sequencing of plasmid / Chapter 2.6.1 --- Gel electrophoresis / Chapter 2.7 --- Northern blot / Chapter 2.7.1 --- Preparation of Northern blot / Chapter 2.7.2 --- Hybridization of Northern blot / Chapter 2.8 --- DIG labeled probes in whole mount in situ hybridization / Chapter 2.8.1. --- Preparation of linear DNA to generate riboprobes / Chapter 2.8.2 --- Preparation of DIG labeled riboprobe / Chapter 2.8.3. --- Preparation of embryo powder / Chapter 2.8.4 --- Absorption of antibody / Chapter 2.8.5 --- Embryo preparation / Chapter 2.8.6 --- Embryos staining / Chapter 2.9 --- Sections from whole mount in situ hybridization / Chapter 2.10 --- The radiolabeled section in situ hybridization / Chapter 2.10.1 --- The preparation of paraffin wax block and sample sections / Chapter 2.10.2 --- Slide pretreatment / Chapter 2.10.3 --- Preparation of probe / Chapter 2.10.4 --- Hybridization / Chapter 2.10.5 --- Washing / Chapter 2.10.6 --- Emulsification and development / Chapter 2.11 --- DIG-label in situ hybridization of frozen / Chapter 2.12 --- H&E staining / Chapter Chapter Three --- Results and Discussion / Chapter 3.1 --- Synthesis of Tlx3 probes for use in the section and whole mount in situ hybridization / Chapter 3.1.1 --- Synthesis of the Tlx cDNA by RT-PCR method / Chapter 3.1.2 --- The Tlx3 genomic clone for detecting the developmental expression of Tlx3 by Northern Blot / Chapter 3.1.3 --- The characterization of Tlx3 cDNAs and the sonic hedgehog cDNA / Chapter 3.2 --- Section in situ hybridization using different probes / Chapter 3.2.1 --- Section in situ hybridization using the transcribed riboprobes of Pax2 cDNA / Chapter 3.2.2 --- The transcribed riboprobes of Tlx genomic clones were used to hybridize the section in situ hybridization / Chapter 3.2.3 --- The in situ hybridization of frozen sections of mouse embryos using the transcribed riboprobes from Tlx3 cDNA subclone / Chapter 3.3 --- Expression pattern of Tlx3 on whole mount embryos using both cDNA and genomic probes / Chapter 3.3.1 --- The expression of Tlx3 on whole mount in situ hybridization of the mouse embryos using the antisense probes from the Tlx3 genomic clone / Chapter 3.3.2 --- Whole mount in situ hybridization of mouse embryo using the transcribed riboprobes of Tlx3 cDNA / Chapter 3.3.3 --- The expression pattern of sonic hedgehog on embryos at 8.5 to9.5 dpc / Conclusion / Future prospective / Appendix / Reference / Acknowledgement

Thesis (M.S.)--West Virginia University, 2002. / Title from document title page. Document formatted into pages; contains viii, 56 p. : ill. (some col.). Vita. Includes abstract. Includes bibliographical references (p. 50-54).

Thesis (Ph. D.)--University of Washington, 1999. / Vita. Includes bibliographical references (leaves 107-120).

Thesis (M.S.)--West Virginia University, 2005. / Title from document title page. Document formatted into pages; contains viii, 99 p. : ill. (some col.). Includes abstract. Includes bibliographical references (p. 82-99).

The polyhomeotic (ph) locus of Drosophila melanogaster has been characterized genetically. Early studies showed that ph is a member of the Polycomb (Pc) group. These genes have similar phenotypes and are required for normal segment determination. Recent analyses of amorphic ph mutations show that the ph locus is complex, has a strong maternal effect and plays a role in cuticular development. To test the function of ph at the molecular level, the cloning of the ph locus was undertaken. One strain had been shown to contain a P element insertion near ph. A genomic library was prepared from this strain and a recombinant phage containing this P element insertion was isolated by transposon tagging. The DNA flanking the insertion was used as a starting point for a chromosomal walk. A series of overlapping phage spanning 170 kilobases was isolated. Southern blot analysis was used to determine the locations of important deficiency breakpoints within the region covered by the walk. A distance of approximately 35 kb was shown to separate the two deficiency breakpoints which include ph. This interval was found to contain rearrangements in four of the seven ph alleles which were examined by Southern blot analysis. The interval also contains a repeated sequence. The relationship between the genetic and molecular structure of ph is discussed. / Science, Faculty of / Zoology, Department of / Graduate

O carcinoma epidermóide de boca, neoplasia maligna de boca mais comum, pode originar-se de lesões potencialmente malignas. O ácido retinóico, que atua no crescimento e diferenciação celular, tem sido comumente estudado como um possível quimioterápico na prevenção dessa progressão. Embora o mecanismo pelo qual o ácido retinóico previne essa progressão, e promove a parada do crescimento celular, não esteja estabelecido, sabe-se que os genes homeobox são importantes alvos do ácido retinóico durante o desenvolvimento embrionário e diferenciação tecidual. Este estudo visa determinar se a modulação da expressão desses genes está envolvida na inibição do crescimento pelo ácido retinóico em carcinoma epidermóide de boca. Para isso, foi realizado PCR array para avaliar a expressão de 84 genes homeobox na linhagem celular de carcinoma epidermóide de boca sensível ao ácido retinóico SSC-25, comparando com a linhagem resistente, SSC-9, após o tratamento com ácido retinóico por sete dias. Os resultados mostraram nove genes com perda de expressão e quatro com alta expressão. A validação por qPCR de 7 desses genes confirmou os resultados. Desses, três genes(ALX1, DLX3, TLX1) foram selecionados para terem a expressão avaliada em amostras tratadas por 3, 5 e 7 dias. O gene ALX1 apresentou baixa expressão apenas no dia 7. O gene DLX3 apresentou baixa expressão no terceiro dia com maior decréscimo no sétimo. Já o gene TLX1, mostrou baixa significativa no quinto dia, com valores semelhantes no sétimo. Os dados mostram genes homebox são modulados pelo ácido retinóico em linhagens de carcinoma epidermóide de boca. No entanto, esses genes não parecem ser alvo direto da inibição do crescimento promovida pelo ácido retinóico. / Oral squamous cell carcinoma, the most frequent oral cancer, may arise from potentially malignant oral lesions. Retinoic acid, which plays a role in cell growth and differentiation, has been frequently studied as a possible chemotherapeutic agent in the prevention of this progression. While the mechanism by which retinoic acid prevents progression and suppresses cell growth has not been completely elucidated, it is known that homeobox genes represent important targets of retinoic acid during embryogenesis and differentiation. The present study aims to determine if modulation of the expression of these genes is involved in inhibition of OSCC cell growth by retinoic acid. In order to achieve this goal a PCR array was performed to evaluate the expression of 84 homeobox genes in retinoic acid sensitive SCC-25 cells compared to retinoic acid resistant SCC-9 cells following treatment with retinoic acid for 7 days. Results showed that 9 homeobox genes are downregulated and 4 are upregulated by retinoic acid. The validation confirmed these results. Three genes (ALX1, DLX3, TLX1) were selected for having their expression evaluated on samples treated with retinoic acid for 3, 5 and 7 days. Three different patterns of gene expression were observed. Gene ALX1 showed down-regulation only on day 7. Homeobox gene DLX3 showed reduced expression on day 3 and decreased expression on day 7. TLX1 showed a substantial down-regulation on day 5 with similar values on. The data presented show that a number of homeobox genes are modulated by retinoic acid in oral squamous cell carcinoma cell lines. However, these genes do not appear to be direct targets of growth suppression trigged by retinoic acid.