Thesis (Ph. D.)--University of Texas at Austin, 2008. / Vita. Includes bibliographical references.

Thesis (M.S.)--Brigham Young University. Dept. of Physiology and Developmental Biology, 2007. / Includes bibliographical references.

- / -

- / -

Made available in DSpace on 2015-06-17T19:34:24Z (GMT). No. of bitstreams: 0 Previous issue date: 2014-02-25. Added 1 bitstream(s) on 2015-06-18T12:48:55Z : No. of bitstreams: 1 000831510.pdf: 612514 bytes, checksum: dc6899b84194e33253d906ed14ea10eb (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / A incidência de câncer de mama (CM) tem aumentado de maneira alarmante nos últimos anos. Diversas pesquisas conduziram à descoberta de amphiregulin (AREG) como um oncogene, tendo sua expressão relacionada com diversos tipos de cânceres. Nosso grupo evidenciou a ação do T3 sobre diferentes genes em células de câncer de mama, por meio de microarray, porém nesse estudo não foi avaliado o mecanismo de ação pelo qual o T3 regula a expressão desses genes e o envolvimento de ER. A relação entre a expressão de AREG e a presença de HT permanece desconhecida, portanto, o objetivo do presente estudo foi verificar a ação nuclear e extra-nuclear do T3 na expressão gênica do oncogene AREG em linhagens celulares de adenocarcinoma de mama, MCF-7 e MDA-MB-231. As linhagens celulares foram submetidas ao tratamento com 10-8M de T3 nos tempos de 10', 30', 1h e 4h, na presença ou ausência de Fulvestrant (ICI 182,780), inibidor de ER, Actinomicina D (ACTD) - inibidor da expressão gênica, Ciclohexamida (CHX), inibidor da síntese protéica, e LY294002 - inibidor da via PI3K. O mRNA de AREG foi analisado pela técnica de RT-PCR. Para a análise dos dados foi utilizado ANOVA complementado com teste de Tukey e adotado significância mínima de 5%. O T3 elevou os níveis de expressão gênica de AREG, em relação ao grupo controle, nas duas linhagens celulares, independente dos tempos de incubação. Nosso trabalho confirma que a expressão gênica de AREG está aumentada na presença T3, em ambas as linhagens e em todos os tempos estudados. Os dados desse estudo indicam que T3 pode agir por meio de ER, diminuindo a expressão de AREG após 30' de tratamento nas células MCF-7 e não alterando a expressão desse gene nas células MDA-MB-231, as quais não possuem ER. Além disso, podemos sugerir que a ação de T3 sobre a expressão gênica de AREG ocorre de forma indireta. O T3 mantém a expressão de AREG mesmo após ... / The incidence of breast cancer (BC) has increased alarmingly in recent years. Several studies have been lead to the discovery of amphiregulin (AREG) as an oncogene, with its expression related to various cancer types. Our group showed the action of T3 on different genes in BC cells using microarray, but the mechanism which T3 regulates the expression of these genes and the involvement of ER have not been evaluated yet. The relationship between the expression of AREG and the presence of TH remains unknown, therefore, the aim of this study was to verify the nuclear and extra-nuclear action of T3 on AREG gene expression in breast adenocarcinoma cell lines, MCF-7 and MDA-MB-231. The cell lines were subjected to treatment with 10-8M T3 at the times 10', 30', 1h and 4h in the presence or the absence of Fulvestrant (ICI 182,780) - ER inhibitor, Actinomycin D (ACTD) - inhibitor of gene expression, cycloheximide (CHX) - inhibitor of protein synthesis, and LY294002 - inhibitor of PI3K pathway. The AREG mRNA levels were analyzed by RT-PCR. For data analysis we used ANOVA followed by the post hoc Tukey's test with a significance level of 5%. T3 increased AREG gene expression levels, compared to the control group in both cell lines, regardless of the incubation times. Our study confirms that AREG gene expression is increased in the presence of T3 in both cell lines and at all the times studied. The data from this study indicate that T3 might act through ER, decreasing AREG expression after 30' treatment in MCF-7 cells and did not alter the expression of this gene in MDA-MB-231 cells, which lack ER. Furthermore, we suggest that T3actionon AREG gene expression occurs indirectly. The T3 maintains AREG expression even after the inhibition of transcription. And, the PI3K pathway activation by T3 is required for the modulation of AREG in both cell lines studied / FAPESP: 2009/15607-2

This thesis describes work done with the thyroid hormone receptor (TR), a nuclear protein which binds to specific DNA sequences and regulates transcription in response to thyroid hormone levels. The studies can be divided into two broad categories: structure/function studies of the TR protein, particularly with regards to DNA binding function; and, structure/function studies of the DNA sequences to which the thyroid hormone receptor binds in order to regulate gene transcription. In order to examine the DNA binding properties of the TR an electrophoretic mobility shift assay (EMSA) was utilized. Conditions of this assay were optimized for the use of in vitro translated TR. Mutant forms of the β-isoform of thyroid hormone receptor were generated using a PCR-based mutagenesis protocol. Each mutant substituted a different residue of the 12 amino acid-long α-recognition helix with alanine. The mutants were analyzed for their abilities to bind to thyroid hormone response elements (TREs), and to activate transcription in transfected eukaryotic cells. The DNA binding results were consistent with a conserved α-helix structure, with conserved function for many residues, that is similar to that of the related receptors for glucocorticoids and estrogen. Only the first of the three non-conserved residues lying in the P-box (EGG), a portion of the recognition α-helix that facilitate differential binding of distinct DNA sequences, disrupted binding when substituted with alanine. The third position of the P-box, when substituted with alanine exhibits an altered ability to bind to certain natural TREs. The mutant form of TR with alanine substituted for the second P-box position displayed only a modest decrease in DNA binding affinity compared to wild-type TR (roughly 3-fold), yet was completely deficient in trans-activation. The structure-function studies of TR binding sites on DNA applied a methylation interference protocol to examine the interactions of TR with direct repeats (DR) of the idealized hexameric sequence, spaced by three to five base-pairs. The interactions of both the TR/TR homodimer and the TR/RXR (9-cis-retinoic acid receptor) heterodimer with the DRs were examined. The methylation interference patterns for the TR/TR homodimer bound to the DR sequences are virtually identical for spacers of four and five base-pairs, but with three base-pairs, there is some evidence that at least one DNA binding domain is misaligned with the DNA to accomodate the unfavourable spacer length. The TR/RXR heterodimer methylation interference pattern is distinct on all three DRs, probably due to the fact that in the heterodimer cooperative intermolecular contacts are made between the DNA binding domains of the two receptors, but only when the spacer distance is four base-pairs. When a poorly conserved everted repeat (EvR) that overlaps the idealized DR is present. the homodimer, but not the heterodimer, binds this cryptic EvR in competition with the DR. The binding modality of the TR homodimer and TR/RXR heterodimer to DRs was reevalutated using point mutants and EMSA. The TR homodimer and TR/RXR heterodimer both bind to idealized direct repeats with DBDs aligned appropriately for a direct repeat; however, evidence is presented that there are certain poorly conserved sequences that are intermediate between DRs and EvRs that are differentially recognized by the TR homodimer and the TR/RXR heterodimer. That is, the homodimer binds with the DBDs aligned appropriately for a EvR, and the heterodimer DBDs are aligned appropriately for a DR. / Graduate

Orientador: Nereu Carlos Prestes / Coorientador: Maria Lucia Gomes Lourenço / Banca: Wilter Ricardo Russiano Vicente / Banca: Eunice Oba / Resumo: Nas cadelas as distocias estão relacionadas a alterações hormonais, nutricionais, endócrinas e hormonais durante a gestação. Desta forma, este estudo irá apresentar os achados clínicos, eletrocardiográficos, bioquímicos (hematócrito, hemoglobina, ureía, creatinina, glicose), eletrolíticos (iCa, Na, K, Cl, TCO2), e hormonais (P4, T3, T4), nas cadelas em eutocia e distocia, dividido em dois momentos; M1 (uma hora antes do parto ou cesariana) e M2 (após o parto ou recuperação anestésica). Utilizou-se 28 cadelas, sendo 22 em distocia e seis em eutocia. A arritmia sinusal foi o ritmo cardíaco predominante no pós-parto das distocias. O momento influenciou na amplitude da onda P e na frequência cardíaca, sendo superiores no M1. Os parâmetros creatinina, hematócrito e a hemoglobina diferiram entre os partos, sendo superiores na eutocia e M1. A elevação das concentrações de P4 foram observadas nos partos distócicos. O T3 demonstrou alteração significativa frente ao momento, sendo superior no M1 em ambos os grupos. O T4 foi superior nas distocias e diferente no M2 entre os grupos. O dióxido de carbono total apresentou diferença significativa entre os momentos, sendo superior no M2. Podemos concluir, o estabelecimento do parto ocorre uma tendência a taquicardia sinusal, bem como o aumento da perfusão sanguínea e função renal. Nas distocias, devido a menor perfusão sanguínea, a intervenção pela cesariana predispõe a diminuição na oxigenação sanguínea no pós-parto. Na vigência dos partos, a elevação das concentrações de progesterona interferiu no estabelecimento das contrações uterinas predispondo a distocia / Abstract: In bitches the maternal of dystocia are related hormonal, endocrinal and nutritional changes during pregnancy. Therefore, this study will presentes clinical findings, eletrocardiographical, biochemical (hematocrit, hemoglobin, urea, creatine, glucose), eletrolytic (iCa, Na, K, Cl, TCO2) and hormonal (P4, T3, T4), in bitches during eutocia and dystocia, divided in two moments, M1 (one hour before parturition or caesarean) and M2 (after parturition or anesthetic recovery). We used 28 bitches, whose 22 in the dystocic and six in the eutocia. The sinus arrhythmia on the eletrocardiographic parameters was significant interval and heart rate. The moment influenced the P wave amplitude and in the heart rate being higher in M1. The parameters creatinine, hematocrit and hemoglobin differences between births, being higher in M1. Rising concentrations of P4 were observed in dystocia. T3 showed significant change across the time, being higher in M1 in both groups. The T4 was higher during dystocia and different in M2 among the groups. The total dioxide carbon showed a significant difference between times, being higher in M2. It follows, the establishment of delivery is a tendency sinus tachycardia, and increased blood perfusion and renal function. In dystocia due to lower blood perfusion, intervention by cesarean predisposes the decrease in blood oxygenation in the post-partum. In the presence of deliveries, the elevation of progesterone interfered with the establishment of uterine contractions predispose to dystocia / Mestre

Made available in DSpace on 2014-10-09T12:26:48Z (GMT). No. of bitstreams: 0 / Made available in DSpace on 2014-10-09T14:06:19Z (GMT). No. of bitstreams: 0 / Dissertacao (Mestrado) / IPEN/D / Instituto de Pesquisas Energeticas e Nucleares - IPEN-CNEN/SP

Made available in DSpace on 2014-10-09T12:47:42Z (GMT). No. of bitstreams: 0 / Made available in DSpace on 2014-10-09T14:08:07Z (GMT). No. of bitstreams: 1 08317.pdf: 3150149 bytes, checksum: 4cea7ea7cd5d6a1b9497c4d9450e06c4 (MD5) / Dissertacao (Mestrado) / IPEN/D / Instituto de Pesquisas Energeticas e Nucleares - IPEN/CNEN-SP

Made available in DSpace on 2014-06-11T19:29:15Z (GMT). No. of bitstreams: 0 Previous issue date: 2013-07-10Bitstream added on 2014-06-13T20:19:13Z : No. of bitstreams: 1 000735996.pdf: 837750 bytes, checksum: 86934181e3622aaf1ea4a9ec2d8615bf (MD5) / Nas cadelas as distocias estão relacionadas a alterações hormonais, nutricionais, endócrinas e hormonais durante a gestação. Desta forma, este estudo irá apresentar os achados clínicos, eletrocardiográficos, bioquímicos (hematócrito, hemoglobina, ureía, creatinina, glicose), eletrolíticos (iCa, Na, K, Cl, TCO2), e hormonais (P4, T3, T4), nas cadelas em eutocia e distocia, dividido em dois momentos; M1 (uma hora antes do parto ou cesariana) e M2 (após o parto ou recuperação anestésica). Utilizou-se 28 cadelas, sendo 22 em distocia e seis em eutocia. A arritmia sinusal foi o ritmo cardíaco predominante no pós-parto das distocias. O momento influenciou na amplitude da onda P e na frequência cardíaca, sendo superiores no M1. Os parâmetros creatinina, hematócrito e a hemoglobina diferiram entre os partos, sendo superiores na eutocia e M1. A elevação das concentrações de P4 foram observadas nos partos distócicos. O T3 demonstrou alteração significativa frente ao momento, sendo superior no M1 em ambos os grupos. O T4 foi superior nas distocias e diferente no M2 entre os grupos. O dióxido de carbono total apresentou diferença significativa entre os momentos, sendo superior no M2. Podemos concluir, o estabelecimento do parto ocorre uma tendência a taquicardia sinusal, bem como o aumento da perfusão sanguínea e função renal. Nas distocias, devido a menor perfusão sanguínea, a intervenção pela cesariana predispõe a diminuição na oxigenação sanguínea no pós-parto. Na vigência dos partos, a elevação das concentrações de progesterona interferiu no estabelecimento das contrações uterinas predispondo a distocia / In bitches the maternal of dystocia are related hormonal, endocrinal and nutritional changes during pregnancy. Therefore, this study will presentes clinical findings, eletrocardiographical, biochemical (hematocrit, hemoglobin, urea, creatine, glucose), eletrolytic (iCa, Na, K, Cl, TCO2) and hormonal (P4, T3, T4), in bitches during eutocia and dystocia, divided in two moments, M1 (one hour before parturition or caesarean) and M2 (after parturition or anesthetic recovery). We used 28 bitches, whose 22 in the dystocic and six in the eutocia. The sinus arrhythmia on the eletrocardiographic parameters was significant interval and heart rate. The moment influenced the P wave amplitude and in the heart rate being higher in M1. The parameters creatinine, hematocrit and hemoglobin differences between births, being higher in M1. Rising concentrations of P4 were observed in dystocia. T3 showed significant change across the time, being higher in M1 in both groups. The T4 was higher during dystocia and different in M2 among the groups. The total dioxide carbon showed a significant difference between times, being higher in M2. It follows, the establishment of delivery is a tendency sinus tachycardia, and increased blood perfusion and renal function. In dystocia due to lower blood perfusion, intervention by cesarean predisposes the decrease in blood oxygenation in the post-partum. In the presence of deliveries, the elevation of progesterone interfered with the establishment of uterine contractions predispose to dystocia