INTRODUÇÃO: O carcinoma adrenocortical é uma neoplasia rara que carreia um prognóstico reservado. Recentemente, uma série de estudos demonstrou o potencial do perfil de miRNAs na diferenciação entre adenomas e carcinomas adrenocorticais, estratificação de risco e prognóstico. Entretanto, pouco se sabe ainda sobre a regulação pós-transcricional de miRNAs. Nesse contexto, o LIN28 é uma proteína ligadora de RNAs altamente conservada que surgiu como um modulador do let-7, uma importante família de miRNAs amplamente conhecida por seus efeitos supressivos tumorais. Além do let-7, o LIN28 também mostrou regular e ser regulado pelo mir-9, mir-30 e mir-125. OBJETIVOS: Analisar a expressão gênica e proteica do LIN28 em uma grande coorte de tumores adrenocorticais (TACs) de adultos e pediátricos, além de investigar a variação no número de cópias dos genes LIN28A e LIN28B e a expressão dos miRNAs regulatórios do LIN28 (família let-7, mir-9, mir-30 e mir-125) em um subgrupo desta coorte. MÉTODOS: A expressão proteica do LIN28 foi avaliada em um total de 266 TACs de adultos (78 adenomas e 188 carcinomas) e 44 pediátricos (35 clinicamente benignos e 9 clinicamente malignos). A expressão dos genes LIN28A e LIN28B foi avaliada em um subgrupo de 86 TACs adultos e pediátricos e a análise da variação no número de cópias destes genes em 58 TACs. O estudo de expressão das famílias dos miRNAs let-7, mir-9, mir-30 e mir-125 foi realizado em 28 carcinomas adrenocorticais de adultos. RESULTADOS: Em adultos, o gene LIN28A mostrou-se hiperexpresso em carcinomas agressivos quando comparado a adenomas [7,0 (0 a 174,3) vs. 3,6 (0 a 18,3); p = 0,006, respectivamente] e observou-se uma tendência a maior expressão quando comparados a carcinomas não agressivos [7,0 (0 a 174,3) vs. 7,1 (0 a 17,1); p = 0,092]. A expressão do LIN28B foi negativa na grande maioria (92%) dos TACs de adultos. Curiosamente, uma imunorreatividade fraca para o LIN28 foi significativamente associada com diminuição da sobrevida livre de doença nessa população (p = 0,01), mas para sobrevida global apenas uma tendência foi observada (p = 0,117). Na análise multivariada, somente o índice Ki67 >= 10% (RR 5,7, 95% IC 3,0-10,8; p= 0,0001) e imunorreatividade fraca para o LIN28 (RR 2,3, 95% IC 1,2-4,4; p = 0,008) foram preditores independentes de recorrência em adultos. De forma interessante, a expressão do mir-9, um regulador negativo do LIN28A/B, foi significativamente maior em carcinomas agressivos quando comparados a não agressivos [2076 (36 a 9307) vs. 133,4 (2,4 a 5193); p = 0,011] e fortemente associada com a redução da sobrevida global (p = 0,01) e livre de doença (p = 0,01). Na população pediátrica, não se observou diferença significativa entre expressão da proteína LIN28, assim como dos genes LIN28A e mir-9, entre tumores clinicamente benignos e malignos. Nas crianças, a hiperexpressão do LIN28B foi significativamente associada com redução da sobrevida livre de doença (p = 0,026), mas não da sobrevida global (p = 0,406). A análise da variação do número de cópias mostrou que somente uma criança com tumor virilizante benigno apresentou amplificação do LIN28B e uma mulher com carcinoma adrenocortical metastático apresentou deleção do LIN28B. Não houve variação no número de cópias para o gene LIN28A. Um índice de Ki67 >= 20% nas crianças foi capaz de discriminar pacientes com pior prognóstico: houve uma associação significativa tanto com diminuição da sobrevida global (p = 0,015) como da sobrevida livre de doença (p = 0,001) em 36 TACs pediátricos com Weiss >- 3. CONCLUSÕES: A imunorreatividade fraca para o LIN28 foi associada à diminuição da sobrevida livre de doença em uma grande coorte de carcinomas adrenocorticais de adultos. O gene LIN28A teve expressão aumentada em carcinomas agressivos de adultos, sugerindo uma regulação pós-transcricional negativa da expressão proteica do LIN28. A hiperexpressão do mir-9, um regulador negativo do LIN28, mostrou-se um importante preditor de desfecho desfavorável nos adultos. Adicionalmente, a hiperexpressão do gene LIN28B mostrou-se um potencial marcador de mau prognóstico na população pediátrica. Um índice de Ki67 >= 10% em adultos e >= 20% em crianças foram associados a mau prognóstico / INTRODUCTION: Adrenocortical carcinoma is a rare neoplasm with overall poor prognosis. Recently, several studies demonstrated the potential of miRNA profiling in differentiating between adrenocortical adenomas and carcinomas, risk stratification and prognosis. Nevertheless, little is known about posttranscriptional regulation of miRNAs. LIN28 is a highly conserved RNA-binding protein that has emerged as a modulator of the processing of let-7, an important family of miRNAs widely known for its tumor-suppressive effects. Besides from let-7, LIN28 has also shown to regulate and be regulated by mir-9, mir-30 and mir-125. OBJECTIVES: To analyze LIN28 gene and protein expression in a large cohort of adult and pediatric adrenocotical tumors (ACTs), and investigate the copy number variation analysis for LIN28A and LIN28B genes and the expression of LIN28 regulatory microRNAs (let-7 family, mir-9, mir-30 e mir-125) in a subgroup of this cohort. METHODS: LIN28 protein expression was assessed in a total of 266 adult (78 adenomas and 188 carcinomas) and 44 pediatric ACTs (35 clinically benign and 9 clinically malignant). LIN28A and LIN28B gene expression was evaluated in a subgroup of 86 adult and pediatric ACTs and copy number variation analysis of these genes in 58 ACTs. The expression of let-7 family, mir-9, mir-30 and mir-125 was performed in 28 adult carcinomas. RESULTS: In adults, LIN28A gene was overexpressed in aggressive carcinomas when compared with adenomas [7.0 fold change (from 0 to 174.3) vs. 3.6 (from 0 to 18.3); p = 0.006, respectively] and a trend towards greaten expression when compared with non-aggressive carcinomas [7.0 (from 0 to 174.3) vs. 7.1 (from 0 to 17.1); p = 0.092]. LIN28B expression was undetectable in the great majority (92%) of adult ACTs. Surprisingly, weak LIN28 staining was significantly associated with reduced disease-free survival in this population (p = 0.01), but for overall survival only a trend was detectable (p= 0.117). In the multivariate analysis, only Ki67 index >- 10% (HR 5.7, 95% CI 3.0-10.8; p = 0,0001) and weak LIN28 staining (HR 2.3, 95% CI 1.2-4.4; p = 0,008) were independent predictors of recurrence in adult patients. Interestingly, mir-9 expression, a negative LIN28A/B regulator, was significantly higher in aggressive than in non-aggressive ACCs [2076 (from 36 to 9307) vs. 133.4 (from 2.4 to 5193); p = 0.011] and was highly associated with reduced overall survival ( p= 0.01) and disease-free survival (p = 0.01). In the pediatric population, no significant difference was observed in the expression of LIN28 protein and LIN28A and mir-9 gene expression between clinically benign and clinically malignant tumors. Additionally. overexpression of LIN28B was significantly associated with reduced disease-free survival (p = 0.026), but not with overall survival (p = 0.406). Copy number variation analysis showed that only a child with a virilizing benign tumor had LIN28B amplification and a woman with a metastatic adrenocortical carcinoma had LIN28B deletion. No LIN28A copy number variation was detected. A Ki67 >= 20% in children was able to discriminate patient with worse prognosis: there was a significant associtation with reduced overall (p = 0,015) and disease-free survival (p = 0,001) in 36 pediatric ACTs with Weiss >- 3. CONCLUSIONS: Weak LIN28 staining was associated with reduced disease-free survival in a large cohort of adult adrenocortical carcinoma. LIN28A had higher expression in aggressive carcinomas in adults, suggesting there might be negative posttranscriptional regulation of LIN28 protein expression. Interestingly, overexpression of mir-9, a negative LIN28A regulator, predicted poor outcome in adult patients. In addition, LIN28B overexpression was an potential marker of poor prognosis in the pediatric population. A Ki67 index >- 10% in adults and >- 20% in children were associated with poor prognosis

Made available in DSpace on 2015-03-26T13:42:32Z (GMT). No. of bitstreams: 1 texto completo.pdf: 806747 bytes, checksum: 15d877e808731540d6a5b0cb3ec099c7 (MD5) Previous issue date: 2007-03-20 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / The plants resist to the infection caused by pathogens using constitutive and induced defenses. The induced defense is activated by the plants when they recognize general elicitors and effector proteins. This recognition leads to the rapid activation of the defense responses, including the synthesis of Pathogenesis-related proteins - PR proteins. There are 17 known PR-protein families (PR-1 to PR-17) that are expressed in response to pathogens and/or chemical inductors. The genes that codes for PR-1 proteins share a high sequence identity and are used as markers of the systemic acquired resistance (SAR). However the biochemical function of these proteins is still unknown. Antimicrobial activities were reported only for basic PR-1 proteins. PR-1 genes are members of gene families in many plant species. Based on sequence analysis of sequences deposited in public database the following possible PR-1 gene family members from tomato plant were identified: PR-1aP4 (accession numbers AJ011520 and M69247); P1p14 (Y08804, M69248 and 68738); PR1A1 (X71592); PR1A2 (Y08844); PR1D (AJ001627) in the NCBI database and two unigenes sequences from the Solanaceae Genomics Network database, SGN-U213451 and SGN- U220473. The genes PR-1aP4 and P1p14 and the unigenes SGN-U213451 and SGN-U220473 encode basic PR-1 proteins and the genes PR1A1, PR1A2 and PR1D encode acid proteins. The gene represented by the unigene SGN-U213451 seems encode the P14c protein, that has a reported antimicrobial activity and until this moment is not in the databases. In this work were developed essays to detect the expression of PR-1 family members by real-time PCR. The analyzed genes can be distinguished by their quantitative and qualitative expression pattern. PR-1aP4, P1p14, PR1A1 and the SGN-U213451 had a higher level of expression in younger leaves in response to A. solani; while, the gene PR1A2 was more induced in the older leaves of these plants, where severity of the disease is greater. The gene represented by the SGN-U220473 did not show induction by A. solani. The P1p14 and PR1A2 expression was induced by benzothiadiazole (commercial product Bion) and jasmonic acid, respectively. Lower gene expression levels were obtained with chemical induction showing that more refined kinetic induction essays of PR-1 genes of the tomato plant in response to chemical inductors are needed in order to establish which specific signaling molecule is able to trigger the expression of each family member. / As plantas resistem à infecção por patógenos utilizando defesas constitutivas e induzidas. A defesa induzida é ativada pelo reconhecimento pelas plantas de elicitores gerais e proteínas de avirulência do patógeno. Este reconhecimento leva à rápida ativação de respostas de defesa, que incluem a síntese de proteínas relacionadas à patogênese (proteínas PR). São conhecidas 17 famílias de proteínas PR (PR-1 a PR-17) que são expressas em resposta a patógenos e ou a indutores químicos. Os genes que codificam proteínas PR-1 constituem famílias gênicas em diversas espécies vegetais. Com base na análise de seqüências depositadas em bancos de dados foram identificados sete possíveis membros da família gênica PR-1 do tomateiro, os genes: PR-1a P4 (números de acessos: AJ011520 e M69247); P1p14 (Y08804, M69248 e 68738); PR1A1 (X71592); PR1A2 (Y08844); PR1D (AJ001627) depositados no NCBI e as seqüências de dois unigenes depositadas no SGN, o SGN-U213451 e SGN-U220473. Os genes PR-1a P4 e P1p14 e os representados pelos unigenes SGN- U213451 e SGN-U220473 codificam proteínas PR-1 básicas, enquanto os genes PR1A1, PR1A2 e PR1D codificam proteínas ácidas. O gene representado pelo unigene SGN-U213451 parece codificar a proteína P14c que apresenta atividade antimicrobiana e até o momento não está anotada nos bancos de dados. Neste trabalho, foram desenvolvidos ensaios para a detecção da expressão desses genes por PCR em tempo real em resposta à infecção por Alternaria solani e a aplicação de indutores químicos. Apenas para o gene PR1D não foi possível obter oligonucleotídeos adequados para efetuar análises específicas de expressão por PCR em tempo real. A análise da cinética de expressão demonstrou que os genes analisados podem ser diferenciados pelo seu padrão qualitativo e quantitativo de expressão. Os genes PR-1a P4, P1p14, PR1A1 e o SGN-U213451 tiveram maior indução da expressão no terço superior de plantas inoculadas com A. solani; enquanto o gene PR1A2 foi mais induzido no terço inferior destas plantas, onde a severidade da doença é maior. Já o gene representado pelo SGN- U220473 não apresentou indução após a inoculação com A. solani. A expressão dos genes P1p14 e PR1A2 foi induzida pela aplicação de benzotiadiazole (produto comercial Bion) e ácido jasmônico, respectivamente. Menores níveis de expressão dos genes foram detectados nos ensaios com indutores químicos comparativamente à indução biótica indicando que ensaios mais refinados de cinética de indução de genes PR-1 do tomateiro em resposta a indutores químicos são necessários para estabelecer quais sinais moleculares induzem a expressão de cada membro desta família.

INTRODUÇÃO: O carcinoma adrenocortical é uma neoplasia rara que carreia um prognóstico reservado. Recentemente, uma série de estudos demonstrou o potencial do perfil de miRNAs na diferenciação entre adenomas e carcinomas adrenocorticais, estratificação de risco e prognóstico. Entretanto, pouco se sabe ainda sobre a regulação pós-transcricional de miRNAs. Nesse contexto, o LIN28 é uma proteína ligadora de RNAs altamente conservada que surgiu como um modulador do let-7, uma importante família de miRNAs amplamente conhecida por seus efeitos supressivos tumorais. Além do let-7, o LIN28 também mostrou regular e ser regulado pelo mir-9, mir-30 e mir-125. OBJETIVOS: Analisar a expressão gênica e proteica do LIN28 em uma grande coorte de tumores adrenocorticais (TACs) de adultos e pediátricos, além de investigar a variação no número de cópias dos genes LIN28A e LIN28B e a expressão dos miRNAs regulatórios do LIN28 (família let-7, mir-9, mir-30 e mir-125) em um subgrupo desta coorte. MÉTODOS: A expressão proteica do LIN28 foi avaliada em um total de 266 TACs de adultos (78 adenomas e 188 carcinomas) e 44 pediátricos (35 clinicamente benignos e 9 clinicamente malignos). A expressão dos genes LIN28A e LIN28B foi avaliada em um subgrupo de 86 TACs adultos e pediátricos e a análise da variação no número de cópias destes genes em 58 TACs. O estudo de expressão das famílias dos miRNAs let-7, mir-9, mir-30 e mir-125 foi realizado em 28 carcinomas adrenocorticais de adultos. RESULTADOS: Em adultos, o gene LIN28A mostrou-se hiperexpresso em carcinomas agressivos quando comparado a adenomas [7,0 (0 a 174,3) vs. 3,6 (0 a 18,3); p = 0,006, respectivamente] e observou-se uma tendência a maior expressão quando comparados a carcinomas não agressivos [7,0 (0 a 174,3) vs. 7,1 (0 a 17,1); p = 0,092]. A expressão do LIN28B foi negativa na grande maioria (92%) dos TACs de adultos. Curiosamente, uma imunorreatividade fraca para o LIN28 foi significativamente associada com diminuição da sobrevida livre de doença nessa população (p = 0,01), mas para sobrevida global apenas uma tendência foi observada (p = 0,117). Na análise multivariada, somente o índice Ki67 >= 10% (RR 5,7, 95% IC 3,0-10,8; p= 0,0001) e imunorreatividade fraca para o LIN28 (RR 2,3, 95% IC 1,2-4,4; p = 0,008) foram preditores independentes de recorrência em adultos. De forma interessante, a expressão do mir-9, um regulador negativo do LIN28A/B, foi significativamente maior em carcinomas agressivos quando comparados a não agressivos [2076 (36 a 9307) vs. 133,4 (2,4 a 5193); p = 0,011] e fortemente associada com a redução da sobrevida global (p = 0,01) e livre de doença (p = 0,01). Na população pediátrica, não se observou diferença significativa entre expressão da proteína LIN28, assim como dos genes LIN28A e mir-9, entre tumores clinicamente benignos e malignos. Nas crianças, a hiperexpressão do LIN28B foi significativamente associada com redução da sobrevida livre de doença (p = 0,026), mas não da sobrevida global (p = 0,406). A análise da variação do número de cópias mostrou que somente uma criança com tumor virilizante benigno apresentou amplificação do LIN28B e uma mulher com carcinoma adrenocortical metastático apresentou deleção do LIN28B. Não houve variação no número de cópias para o gene LIN28A. Um índice de Ki67 >= 20% nas crianças foi capaz de discriminar pacientes com pior prognóstico: houve uma associação significativa tanto com diminuição da sobrevida global (p = 0,015) como da sobrevida livre de doença (p = 0,001) em 36 TACs pediátricos com Weiss >- 3. CONCLUSÕES: A imunorreatividade fraca para o LIN28 foi associada à diminuição da sobrevida livre de doença em uma grande coorte de carcinomas adrenocorticais de adultos. O gene LIN28A teve expressão aumentada em carcinomas agressivos de adultos, sugerindo uma regulação pós-transcricional negativa da expressão proteica do LIN28. A hiperexpressão do mir-9, um regulador negativo do LIN28, mostrou-se um importante preditor de desfecho desfavorável nos adultos. Adicionalmente, a hiperexpressão do gene LIN28B mostrou-se um potencial marcador de mau prognóstico na população pediátrica. Um índice de Ki67 >= 10% em adultos e >= 20% em crianças foram associados a mau prognóstico / INTRODUCTION: Adrenocortical carcinoma is a rare neoplasm with overall poor prognosis. Recently, several studies demonstrated the potential of miRNA profiling in differentiating between adrenocortical adenomas and carcinomas, risk stratification and prognosis. Nevertheless, little is known about posttranscriptional regulation of miRNAs. LIN28 is a highly conserved RNA-binding protein that has emerged as a modulator of the processing of let-7, an important family of miRNAs widely known for its tumor-suppressive effects. Besides from let-7, LIN28 has also shown to regulate and be regulated by mir-9, mir-30 and mir-125. OBJECTIVES: To analyze LIN28 gene and protein expression in a large cohort of adult and pediatric adrenocotical tumors (ACTs), and investigate the copy number variation analysis for LIN28A and LIN28B genes and the expression of LIN28 regulatory microRNAs (let-7 family, mir-9, mir-30 e mir-125) in a subgroup of this cohort. METHODS: LIN28 protein expression was assessed in a total of 266 adult (78 adenomas and 188 carcinomas) and 44 pediatric ACTs (35 clinically benign and 9 clinically malignant). LIN28A and LIN28B gene expression was evaluated in a subgroup of 86 adult and pediatric ACTs and copy number variation analysis of these genes in 58 ACTs. The expression of let-7 family, mir-9, mir-30 and mir-125 was performed in 28 adult carcinomas. RESULTS: In adults, LIN28A gene was overexpressed in aggressive carcinomas when compared with adenomas [7.0 fold change (from 0 to 174.3) vs. 3.6 (from 0 to 18.3); p = 0.006, respectively] and a trend towards greaten expression when compared with non-aggressive carcinomas [7.0 (from 0 to 174.3) vs. 7.1 (from 0 to 17.1); p = 0.092]. LIN28B expression was undetectable in the great majority (92%) of adult ACTs. Surprisingly, weak LIN28 staining was significantly associated with reduced disease-free survival in this population (p = 0.01), but for overall survival only a trend was detectable (p= 0.117). In the multivariate analysis, only Ki67 index >- 10% (HR 5.7, 95% CI 3.0-10.8; p = 0,0001) and weak LIN28 staining (HR 2.3, 95% CI 1.2-4.4; p = 0,008) were independent predictors of recurrence in adult patients. Interestingly, mir-9 expression, a negative LIN28A/B regulator, was significantly higher in aggressive than in non-aggressive ACCs [2076 (from 36 to 9307) vs. 133.4 (from 2.4 to 5193); p = 0.011] and was highly associated with reduced overall survival ( p= 0.01) and disease-free survival (p = 0.01). In the pediatric population, no significant difference was observed in the expression of LIN28 protein and LIN28A and mir-9 gene expression between clinically benign and clinically malignant tumors. Additionally. overexpression of LIN28B was significantly associated with reduced disease-free survival (p = 0.026), but not with overall survival (p = 0.406). Copy number variation analysis showed that only a child with a virilizing benign tumor had LIN28B amplification and a woman with a metastatic adrenocortical carcinoma had LIN28B deletion. No LIN28A copy number variation was detected. A Ki67 >= 20% in children was able to discriminate patient with worse prognosis: there was a significant associtation with reduced overall (p = 0,015) and disease-free survival (p = 0,001) in 36 pediatric ACTs with Weiss >- 3. CONCLUSIONS: Weak LIN28 staining was associated with reduced disease-free survival in a large cohort of adult adrenocortical carcinoma. LIN28A had higher expression in aggressive carcinomas in adults, suggesting there might be negative posttranscriptional regulation of LIN28 protein expression. Interestingly, overexpression of mir-9, a negative LIN28A regulator, predicted poor outcome in adult patients. In addition, LIN28B overexpression was an potential marker of poor prognosis in the pediatric population. A Ki67 index >- 10% in adults and >- 20% in children were associated with poor prognosis

Hyaluronic acid (HA) is an important substance, which is mostly used in pharmaceutical and cosmetic industry. This substance is commonly found in the human body. HA is one of the factors contributing to virulence of microorganisms. Some bacterial strains produce hyaluronic acid in the form of a mucoid capsule that encapsulates the cell to protect bacteria against the immune system of the host organism. One of the main producers is the bacterial strain Streptococcus equi subsp. zooepidemicus. Contipro a.s. uses the strain CO4A to produce hyaluronic acid in large scale. The production strain was obtained by random mutagenesis by UV light. The aim of the work was to study changes in the genome, which led to a significant increase in hyaluronic acid production, using DNA microarray and real-time PCR (qPCR). The genome of the strain CO4A was sequenced and compared to reference ATCC35246 [1]. The size of the genome is 2,167,251 bp and 83 relevant variants (59 SNV and 34 indels) have been identified. Variants in coding regions were annotated and amino acid sequence changes were determined. In SNV mutations there was a change in the amino acid sequence in 45 cases. The change was identified in every case of indel mutations. The expression level of selected groups of genes was monitored in both strains by the method of DNA microarrays. A cascade of increased expression level of amino sugar metabolism genes leading to the synthesis of UDP-N-acetyl glucosamine was observed in strain CO4A (the increase in expression level of these genes compared to ATCC35246 was on average 28 %). Subsequently, the expression of selected genes was verified by qPCR. There was no significant difference in the expression level of the has operon genes of both strains. The effect of supplementation of the culture medium with N-acetylglucosamine (GlcNAc), which is one of the precursors of HA synthesis, was also studied by qPCR. A positive effect of the supplementation of the culture medium with external GlcNAc in the CO4A strain has been recorded. Also, the supplementation has positive effect on the yield of HA from the medium (increase in yield was on average by 17 %). GlcNAc has been shown to have a positive effect on the yield of HA in ATCC35246 strain as well (increase in yield was 9 % on average), but no significant changes in the expression levels were found in selected groups of genes in ATCC35246.

The staphylococcal protein VgaA belongs to ARE ABCF family, which confers resistance to ribosome binding antibiotics by the target protection mechanism. VgaA confers resistance to lincosamides, streptogramins A and pleuromutilins and thus provides the so-called LSAP resistance phenotype. The expression of resistance genes often reduces fitness in the absence of an antibiotic, therefore the expression of resistance genes is often tightly controlled and triggered only in response to the presence of an antibiotic to which the protein confers resistance. The inducible expression has also been observed for the vgaA gene, nevertheless, its mechanism has not been elucidated. In the diploma thesis, it was shown that the vgaALC gene from Staphylococcus haemolyticus is regulated by ribosome-mediated attenuation. The mechanism is based on the detection of translation inhibitors via a ribosome translating a special regulatory open reading frame (uORF), which is part of an attenuator located in the 5' untranslated region of the mRNA. The vgaALC gene is regulated at the transcriptional level in response to LSAP antibiotics. Antibiotic specificity of induction is affected not only by the nature of the peptide encoded by uORF but also by the antibiotic specificity of the resistance protein. Fluorescence microscopy...

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Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Šárka Strachoňová Supervisor: PharmDr. Lukáš Červený, Ph.D. Title of diploma thesis: Studium of gene regulation of nucleoside transporters in BeWo cell line Nucleoside transporters (NTs) localized in syncytiotrophoblast control placental uptake of nucleosides. Dysregulation of NTs can disrupt nucleoside homeostasis with a negative consequences on placental and fetal development and can lead to a change in placental pharmacokinetics of nucleoside-derived drugs. Therefore, understanding the expression and function of NTs is necessary for effective and safe pharmacotherapy during pregnancy. The aim of this diploma thesis was to study the adenylate cyclase (AC) activated regulatory pathways of gene expression of concentrative nukleoside transporter 2 (CNT2). For this purpose, qRT-PCR and in vitro accumulation assays using the model substrate [3 H]-adenosine were employed. The human placental choriocarcinoma-derived BeWo cell line has been exposed to an AC activator, forskolin (50 µM), and/or inhibitors of AC/cAMP/PKA, AC/cAMP/MAPK (MEK1/2, p38 MAPK) signaling pathways, PKA inhibitor, KT 5720 (5 μM), an inhibitor of MEK1/2, U0126 (10 μM) and an inhibitor of p38 MAPK, SB202190 (10 μM). The...

O operon groESL de C. crescentus apresenta dupla regulação. A indução deste operon por choque térmico é dependente do fator sigma de choque térmico σ32. A temperaturas fisiológicas, a expressão de groESL apresenta regulação temporal durante o ciclo celular da bactéria e o controle envolve a proteína repressora HrcA e o elemento CIRCE (controlling inverted repeat of chaperonin expression). Para estudar a atividade da proteína repressora in vitro, produzimos e purificamos de E. coli a HrcA de C. creseentus contendo uma cauda de histidinas e a ligação especifica ao elemento CIRCE foi analisada em ensaios de migração retardada em gel de poliacrilamida (EMRGP). A quantidade de DNA retardada pela ligação a HrcA aumentou significativamente na presença de GroES/GroEL, sugerindo que estas proteínas modulam a atividade de HrcA. Corroboração desta modulação foi obtida analisando fusões de transcrição da região regulatória de groESL com o gene lacZ, em células de C. crescentus produzindo diferentes quantidades de GroES/EL. HrcA contendo as substituições Pro81 AJa e Arg87Ala, aminoácidos que se localizam no domínio putativo de ligação ao DNA da proteína, mostraram ser deficientes na ligação a CIRCE, tanto in vitro como in vivo. Em adição, HrcA Ser56Ala expressa na mesma célula juntamente com a proteína selvagem produziu um fenótipo dominante-negativo, indicando que a HrcA de C. crescentus liga-se a CIRCE como um oligômero, provavelmente um dímero. As tentativas de obtenção de mutantes nulos para os genes groESL ou dnaKJ falharam, indicando que as proteínas GroES/GroEL e DnaK/DnaJ são essenciais em C. crescentus, mesmo a temperaturas normais. Foram então construídas no laboratório as linhagens mutantes condicionais SG300 e SG400 de C. crescentus, onde a expressão de groESL e de dnaKJ, respectivamente, está sob controle de um promotor induzido por xilose (PxyIX). Estas linhagens foram caracterizadas quanto á sua morfologia em condições permissivas ou restritivas, assim como quanto à capacidade de sobrevivência frente a vários tipos de estresse. As células da linhagem SG300, exauridas de GroES/GroEL, são resistentes ao choque térmico a 42°C e são capazes de adquirir alguma termotolerância. Entretanto, estas células são sensíveis aos estresses oxidativo, salino e osmótico. As células da linhagem SG400, exauridas de DnaKlJ, são sensíveis ao choque térmico, à exposição a etanol e ao congelamento, e são incapazes de adquirir termotolerância. Além disso, tanto as células exauridas de GroES/GroEL quanto as exauridas de DnaK/DnaJ apresentam problemas na sua morfologia. As células de SG300 exauridas de GroES/GroEL formam filamentos longos que possuem constrições fundas e irregulares. As células de SG400 exauridas de DnaK/DnaJ são apenas um pouco mais alongadas que as células pré-divisionais selvagens e a maioria das células não possuem septo. Estas observações indicam bloqueio da divisão celular, que deve ocorrer em diferentes estágios em cada linhagem. / In Caulobacter crescentus, the groESL operon presents a dual type of control. Heat shock induction of the operon is dependent on the heat shock sigma factor σ-32. At physiological temperatures, groESL expression is cell cycle regulated and the control involves the repressor protein HrcA and the element CIRCE (controlling inverted repeat of chaperonin ~xpression). To study the activity of HrcA in vitro, we produced and purified from E. coli a histidine-tagged version of the protein, and specific binding to the CIRCE element was analyzed in electrophoretic mobility shift assays (EMSA). The amount of retarded DNA increased significantly in the presence of GroES/GroEL, suggesting that these proteins modulate HrcA activity. Further evidence of this modulation was obtained using lacZ transcription fusions with the groESL regulatory region in C. crescentus cells producing different amounts of GroES/GroEL. The mutants proteins HrcA Pro81Ala and HrcA Arg87Ala, that contain amino acid substitutions in the putative DNA-bindíng domain of the protein, were found to be deficient in binding to CIRCE in vitro and in vivo. Furthermore, HrcA Ser56Ala expressed together with the wild type protein within the same cell, produced a dominant-negative phenotype, indicating that C. crescentus HrcA binds to CIRCE in an oligomeric form, most likely as a dimer. Attempts to obtain null mutants for groESL or dnaKJ were unsuccessful indicating the importance of GroES/GroEL and DnaK/lDnaJ to the survival of C. crescentus cells. Conditional mutants were then constructed in our laboratory in which groESL and dnaKJ expression is under the control ofaxylose inducible promoter (PxyIX) , giving rise to strains SG300 and SG400, respectively. These strains were characterized in regard to their morphology under permissive and restrictive conditions, as well as their viability under different types of environmental stresses. SG300 cells depleted of GroES/GroEL are resistant to heat shock at 42°C and can acquire some thermotolerance, but they are sensitive to oxidative, saline and osmotic stresses. SG400 cells depleted of DnaKlJ are quite sensitive to heat shock, ethanol and freezing, and are unable of acquiring thermotolerance. Cells depleted of either GroES/EL or DnaKlJ also present morphological problems. SG300 cells depleted of GroES/EL form long and pinched filaments. SG400 cells depleted of DnaKlJ are only somewhat more elongated than wild-type predivisional cells and most cells do not present septum. These observations indicate a cell division arrest, which should occur at different stages in each strain.

Die Aufklärung der Mechanismen zur Kontrolle der Genexpression ist eines der wichtigsten Probleme der modernen Molekularbiologie. Detaillierte experimentelle Untersuchungen sind enorm aufwändig aufgrund der komplexen und kombinatorischen Wechselbeziehungen der beteiligten Moleküle. Infolgedessen sind bioinformatische Methoden unverzichtbar. Diese Dissertation stellt drei Methoden vor, die die Vorhersage der regulatorischen Elementen der Gentranskription verbessern. Der erste Ansatz findet Bindungsstellen, die von den Transkriptionsfaktoren erkannt werden. Dieser sucht statistisch überrepräsentierte kurze Motive in einer Menge von Promotersequenzen und wird erfolgreich auf das Genom der Bäckerhefe angewandt. Die Analyse der Genregulation in höheren Eukaryoten benötigt jedoch fortgeschrittenere Techniken. In verschiedenen Datenbanken liegen Hunderte von Profilen vor, die von den Transkriptionsfaktoren erkannt werden. Die Ähnlichkeit zwischen ihnen resultiert in mehrfachen Vorhersagen einer einzigen Bindestelle, was im nachhinein korrigiert werden muss. Es wird eine Methode vorgestellt, die eine Möglichkeit zur Reduktion der Anzahl von Profilen bietet, indem sie die Ähnlichkeiten zwischen ihnen identifiziert. Die komplexe Natur der Wechselbeziehung zwischen den Transkriptionsfaktoren macht jedoch die Vorhersage von Bindestellen schwierig. Auch mit einer Verringerung der zu suchenden Profile sind die Resultate der Vorhersagen noch immer stark fehlerbehafted. Die Zuhilfenahme der unabhängigen Informationsressourcen reduziert die Häufigkeit der Falschprognosen. Die dritte beschriebene Methode schlägt einen neuen Ansatz vor, die die Gen-Anotation mit der Regulierung von multiplen Transkriptionsfaktoren und den von ihnen erkannten Bindestellen assoziiert. Der Nutzen dieser Methode wird anhand von verschiedenen wohlbekannten Sätzen von Transkriptionsfaktoren demonstriert. / Understanding the mechanisms which control gene expression is one of the fundamental problems of molecular biology. Detailed experimental studies of regulation are laborious due to the complex and combinatorial nature of interactions among involved molecules. Therefore, computational techniques are used to suggest candidate mechanisms for further investigation. This thesis presents three methods improving the predictions of regulation of gene transcription. The first approach finds binding sites recognized by a transcription factor based on statistical over-representation of short motifs in a set of promoter sequences. A succesful application of this method to several gene families of yeast is shown. More advanced techniques are needed for the analysis of gene regulation in higher eukaryotes. Hundreds of profiles recognized by transcription factors are provided by libraries. Dependencies between them result in multiple predictions of the same binding sites which need later to be filtered out. The second method presented here offers a way to reduce the number of profiles by identifying similarities between them. Still, the complex nature of interaction between transcription factors makes reliable predictions of binding sites difficult. Exploiting independent sources of information reduces the false predictions rate. The third method proposes a novel approach associating gene annotations with regulation of multiple transcription factors and binding sites recognized by them. The utility of the method is demonstrated on several well-known sets of transcription factors. RNA interference provides a way of efficient down-regulation of gene expression. Difficulties in predicting efficient siRNA sequences motivated the development of a library containing siRNA sequences and related experimental details described in the literature. This library, presented in the last chapter, is publicly available at http://www.human-sirna-database.net

Thesis (Ph. D.)--University of Virginia, 2008. / Includes bibliographical references. Also available online through Digital Dissertations.