Bacterial regulatory small RNAs (sRNAs) act as crucial regulators in central carbon metabolism by modulating translation initiation and/or degradation of target mRNAs in metabolic pathways. This work demonstrates that a noncoding sRNA, SdhX, is produced by RNase E-dependent processing from the 3 ́ untranslated region (3 ́ UTR) of an operon encoding three enzymes of the tricarboxylic acid (TCA) cycle. In Escherichia coli, SdhX negatively regulates ackA (encoding an enzyme critical for degradation of the signaling molecule acetyl phosphate) without affecting the downstream pta gene that encodes the enzyme critical for acetyl phosphate synthesis. SdhX abundance is tightly coupled to the transcription signals of the TCA cycle genes but escapes all known post-transcriptional regulation. Therefore, SdhX expression directly correlates with transcriptional input to the TCA cycle, providing an effective mechanism for the cell to link the TCA cycle with acetate metabolism pathways. / Doctorat en Sciences / info:eu-repo/semantics/nonPublished

La kinase dépendante des cyclines 5 (Cdk5) est un facteur de sensibilité aux inhibiteurs de PARP et aux rayonnements ionisants (RI), elle est nécessaire pour le point de contrôle du cycle cellulaire en phase S. Cependant, elle n’est pas directement impliquée dans la réparation des cassures de brin d’ADN, suggérant un rôle dans les étapes plus précoces de la signalisation des dommages. Nous rapportons ici que des cellules HeLa déplétées pour Cdk5 (Cdk5 KD) montrent une grande sensibilité aux RI surtout lorsqu'elles sont irradiées en phase S, au 5-Fluoro-Uracile, à la 6-Thioguanine et à une exposition chronique à l'hydroxyurée (HU). Les cellules Cdk5 KD montrent une altération de la dynamique de la phase S causée par une vitesse de réplication plus lente et une réduction des origines actives par mégabase d'ADN. En revanche, après un traitement au HU, ces cellules sortent plus rapidement du blocage en phase S. Ceci s’accompagne d’une déficience de la phosphorylation de RPA-32 sur les sérines 29 et 33 et de SMC1 sur la sérine 966 ainsi que d’une réduction du niveau de dommages de l'ADN évalués par le test des comètes alcalines, de l’intensité du signal gamma-H2AX, des foyers RPA, Rad51 et RPA sur les sérines 4 et 8 ainsi que du niveau d'échanges de chromatides sœurs. Des essais kinase in vitro couplés à la spectrométrie de masse ont montré que Cdk5 peut phosphoryler RPA-32 sur ses sérines 23, 29, et 33. De plus, des niveaux inférieurs d’expression de Cdk5 ont été associés à une meilleure survie sans métastases chez des patientes atteintes d’un cancer du sein et à une réduction de la survie des cellules de tumeurs du sein déplétées pour Cdk5 après un traitement aux RI et en présence d’un inhibiteur de PARP. Globalement, ces résultats montrent que Cdk5 est nécessaire pour la réplication basale et l'activation du point de contrôle en phase S en réponse à un stress réplicatif, ouvrant des perspectives cliniques intéressantes pour améliorer la destruction des cellules tumorales dans certaines populations de patientes atteintes de cancer du sein grâce à des agents qui génèrent un stress réplicatif. / Cyclin dependent kinase 5 (Cdk5) is a determinant of sensitivity to PARP inhibitors and ionizing radiation (IR) and is required for the intra-S DNA damage checkpoint. It is not however directly implicated in strand break repair suggesting a role in the earlier steps of checkpoint activation. We report here that Cdk5-Depleted (Cdk5-KD) HeLa cells show higher sensitivity to IR when irradiated in S-Phase, and to chronic hydroxyurea (HU) exposure, 5-Fluorouracil and 6-Thioguanine. Cdk5-KD cells show altered basal S-Phase dynamics caused by a slower replication velocity and fewer active origins per megabase of DNA, however they show a faster recovery from an HU block. This was accompanied by impaired RPA-32 priming serine 29 and serine 33 phosphorylations and SMC1-Serine 966 phosphorylation as well as lower levels of DNA damage assessed by the alkaline Comet assay, gamma-H2AX signal intensity, RPA and Rad51 foci and RPA-32 serine 4 and serine 8 phosphorylation and levels of sister chromatid exchanges. In vitro kinase assays coupled with mass spectrometry showed that Cdk5 can phosphorylate RPA-32 on serines 23, 29, and 33. In addition lower Cdk5 levels were associated with longer metastasis free survival in breast cancer patients and lower cell survival in Cdk5 depleted breast tumor cells after treatment with IR and a PARP inhibitor. Taken together, these results show that Cdk5 is necessary for basal replication and replication stress checkpoint activation and opens up interesting clinical opportunities to enhance tumor cell killing in certain populations of breast cancer patients through agents that generate replication stress.

This dissertation thesis consists of a series of chapters that are interwoven by solving interesting biological problems, employing various computational methodologies. These techniques provide meaningful physical insights to promote the scientific fields of interest. Focus of chapter 1 concerns, the importance of computational tools like docking studies in advancing structure based drug design processes. This chapter also addresses the prime concerns like scoring functions, sampling algorithms and flexible docking studies that hamper the docking successes. Information about the different kinds of flexible dockings in terms of accuracy, time limitations and success studies are presented. Later the importance of Induced fit docking studies was explained in comparison to traditional MD simulations to predict the absolute binding modes. Chapter 2 and 3 focuses on understanding, how sickle cell disease progresses through the production of sickled hemoglobin and its effects on sickle cell patients. And how, hydroxyurea, the only FDA approved treatment of sickle cell disease acts to subside sickle cell effects. It is believed the primary mechanism of action is associated with the pharmacological elevation of nitric oxide in the blood, however, the exact details of this mechanism is still unclear. HU interacts with oxy and deoxyHb resulting in slow NO production rates. However, this did not correlate with the observed increase of NO concentrations in patients undergoing HU therapy. The discrepancy can be attributed to the interaction of HU competing with other heme based enzymes such as catalase and peroxidases. In these two chapters, we investigate the atomic level details of this process using a combination of flexible-ligand / flexible-receptor virtual screening (i.e. induced fit docking, IFD) coupled with energetic analysis that decomposes interaction energies at the atomic level. Using these tools we were able to elucidate the previously unknown substrate binding modes of a series of hydroxyurea analogs to human hemoglobin, catalase and the concomitant structural changes of the enzymes. Our results are consistent with kinetic and EPR measurements of hydroxyurea-hemoglobin reactions and a full mechanism is proposed that offers new insights into possibly improving substrate binding and/or reactivity. Finally in chapter 4, we have developed a 3D bioactive structure of O6-alkylguanine-DNA alkyltransferase (AGT), a DNA repair protein using Monte Carlo conformational search process. It is known that AGT prevents DNA damage, mutations and apoptosis arising from alkylated guanines. Various Benzyl guanine analouges of O6- methylguanine were tested for activity as potential inhibitors. The nature and position of the substitutions methyl and aminomethyl profoundly affected their activity. Molecular modeling of their interactions with alkyltransferase provided a molecular explanation for these results. The square of the correlation coefficient (R2 ) obtained between E-model scores (obtained from GLIDE XP/QPLD docking calculations) vs log(ED)values via a linear regression analysis was 0.96. The models indicate that the ortho-substitution causes a steric clash interfering with binding, whereas the meta-aminomethyl substitution allows an interaction of the amino group to generate an additional hydrogen bond with the protein. Using this model for virtually screening studies resulted in identification of seven lead compounds with novel scaffolds from National Cancer Institute Diversity Set2.

Οι αιμοσφαιρινοπάθειες, συμπεριλαμβανομένης της β-θαλασσαιμίας και της δρεπανοκυτταρικής αναιμίας, συγκαταλέγονται ανάμεσα στις πιο κοινές μονογονιδιακές αναταραχές παγκοσμίως, που χρήζουν αποτελεσματικής θεραπευτικής στρατηγικής. Σήμερα, η επανενεργοποίηση των γονιδίων της γ-σφαιρίνης φαίνεται να είναι μια ενδιαφέρουσα θεραπευτική προσέγγιση για τους ασθενείς που πάσχουν από β-τύπου αιμοσφαρινοπάθειες. / Hemoglobinopathies are amongst the most common single gene disorders worldwide, including beta-thalassemia and sicle cell disease (SCD), seeking for therapeutic intervention. The reactivation of the human gamma-globin genes is considered to be a therapeutic strategy for beta-type hemoglobinopathies patients.

Thesis (Ph. D.)--University of Washington, 2004. / Vita. Includes bibliographical references (leaves 71-80).

Thesis (Ph.D.)--University of Florida, 2004. / Typescript. Title from title page of source document. Document formatted into pages; contains 121 pages. Includes Vita. Includes bibliographical references.

Orientador: Eduardo Alves de Almeida / Coorientador: Claudia Regina Bonini Domingos / Banca: Gustavo Orlando Bonilla Rodriguez / Banca: Dorotéia Rossi Silva Souza / Resumo: A anemia falciforme (AF) apresenta fisiopatologia que envolve múltiplas alterações nos eritrócitos falcêmicos, episódios vasoclusivos, hemólise, ativação de mediadores inflamatórios, disfunção das células endoteliais e estresse oxidativo. Estes eventos dificultam o tratamento e culminam no desenvolvimento de manifestações como anemia, crises de dor e insuficiência de múltiplos órgãos. Portanto, o presente estudo objetivou, em portadores da AF, avaliar marcadores do estresse oxidativo e da capacidade antioxidante, correlacionando-os ao tratamento com hidroxiureia (HU), aos haplótipos da β-globina e aos polimorfismos da glutationa S-transferase (GSTT1, GSTM1 e GSTP1), em comparação com os resultados do grupo controle (GC). Os grupos estudados se compuseram de 48 indivíduos sem hemoglobinopatias (GC), pacientes com AF tratados com HU [AF (+HU), n=13] e pacientes não tratados [AF (-HU); n=15], após consentimento informado. Para a composição dos grupos utilizamos métodos citológicos, eletroforéticos, cromatográficos, moleculares e informações de prontuários. A pesquisa dos polimorfismos de GSTM1 e GSTT1 foi realizada por meio de PCR-MULTIPLEX, enquanto o polimorfismo GSTP1 por PCR-RFLP. Os parâmetros bioquímicos foram avaliados por meio de métodos espectrofotométricos [TBARS, TEAC, atividade das enzimas catalase (CAT) e GST] e cromatográficos [glutationa (GSH)]. Os níveis de Hb Fetal (Hb F) verificados no grupo AF (+HU) (10,9%) confirmaram seu já bem descrito efeito farmacológico da HU, porém, o grupo AF (-HU) também apresentou níveis elevados de Hb F (6,1%) que podem ter sido influenciados por fatores genéticos não considerados neste estudo. Encontramos maior frequência do haplótipo Bantu (48,2%), seguido pelo Benin (32,1%) e, também, haplótipo Camarões... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Sickle cell anemia (SCA) shows a pathophysiology that involves multiple changes in the sickle cell erythrocytes, vaso-occlusive episodes, hemolysis, activation of inflammatory mediators, endothelial cell dysfunction and oxidative stress. These events complicate the treatment and culminate in the manifestations development such as anemia, pain crises and multiorgan dysfunction. Therefore, the aim of this study was to evaluate, in SCA patients, oxidative stress and antioxidant capacity markers, correlating them to treatment with hydroxyurea (HU), the β-globin haplotypes and glutathione S-transferase polymorphisms (GSTT1, GSTM1 and GSTP1) in comparison to a control group (CG). The groups were composed of 48 individuals without hemoglobinopathies (CG), SCA patients treated with HU [AF (+ HU), n = 13] and untreated patients [AF (-HU), n = 15], after informed consent . For the groups composition we used cytological, electrophoretic, chromatographic and molecular methods and information from medical records. The GSTM1 and GSTT1 polymorphisms study was performed by multiplex PCR, while the GSTP1 polymorphism by PCR-RFLP. Biochemical parameters were measured using spectrophotometric methods [TBARS, TEAC and catalase (CAT) and GST activities] and chromatographic method [glutathione (GSH)]. The Fetal Hb (Hb F) levels observed in the SCA (+HU) group (10.9%) confirmed their already welldescribed pharmacological effect of HU, however, the SCA (-HU) group also had high Hb F levels (6.1%) that may have been influenced by genetic factors not targeted in this study. We found a higher frequency of Bantu haplotype (48.2%), followed by Benin (32.1%) and also Cameroon haplotype, rare in our population and 19.7% of atypical haplotypes. The Bantu haplotype presence was related to higher lipid peroxidation levels in patients, but also, it confered the differential response to HU treatment... (Complete abstract click electronic access below) / Mestre

Made available in DSpace on 2014-06-11T19:26:03Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-02-23Bitstream added on 2014-06-13T18:53:57Z : No. of bitstreams: 1 belinijunior_e_me_sjrp.pdf: 995631 bytes, checksum: d866ab976032b462435b7ca7899faf6d (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / A causa principal da doença falciforme (DF) é uma mutação pontual no gene β globina que codifica uma hemoglobina (Hb) com características diferentes da Hb normal, denominada de Hb S. Sua fisiopatologia multifacetada envolve múltiplas alterações nos eritrócitos falcêmicos, episódios vaso-oclusivos, hemólise, ativação de mediadores inflamatórios, disfunção das células endoteliais e estresse oxidativo. Com isso, há uma diminuição do fluxo sanguíneo e obstrução da microcirculação acarretando aos portadores, anemia, crises de dor e insuficiência de múltiplos órgãos. Visto que o excesso de espécies reativas de oxigênio é citotóxico modificando vários mecanismos celulares e, consequentemente, ocasionando danos aos órgãos, pretendeu-se avaliar a capacidade antioxidante e o estresse oxidativo em DF, correlacionando com os genótipos da DF, haplótipos do cluster β globina e o uso de medicação específica. Em um estudo longitudinal, foram avaliadas, para o Tempo 1 (T1) de análise, 69 amostras de sangue e, para o Tempo 2 (T2), 55 amostras de DF do Hemocentro de São José do Rio Preto (SJRP) e do Hemocentro da Santa Casa de São Paulo (SP). As amostras foram submetidas a testes eletroforéticos, cromatográficos e moleculares para a identificação dos genótipos da DF e haplótipos do cluster , bem como dosagens bioquímicas para determinação das espécies reativas ao ácido tiobarbitúrico (TBARS) e da capacidade antioxidante em equivalência ao Trolox (TEAC). Por meio da caracterização genotípica da DF, encontramos um maior número de Hb SS, tanto em SP quanto em SJRP. Houve maior frequência da interação Hb S/Beta talassemia em SJRP (10,9%) com as mutações características de países mediterrâneos como a CD39 e IVS-I-110. O haplótipo Bantu foi mais frequente nos dois grupos (60,6%); o haplótipo II esteve associado à mutação... / The main cause of sickle cell disease (SCD) is a single β-globin gene mutation that encodes a hemoglobin (Hb) with different characteristics from normal Hb, called Hb S. This disease is characterized by presenting a multifaceted pathophysiology which involves multiple changes in sickle erythrocytes, vaso-occlusive episodes, hemolysis, inflammatory mediators activation, oxidative stress and endothelical cells dysfunction. Thereby, there is a decrease in blood flow and obstruction of the microcirculation leading the patients to anemia, pain crises and failure of multiple organs. Since the excess of reactive oxygen species is cytotoxic, modifying various cellular mechanisms, and thus, causing damage to organs, the aim of this work was to assess the antioxidant capacity and oxidative stress in SCD, correlating with SCD genotypes, cluster β-globin haplotypes and specific medication. In a longitudinal study were evaluated for the T1: 69 samples of peripheral blood, and for the T2: 55 samples of the SCD patients from Blood Center of the São José do Rio Preto (SJRP) and the Blood Center of the Santa Casa Medical School of São Paulo (SP). We performed electrophoretics, chromatographic and molecular assays for SCD genotypes and cluster β-globin identification, such as biochemical measurements for the determination of thiobarbituric acid reactive species (TBARS) and Trolox equivalence antioxidant capacity (TEAC). Through SCD genetic characterization, we found a greater number of Hb SS in both SP and in SJRP. There was a higher frequency of interaction Hb S/Beta-thalassemia in SJRP (10.9%) with typical mutation of Mediterranean countries such as CD39 and IVS-I -110. The Bantu haplotype was more frequent in both groups (60.6%); the haplotype II was associated with CD39 mutation and the haplotype I with the IVS-I-110. We found a patient with Cameroon haplotype, rare in the Brasilian... (Complete abstract click electronic access below)

Made available in DSpace on 2014-06-11T19:26:05Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-02-16Bitstream added on 2014-06-13T20:33:44Z : No. of bitstreams: 1 silva_dgh_me_sjrp.pdf: 1493161 bytes, checksum: 255037ed9cce3a4f0a19a45e45dfd23d (MD5) / A anemia falciforme (AF) apresenta fisiopatologia que envolve múltiplas alterações nos eritrócitos falcêmicos, episódios vasoclusivos, hemólise, ativação de mediadores inflamatórios, disfunção das células endoteliais e estresse oxidativo. Estes eventos dificultam o tratamento e culminam no desenvolvimento de manifestações como anemia, crises de dor e insuficiência de múltiplos órgãos. Portanto, o presente estudo objetivou, em portadores da AF, avaliar marcadores do estresse oxidativo e da capacidade antioxidante, correlacionando-os ao tratamento com hidroxiureia (HU), aos haplótipos da β-globina e aos polimorfismos da glutationa S-transferase (GSTT1, GSTM1 e GSTP1), em comparação com os resultados do grupo controle (GC). Os grupos estudados se compuseram de 48 indivíduos sem hemoglobinopatias (GC), pacientes com AF tratados com HU [AF (+HU), n=13] e pacientes não tratados [AF (-HU); n=15], após consentimento informado. Para a composição dos grupos utilizamos métodos citológicos, eletroforéticos, cromatográficos, moleculares e informações de prontuários. A pesquisa dos polimorfismos de GSTM1 e GSTT1 foi realizada por meio de PCR-MULTIPLEX, enquanto o polimorfismo GSTP1 por PCR-RFLP. Os parâmetros bioquímicos foram avaliados por meio de métodos espectrofotométricos [TBARS, TEAC, atividade das enzimas catalase (CAT) e GST] e cromatográficos [glutationa (GSH)]. Os níveis de Hb Fetal (Hb F) verificados no grupo AF (+HU) (10,9%) confirmaram seu já bem descrito efeito farmacológico da HU, porém, o grupo AF (-HU) também apresentou níveis elevados de Hb F (6,1%) que podem ter sido influenciados por fatores genéticos não considerados neste estudo. Encontramos maior frequência do haplótipo Bantu (48,2%), seguido pelo Benin (32,1%) e, também, haplótipo Camarões... / Sickle cell anemia (SCA) shows a pathophysiology that involves multiple changes in the sickle cell erythrocytes, vaso-occlusive episodes, hemolysis, activation of inflammatory mediators, endothelial cell dysfunction and oxidative stress. These events complicate the treatment and culminate in the manifestations development such as anemia, pain crises and multiorgan dysfunction. Therefore, the aim of this study was to evaluate, in SCA patients, oxidative stress and antioxidant capacity markers, correlating them to treatment with hydroxyurea (HU), the β-globin haplotypes and glutathione S-transferase polymorphisms (GSTT1, GSTM1 and GSTP1) in comparison to a control group (CG). The groups were composed of 48 individuals without hemoglobinopathies (CG), SCA patients treated with HU [AF (+ HU), n = 13] and untreated patients [AF (-HU), n = 15], after informed consent . For the groups composition we used cytological, electrophoretic, chromatographic and molecular methods and information from medical records. The GSTM1 and GSTT1 polymorphisms study was performed by multiplex PCR, while the GSTP1 polymorphism by PCR-RFLP. Biochemical parameters were measured using spectrophotometric methods [TBARS, TEAC and catalase (CAT) and GST activities] and chromatographic method [glutathione (GSH)]. The Fetal Hb (Hb F) levels observed in the SCA (+HU) group (10.9%) confirmed their already welldescribed pharmacological effect of HU, however, the SCA (-HU) group also had high Hb F levels (6.1%) that may have been influenced by genetic factors not targeted in this study. We found a higher frequency of Bantu haplotype (48.2%), followed by Benin (32.1%) and also Cameroon haplotype, rare in our population and 19.7% of atypical haplotypes. The Bantu haplotype presence was related to higher lipid peroxidation levels in patients, but also, it confered the differential response to HU treatment... (Complete abstract click electronic access below)

Orientador: Claudia Regina Bonini Domingos / Banca: Dorotéia Rossi Silva Souza / Banca: Ivan de Lucena Angulo / Resumo: A causa principal da doença falciforme (DF) é uma mutação pontual no gene β globina que codifica uma hemoglobina (Hb) com características diferentes da Hb normal, denominada de Hb S. Sua fisiopatologia multifacetada envolve múltiplas alterações nos eritrócitos falcêmicos, episódios vaso-oclusivos, hemólise, ativação de mediadores inflamatórios, disfunção das células endoteliais e estresse oxidativo. Com isso, há uma diminuição do fluxo sanguíneo e obstrução da microcirculação acarretando aos portadores, anemia, crises de dor e insuficiência de múltiplos órgãos. Visto que o excesso de espécies reativas de oxigênio é citotóxico modificando vários mecanismos celulares e, consequentemente, ocasionando danos aos órgãos, pretendeu-se avaliar a capacidade antioxidante e o estresse oxidativo em DF, correlacionando com os genótipos da DF, haplótipos do cluster β globina e o uso de medicação específica. Em um estudo longitudinal, foram avaliadas, para o Tempo 1 (T1) de análise, 69 amostras de sangue e, para o Tempo 2 (T2), 55 amostras de DF do Hemocentro de São José do Rio Preto (SJRP) e do Hemocentro da Santa Casa de São Paulo (SP). As amostras foram submetidas a testes eletroforéticos, cromatográficos e moleculares para a identificação dos genótipos da DF e haplótipos do cluster , bem como dosagens bioquímicas para determinação das espécies reativas ao ácido tiobarbitúrico (TBARS) e da capacidade antioxidante em equivalência ao Trolox (TEAC). Por meio da caracterização genotípica da DF, encontramos um maior número de Hb SS, tanto em SP quanto em SJRP. Houve maior frequência da interação Hb S/Beta talassemia em SJRP (10,9%) com as mutações características de países mediterrâneos como a CD39 e IVS-I-110. O haplótipo Bantu foi mais frequente nos dois grupos (60,6%); o haplótipo II esteve associado à mutação... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The main cause of sickle cell disease (SCD) is a single β-globin gene mutation that encodes a hemoglobin (Hb) with different characteristics from normal Hb, called Hb S. This disease is characterized by presenting a multifaceted pathophysiology which involves multiple changes in sickle erythrocytes, vaso-occlusive episodes, hemolysis, inflammatory mediators activation, oxidative stress and endothelical cells dysfunction. Thereby, there is a decrease in blood flow and obstruction of the microcirculation leading the patients to anemia, pain crises and failure of multiple organs. Since the excess of reactive oxygen species is cytotoxic, modifying various cellular mechanisms, and thus, causing damage to organs, the aim of this work was to assess the antioxidant capacity and oxidative stress in SCD, correlating with SCD genotypes, cluster β-globin haplotypes and specific medication. In a longitudinal study were evaluated for the T1: 69 samples of peripheral blood, and for the T2: 55 samples of the SCD patients from Blood Center of the São José do Rio Preto (SJRP) and the Blood Center of the Santa Casa Medical School of São Paulo (SP). We performed electrophoretics, chromatographic and molecular assays for SCD genotypes and cluster β-globin identification, such as biochemical measurements for the determination of thiobarbituric acid reactive species (TBARS) and Trolox equivalence antioxidant capacity (TEAC). Through SCD genetic characterization, we found a greater number of Hb SS in both SP and in SJRP. There was a higher frequency of interaction Hb S/Beta-thalassemia in SJRP (10.9%) with typical mutation of Mediterranean countries such as CD39 and IVS-I -110. The Bantu haplotype was more frequent in both groups (60.6%); the haplotype II was associated with CD39 mutation and the haplotype I with the IVS-I-110. We found a patient with Cameroon haplotype, rare in the Brasilian... (Complete abstract click electronic access below) / Mestre