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Legionella pneumophila, an intracellular pathogen of protozoa, is well known for its dimorphic life cycle that alternates between the vegetative replicative form (RF) and highly infectious cyst-like form (CLF). To this date several virulence factors including LpRpoS, LpIHF, and the Dot/Icm secretion system have been found to be required for the survival of L. pneumophila in macrophage and protozoa. Here we have identified and characterised Lpg1967, an orthologue of Pseudomonas PsrA in L. pneumophila. PsrA (Lpg1967) was found to regulate the expression of previously known virulence factors such non-coding RNAs, RsmY/Z, RpoS, LpIHF, flagella and Dot/Icm Type IV secretion system. In addition, the ΔpsrA mutant strain was unable to establish Legionella-containing vacuole and thus displayed a severe growth defect in the U937 derived macrophage cell line. Thus, PsrA was found to play an important role in controlling the regulatory cascade governing virulence in L. pneumophila. / October 2014

DNA double-strand breaks (DSBs), where both strands of the DNA duplex are simultaneously fractured, are considered the most lethal type of DNA damage. The conserved Mre11-Rad50 DNA repair complex enables the catalytic activities of the Mre11 nuclease and the Rad50 ATPase to function together to coordinate the recognition and processing of DSBs prior to the recruitment of long-range end-resection machinery required to trigger the DSB repair by the homologous recombination (HR) pathway. Fast-scan atomic force microscopy (AFM) in fluid conditions was primarily used to explore the architectural arrangement, DNA binding and processing machinery of the Mre11-Rad50 complex from the thermophilic crenarchaeote Sulfolobus acidocaldarius. The structural analysis identified four distinct architectural arrangements and demonstrates the key role of the Rad50 zinc hooks in the oligomerisation of the complex. AFM imaging showed a dynamic and Velcro-like interplay between Mre11-Rad50 protein complexes and the DNA double-helix using the Rad50 coiled-coils in a novel mode of DNA binding. The complex appears to use the Rad50 zinc hook region to bind to and track along dsDNA for broken DNA-terminals. Furthermore, the present study shows that this archaeal complex can drive extensive ATP-dependent unwinding of DNA templates. It is the first time that such unwinding has been observed in a single molecule study. These observations reveal novel activities leading to the proposal of a new model for Mre11-Rad50 action during DSB repair. AFM was also used to visualise the structure and activity of the HerA-NurA protein complex, which has been predicted to combine the activity of the NurA nuclease and hexameric HerA-translocase to generate long single-stranded DNA overhangs essential for DSB repair by HR in archaea. The present data verify and clarify the presumed biological role of this complex. Overall, the present study provides new insights into the initial steps of DNA DSB repair by the HR pathway and, most importantly, the detection of the broken ends.

This dissertation focuses on rationalised DNA design as a tool for the discovery and development of new therapeutic entities, as well as understanding the biological function of DNA beyond the storage of genetic information. The study is comprised of two main areas of study: (i) the use of DNA as a coding unit to illustrate the relationship between code-diversity and dynamics of self-assembly; and (ii) the use of DNA as an active unit that interacts and regulates a target protein. In the study of DNA as a coding unit in code-diversity and dynamics of self-assembly, we developed the DNA-Based Diversity Modelling and Analysis (DDMA) method. Using Polymerase Chain Reaction (PCR) and Real Time Polymerase Chain Reaction (RT-PCR), we studied the diversity and evolution of synthetic oligonucleotide populations. The manipulation of critical conditions, with monitoring and interpretation of their effects, lead to understanding how PCR amplification unfolding could reshape a population. This new take on an old technology has great value for the study of: (a) code-diversity, convenient in a DNA-based selection method, so semi-quantitation can evaluate a selection development and the population\'s behaviour can indicate the quality; (b) self-assembly dynamics, for the simulation of a real evolution, emulating a society where selective pressures direct the population's adaptation; and (c) development of high-entropy DNA structures, in order to understand how similar unspecific DNA structures are formed in certain pathologies, such as in auto-immune diseases. To explore DNA as an active unit in Tumour Necrosis Factor α (TNF-α) interaction and activity modulation, we investigate DNA's influence on its spatial conformation by physical environment regulation. Active TNF-α is a trimer and the protein-protein interactions between its monomers are a promising target for drug development. It has been hypothesised that TNF-α forms a very intricate network after its activation between its subunits and receptors, but the mechanism is still not completely clear. During our research, we estimate the non-specific DNA binding to TNF-α in the low micro-molar range. Cell toxicity assays confirm this interaction, where DNA consistently enhances TNF-α's cytotoxic effect. Further binding and structural studies lead to the same conclusion that DNA binds and interferes with TNF-α structure. From this protein-DNA interaction study, a new set of tools to regulate TNF-α's biological activity can be developed and its own biology can be unveiled.

Esta tese trata do desenvolvimento de métodos bioanalíticos, baseados na técnica de eletroforese capilar, para duas aplicações distintas: a análise de complexos formados pela ligação entre proteínas e DNA e detecção e monitoramento de hidrólise de GTP catalisada por enzimas. No primeiro capítulo descreve-se uma investigação sobre a viabilidade de ensaios tipo EMSA (?electrophoretic mobility shift assay?) em chips com microcanais para eletroforese. Os fatores de transcrição purificados c-Jun(AP1) e p-50(NFkB) foram usados nos estudos de ligação a sondas de DNA fita dupla contendo as seqüências consenso de ligação dos fatores AP1, NFkB e AP2. As sondas de DNA sintéticas continham como modificação a marcação com o corante fluorescente Cy5 ligado à extremidade 5?, sendo que as mesmas seqüências não marcadas foram usadas para experimentos de competição. Ensaios tipo EMSA em chip puderam ser realizados em cerca de 2 h com baixo consumo de amostra e sem a necessidade de usar marcação com material radioativo. A sondas de DNA e os complexos formados nas reações de ligação foram analisados no Bioanalyzer usando tanto o procedimento padrão para a análise de DNA quanto um protocolo modificado. Nesta modificação não foi usado corante de intercalação mas 4,9 nM de Cy5-dCTP que foi adicionado ao gel, permitindo apenas a detecção de DNA previamente marcado. Apesar da necessidade de ajustes no método para cada proteína testada, foi mostrado o potencial de se substituir o método de EMSA em gel por métodos baseados em eletroforese em chip. Um experimento de competição foi realizado com sucesso mostrando a ligação do fator de transcrição p-50 à sonda contendo a seqüência consenso NFkB. Este experimento foi considerado como prova de princípio para a hipótese estudada. No segundo capítulo relata-se o desenvolvimento de um método para a detecção e monitoramento in vitro de atividade nucleotídeo-trifosfatase. O método que se mostrou robusto e reprodutível foi aplicado para investigar a atividade GTPase de uma proteína recombinante contendo o domínio catalítico de uma septina humana SEPT4 / Bradeiona (GST-rDGTPase). O exemplo de aplicação demonstra que a técnica de eletroforese capilar pode substituir o método tradicionalmente usado com marcação radioativa para detecção de atividade GTPase inclusive em estudos de cinética enzimática. Os parâmetros cinéticos determinados para a GST-rDGTPase foram: vmax = 1.7 μ M min-1 ± 0.1 and Km = 1.0 mM ± 0.3; kcat = 9 x 10-3 SM-1. O efeito de co-fatores como Mg2+ and Mn2+ também foi estudado. O método analítico descrito também se mostrou útil para a análise de di- e trifosfatos de outros nucleotídeos. / This Thesis concerns on the development of capillary electrophoresis-based bioanalytical methods for two distinct applications: DNA-protein binding analysis and monitoring of enzyme-catalyzed GTP hydrolysis. In the first chapter, the feasibility of on-chip electrophoretic mobility-shift assays (EMSA) is investigated. Purified transcription factors c-Jun(AP1) and p-50(NFkB) were used for binding studies to dsDNA probes containing the consensus sequences from AP1, NFkB and AP2 regulatory sequences. DNA probes were modified at the 5?-end with the Cy5 and unlabeled oligos were used for competition experiments. On-chip-EMSA could be carried out within ca. 2 h with low sample consumption and no need to handle radioactive material. Both, the dsDNA probes and the shifted oligos from binding reactions were analyzed on the ?2100 Bioanalyzer? using either, the standard procedure for DNA analysis or a modified protocol, in which no intercalating dye was used. Instead, 4.9 nM Cy5-dCTP was added to the gel matrix allowing the detection of only Cy5-labeled DNA. Despite the need of specific adjustments for each protein, we have shown the potential for replacing slab gel-based EMSA for on-chip methods. A competition experiment to show sequence specific binding of the transcription factor p-50 to the consensus sequence NFkB is presented as a proof of principle. In chapter II, a capillary electrophoresis-based method for in vitro detection and monitoring of nucleotide-triphosphatase activity is described. This robust and reproducible method was used to investigate GTPase activity of a recombinant protein construct containing the catalytic domain of Human SEPT4 /Bradeion ? (GST-rDGTPase). The application example demonstrates that the capillary electrophoresis technique can replace classical radioactive methods for GTPase activity assays and may be used as a routine analytical tool. Enzyme kinetics of GST-rDGTPase was studied and yielded the following kinetic parameters: vmax = 1.7 μM min-1 ± 0.1 and Km = 1.0 mM ± 0.3; kcat = 9 x 10-3 s-1. In addition the effect of co-factors such as Mg2+ and Mn2+ in the catalytic activity was investigated. The described analytical method was also shown to be useful to analyze di- and triphosphated forms of other nucleotides.

Dans cette thèse, nous avons construit et optimisé des méthodes de microscopie de fluorescence super-résolue stochastique, polarisée et quantitative qui nous permettent d'imager l'orientation moléculaire dans des environnements dynamiques et statiques a l’échelle de la molécule unique et avec une résolution nanoscopique. En utilisant un montage de microscopie super-résolue à lecture stochastique en combinaison avec une détection polarisée, nous avons pu reconstruire des images d'anisotropie de fluorescence avec une résolution spatiale de 40 nm. En particulier, nous avons pu imager l'ordre orientationnel d'assemblages biomoléculaires et cellulaires. Pour l'imagerie cellulaire, nous avons pu étudier la capacité d'étiquettes de marquer fluorophoresde reporter quantifier l'orientation moléculaire dans l'actine et les microtubules dans des cellules fixées. Nous avons également mis à profit la meilleure résolution et la détection polarisée pour étudier l'ordre moléculaire d’agrégats d’amyloïdes a l’échelle nanoscopique. Enfin, nous avons étudié l'interaction de la protéine de réparation RAD51 avec l'ADN par microscopie de fluorescence polarisée super-résolue pour quantifier l'ordre orientationnel de l'ADN et de la protéine RAD51 afin de comprendre la recombinaison homologue du mécanisme de réparation de l'ADN. / .In this thesis we built and optimized quantitative polarized stochastic super-resolution fluorescence microscopy techniques that enabled us to image molecular orientation behaviors in static and dynamic environments at single molecule level and with nano-scale resolution. Using a scheme of stochastic read-out super resolution microscopy in combination with polarized detection, we can reconstruct fluorescence anisotropy images at a spatial resolution of 40 nm. In particular, we have been able to use the techniques to quantify the molecular orientationalorder in cellular and bio-molecular assemblies. For cellular imaging, we could quantify the ability of fluorophore labels to report molecular orientation of actin and microtubules in fixed cells. Furthermore, we used the improvements of resolution and polarization detection to study molecular order of amyloid aggregates at a nanoscopic scale. Also, we studied repair protein RAD51` s interaction with DNA by using dual color polarized fluorescence microscopy, to quantify the orientational order of DNA and RAD51 to understand the homologous recombination of DNA repair mechanism.

Orientador: Maria Isabel Nogueira Cano / Resumo: Entre as espécies do gênero Leishmania estão os protozoários que causam leishmaniose, uma doença tropical negligenciada endêmica em muitos países, incluindo o Brasil. Métodos de controle e tratamento ainda são ineficientes e a resistência a drogas é um desafio. Por isso, pesquisas para entender melhor a biologia molecular desses parasitos são encorajadas. Uma possível estratégia para isso, é o estudo dos telômeros, estrutura fundamental para a homeostase do genoma. Os telômeros são estruturalmente diferentes do resto do cromossomo, e contam com proteínas específicas que realizam sua manutenção. A Replication Protein A subunit 1 (RPA-1) é uma proteína que interage de DNA de simples fita que tem diversas funções relacionadas com o metabolismo do DNA eucarioto, incluindo os telômeros. A RPA-1 é parte de um complexo heterotrimérico conservado nos eucariotos, incluindo Leishmania spp.. Recentemente nós mostramos por modelagem molecular que a estrutura terciária da LaRPA-1 difere dos seus ortólogos em humanos e leveduras, além de mostrar interações específicas nos telômeros dos parasitos, que na ausência de homólogos canônicos para telomere-end binding protein (TEP) elegem a LaRPA-1 como um potencial candidato para essa função. Neste trabalho, avaliamos a capacidade da LaRPA-1 como uma TEP, cujo papel principal é proteger a extremidade 3' dos telômeros de ataques por exonucleases. Uma busca estrutural por proteínas que compartilham com as TEP domínios de interação proteína-DNA, mos... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Among the protozoa parasites of the Leishmania genus are the causative agents of leishmaniasis, a neglected tropical disease endemic in many countries, including Brazil. Disease control and treatment are still inefficient and parasite drug resistance is a challenge. Therefore, efforts for the establishment of intensive research to better understand the molecular biology of these parasites are encouraged. One possible strategy is to study parasite telomeres, a vital chromosome structure important to maintain genome homeostasis. Telomeres are significantly different from the rest of the chromosome and are associated with proteins involved in their maintenance. Replication Protein A subunit 1 (RPA1), a single-stranded DNA-binding protein that plays multiple roles in eukaryotic DNA metabolism, including telomeres, is part of a conserved heterotrimeric complex which is present in most eukaryotes including Leishmania spp. Recently, using molecular dynamics simulations we have shown that the tertiary structure of LaRPA-1 differs from human and yeast RPA-1 and that it also shows parasitespecific interactions with telomeric DNA. In the absence of real homologues to telomere-end binding proteins, LaRPA-1 could be considered a potential candidate. If LaRPA-1 is a telomere-end binding protein, one of its main role would be to protect the telomeric 3`-end termini from nuclease attack. A structural search for proteins that share with the TEP domains of protein-DNA interaction, showed that ... (Complete abstract click electronic access below) / Mestre

<p>The mouse xenobiotic-inducible <i>Cyp2a5</i> gene is both transcriptionally and posttranscriptionally regulated. One of the most potent <i>Cyp2a5</i> inducers, the hepatotoxin pyrazole, increases the CYP2A5 mRNA half-life. The induction is accomplished through the interaction of a pyrazole-inducible protein with a 71 nt long, putative hairpin-loop region in the 3' UTR of the CYP2A5 mRNA.</p><p>The aims of this thesis have been to identify the pyrazole-inducible protein, to investigate its role in the <i>Cyp2a5</i> expression and the significance of the 71 nt hairpin-loop region for the <i>Cyp2a5</i> expression, and to examine a possible coupling between transcriptional and posttranscriptional processes in <i>Cyp2a5</i> expression.</p><p>The pyrazole-inducible protein was identified as the heterogeneous nuclear ribonucleoprotein (hnRNP) A1. Studies performed in mouse primary hepatocytes overexpressing hnRNP A1, and in mouse erythroleukemia derived cells lacking hnRNP A1, revealed that the 71 nt region in the 3' UTR of the CYP2A5 mRNA is essential for <i>Cyp2a5</i> expression.</p><p>The hnRNP A1 is a multifunctional nucleocytoplasmic shuttling protein, with the ability to bind both RNA and DNA. These properties make it an interesting candidate mediating a coupling between nuclear and cytoplasmic gene regulatory events, which was investigated for the <i>Cyp2a5</i>. In conditions of cellular stress hnRNP A1 translocates from the nucleus to the cytoplasm. The accumulation of cytoplasmic hnRNP A1 after RNA polymerase II transcription inhibition, resulted in an increased binding of hnRNP A1 to the CYP2A5 mRNA, parallel with a stabilization of the CYP2A5 mRNA.</p><p>Treating primary mouse hepatocytes with phenobarbital (PB), a <i>Cyp2a5</i> transcriptional inducer, resulted in a mainly nuclear localization of the hnRNP A1. Electrophoretic mobility shift assays with nuclear extracts from control or PB-treated mice, revealed that hnRNP A1 interacts with two regions in the <i>Cyp2a5</i> proximal promoter, and that the interaction to one of the regions was stimulated by PB treatment.</p><p>In conclusion, the change in hnRNP A1 subcellular localization after transcriptional inhibition or activation, together with the effects on the interaction of hnRNP A1 with the CYP2A5 mRNA and <i>Cyp2a5</i> promoter, suggest that hnRNP A1 could couple the nuclear and cytoplasmic events of the <i>Cyp2a5</i> expression.</p><p>The presented studies are the first showing involvement of an hnRNP protein in the regulation of a <i>Cyp</i> gene. Moreover, it is the first time an interconnected transcriptional and posttranscriptional regulation has been suggested for a member of the <i>Cyp</i> gene family.</p>

The mouse xenobiotic-inducible Cyp2a5 gene is both transcriptionally and posttranscriptionally regulated. One of the most potent Cyp2a5 inducers, the hepatotoxin pyrazole, increases the CYP2A5 mRNA half-life. The induction is accomplished through the interaction of a pyrazole-inducible protein with a 71 nt long, putative hairpin-loop region in the 3' UTR of the CYP2A5 mRNA. The aims of this thesis have been to identify the pyrazole-inducible protein, to investigate its role in the Cyp2a5 expression and the significance of the 71 nt hairpin-loop region for the Cyp2a5 expression, and to examine a possible coupling between transcriptional and posttranscriptional processes in Cyp2a5 expression. The pyrazole-inducible protein was identified as the heterogeneous nuclear ribonucleoprotein (hnRNP) A1. Studies performed in mouse primary hepatocytes overexpressing hnRNP A1, and in mouse erythroleukemia derived cells lacking hnRNP A1, revealed that the 71 nt region in the 3' UTR of the CYP2A5 mRNA is essential for Cyp2a5 expression. The hnRNP A1 is a multifunctional nucleocytoplasmic shuttling protein, with the ability to bind both RNA and DNA. These properties make it an interesting candidate mediating a coupling between nuclear and cytoplasmic gene regulatory events, which was investigated for the Cyp2a5. In conditions of cellular stress hnRNP A1 translocates from the nucleus to the cytoplasm. The accumulation of cytoplasmic hnRNP A1 after RNA polymerase II transcription inhibition, resulted in an increased binding of hnRNP A1 to the CYP2A5 mRNA, parallel with a stabilization of the CYP2A5 mRNA. Treating primary mouse hepatocytes with phenobarbital (PB), a Cyp2a5 transcriptional inducer, resulted in a mainly nuclear localization of the hnRNP A1. Electrophoretic mobility shift assays with nuclear extracts from control or PB-treated mice, revealed that hnRNP A1 interacts with two regions in the Cyp2a5 proximal promoter, and that the interaction to one of the regions was stimulated by PB treatment. In conclusion, the change in hnRNP A1 subcellular localization after transcriptional inhibition or activation, together with the effects on the interaction of hnRNP A1 with the CYP2A5 mRNA and Cyp2a5 promoter, suggest that hnRNP A1 could couple the nuclear and cytoplasmic events of the Cyp2a5 expression. The presented studies are the first showing involvement of an hnRNP protein in the regulation of a Cyp gene. Moreover, it is the first time an interconnected transcriptional and posttranscriptional regulation has been suggested for a member of the Cyp gene family.

Submitted by Gabriel Arantes Galvão Dias dos Santos (arantes_gabriel@hotmail.com) on 2018-05-14T15:21:05Z No. of bitstreams: 1 Dissertação pós defesa.pdf: 3303163 bytes, checksum: 01b3ceab15b6016a9aaca279d39ebdc5 (MD5) / Approved for entry into archive by Sulamita Selma C Colnago null (sulamita@btu.unesp.br) on 2018-05-14T17:46:54Z (GMT) No. of bitstreams: 1 santos_gagd_me_bot.pdf: 3303163 bytes, checksum: 01b3ceab15b6016a9aaca279d39ebdc5 (MD5) / Made available in DSpace on 2018-05-14T17:46:54Z (GMT). No. of bitstreams: 1 santos_gagd_me_bot.pdf: 3303163 bytes, checksum: 01b3ceab15b6016a9aaca279d39ebdc5 (MD5) Previous issue date: 2018-04-27 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Entre as espécies do gênero Leishmania estão os protozoários que causam leishmaniose, uma doença tropical negligenciada endêmica em muitos países, incluindo o Brasil. Métodos de controle e tratamento ainda são ineficientes e a resistência a drogas é um desafio. Por isso, pesquisas para entender melhor a biologia molecular desses parasitos são encorajadas. Uma possível estratégia para isso, é o estudo dos telômeros, estrutura fundamental para a homeostase do genoma. Os telômeros são estruturalmente diferentes do resto do cromossomo, e contam com proteínas específicas que realizam sua manutenção. A Replication Protein A subunit 1 (RPA-1) é uma proteína que interage de DNA de simples fita que tem diversas funções relacionadas com o metabolismo do DNA eucarioto, incluindo os telômeros. A RPA-1 é parte de um complexo heterotrimérico conservado nos eucariotos, incluindo Leishmania spp.. Recentemente nós mostramos por modelagem molecular que a estrutura terciária da LaRPA-1 difere dos seus ortólogos em humanos e leveduras, além de mostrar interações específicas nos telômeros dos parasitos, que na ausência de homólogos canônicos para telomere-end binding protein (TEP) elegem a LaRPA-1 como um potencial candidato para essa função. Neste trabalho, avaliamos a capacidade da LaRPA-1 como uma TEP, cujo papel principal é proteger a extremidade 3' dos telômeros de ataques por exonucleases. Uma busca estrutural por proteínas que compartilham com as TEP domínios de interação proteína-DNA, mostrou que no genoma de Leishmania spp. não existem homólogos estruturais para as mesmas. Aqui mostramos por diferentes abordagens que a LaRPA-1 tem a capacidade de interagir com no mínimo uma repetição telomérica e também é capaz de proteger in vitro a simples fita telomérica rica em G (5’ TTAGGG 3’) da digestão por Exonuclease I bacteriana cuja atividade é no sentido 3’-5’. Somando esses dados, com dados anteriores que mostram que a LaRPA-1 tem preferência pela fita telomérica rica em G e o fato dela ter sido co-purificada com a atividade de telomerase sugerem fortemente que ela está diretamente relacionada com a manutenção da maquinaria telomérica, podendo inclusive ser considerada a principal ligante de simples fita telomérica rica em G (3’ G-overhang) em Leishmania spp. / Among the protozoa parasites of the Leishmania genus are the causative agents of leishmaniasis, a neglected tropical disease endemic in many countries, including Brazil. Disease control and treatment are still inefficient and parasite drug resistance is a challenge. Therefore, efforts for the establishment of intensive research to better understand the molecular biology of these parasites are encouraged. One possible strategy is to study parasite telomeres, a vital chromosome structure important to maintain genome homeostasis. Telomeres are significantly different from the rest of the chromosome and are associated with proteins involved in their maintenance. Replication Protein A subunit 1 (RPA1), a single-stranded DNA-binding protein that plays multiple roles in eukaryotic DNA metabolism, including telomeres, is part of a conserved heterotrimeric complex which is present in most eukaryotes including Leishmania spp. Recently, using molecular dynamics simulations we have shown that the tertiary structure of LaRPA-1 differs from human and yeast RPA-1 and that it also shows parasitespecific interactions with telomeric DNA. In the absence of real homologues to telomere-end binding proteins, LaRPA-1 could be considered a potential candidate. If LaRPA-1 is a telomere-end binding protein, one of its main role would be to protect the telomeric 3`-end termini from nuclease attack. A structural search for proteins that share with the TEP domains of protein-DNA interaction, showed that in the genome of Leishmania spp. there are no structural homologues for them. In this work, we show by different methods, that in vitro LaRPA-1 can bind at least one telomeric repeat and it can also protect the telomeric G-rich sequence (5’ TTAGGG 3’) from the bacterial 3’-5’Exonuclease I digestion. These data compiled to previous data showing that LaRPA-1 preferentially binds the G-rich telomeric DNA and that it co-purifies with telomerase activity strongly suggest that LaRPA-1 is directly involved with parasite telomere maintenance and, possibly, is the main G-rich single-stranded (3’ G-overhang) telomere-binding protein in Leishmania spp.