Würzburg, Univ., Diss., 2003.

Tierärztl. Hochsch., Diss., 2003--Hannover.

Techn. Universiẗat, Diss., 2004--Berlin.

Resumo: O câncer de mama é o tipo mais comum e a principal causa de morte por câncer entre as mulheres no mundo todo. As metástases contribuem com 90% das causas de morte por câncer. O estudo de novos marcadores moleculares de câncer estão em amplo e atual desenvolvimento, tanto para diagnóstico como prognóstico da doença. Diversos estudos tem mostrado o papel de eventos epigenéticos entre eles a metilação do DNA, em regiões promotoras de genes como um evento importante no processo de tumorigênese em diversos tipos de câncer. No câncer de mama já foram descritos um grande número de genes envolvidos com controle da proliferação, adesão e migração celular que podem ser silenciados por mecanismos epigenéticos. O gene CXCL12 codifica para uma quimiocina ou citocina quimiotática, que é comprovadamente silenciado por metilação de sua região promotora em câncer gástrico, de cólon intestinal e de mama. O produto deste gene esta envolvido no processo migratório como molécula ligante de células tumorais que superexpressam o receptor CXCR4 e que migram e colonizam secundariamente pulmões ossos, fígado ou linfonodos que tem altos níveis de CXCL12. Nesse trabalho estudamos detalhadamente a região promotora do gene CXCL12 que contém 5 regiões ricas em dinucleotídeos CpGs (ilhas de CpG). As ilhas de CpG 1 ,2, 3 e 5 foram amplificadas, clonadas e sequenciadas a partir de DNA de sete linhagens tumorais de mama. A ausência ou presença de metilação do DNA de cada região foi correlacionado com a presença ou ausência de expressão do gene CXCL12. A ilha de CpG 1 apresentou-se hipermetilada em linhagens que expressam CXCL12, sendo assim essa região parece não participar do processo de silenciamento desse gene nessas linhagens. A ilha de CpG 2 que contém 1346 pb foi analisada em 2 partes sendo que a parte inicial que contém o nucleotídeo +1 apresentou-se fortemente correlacionada com a expressão do gene. Somente uma linhagem imortalizada, a HB4a apresentou 53,25% de metilação global, o que poderia interferir na expressão do gene. A segunda parte da ilha 2, assim como a ilha de CpG 3 e 5 apresentaram-se hipermetiladas nas linhagens que expressam o gene, e portanto essas regiões provavelmente não interferem na expressão do gene CXCL12 nessas linhagens tumorais. Por outro lado a ilha de CpG 4 localizada a cerca de 1500 pb de distância do início de transcrição e fora da provável região promotora, apresentou-se pouco metilada nas linhagens que expressam o gene e hipermetilada nas linhagens que não expressam CXCL12. Concluímos que as regiões denominadas ilhas de CpG 1 o terço final da Ilha de CpG 2, ilha de CpG 3 e 5 estão metiladas em linhagens que expressam o gene CXCL12 e assim sendo, não são importantes no processo de regulação da expressão desse gene. A Ilha de CpG 4 é a única região diferencialmente metilada e que parece atuar de modo a sinalizar o silenciamento do gene CXCL12.

DNA vaccines offer unique promise as a means of generating immunity against infectious and malignant disease. Unfortunately a number of obstacles, including rapid degradation of naked plasmid DNA (pDNA), poor cellular uptake by antigen presenting cells (APCs) and subsequent low levels of gene expression have limited the ability of DNA vaccines to raise sufficient immune responses towards the target antigen. This thesis is focused on investigating the immunostimulatory potential of liposomal nanoparticulate (LN) formulations of pDNA (stabilized plasmid lipid particles; SPLP) and cytosine-guanine oligodeoxynucleotides (CpG-ODN; LN CpG-ODN), and examining their ability to act together as a non-viral DNA vaccine in attempt to address the shortcomings of current DNA vaccine approaches. One focus of this thesis concerns investigating the immunostimulatory activity of LN formulations of CpG-ODN and pDNA. It is shown that despite dramatic differences in pharmacokinetics and biodistribution of LN CpG-ODN following intravenous (i.v.) and subcutaneous (s.c.) administration the resultant immune response is very similar, which is concluded to be due to the intrinsic ability of APCs to sequester LN CpG- ODN. In addition, it is demonstrated that lipid encapsulation dramatically enhances the immunostimulatory potential of pDNA and it is observed that SPLP maintains immunostimulatory activity in Toll-like receptor 9 (TLR9) knock-out mice. Together theses findings highlight the need for DNA-based therapies to consider both TLR9-dependent and -independent immunostimulatory activities of pDNA when constructing non-viral vectors. Furthermore, a new role for SPLP as a non-viral gene delivery vehicle for the generation of a systemically administered genetic vaccine in the presence of LN CpG-ODN is introduced. The ability of vaccination with SPLP to act prophylactically, to protect mice from tumour challenge, and therapeutically, in a novel vaccination strategy where the antigen is expressed at the tumour site as a result of SPLP-mediated transfection, is explored, demonstrating that in the presence of LN CpG-ODN SPLP possesses potential as a non-viral delivery system for DNA-based cancer vaccines. In summary, this work represents a substantial advance in the understanding of the immunostimulatory potential of both SPLP and LN CpG-ODN and provides insight into their ability to work together as a non-viral DNA vaccine. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

With the advent of high-throughput genomics, biological big data brings challenges to scientists in handling, analyzing, processing and mining this massive data. In this new interdisciplinary field, diverse theories, methods, tools and knowledge are utilized to solve a wide variety of problems. As an exploration, this dissertation project is designed to combine concepts and principles in multiple areas, including signal processing, information-coding theory, artificial intelligence and cloud computing, in order to solve the following problems in computational biology: (1) comparative gene structure detection, (2) DNA sequence annotation, (3) investigation of CpG islands (CGIs) for epigenetic studies. Briefly, in problem #1, sequences are transformed into signal series or binary codes. Similar to the speech/voice recognition, similarity is calculated between two signal series and subsequently signals are stitched/matched into a temporal sequence. In the nature of binary operation, all calculations/steps can be performed in an efficient and accurate way. Improving performance in terms of accuracy and specificity is the key for a comparative method. In problem #2, DNA sequences are encoded and transformed into numeric representations for deep learning methods. Encoding schemes greatly influence the performance of deep learning algorithms. Finding the best encoding scheme for a particular application of deep learning is significant. Three applications (detection of protein-coding splicing sites, detection of lincRNA splicing sites and improvement of comparative gene structure identification) are used to show the computing power of deep neural networks. In problem #3, CpG sites are assigned certain energy and a Gaussian filter is applied to detection of CpG islands. By using the CpG box and Markov model, we investigate the properties of CGIs and redefine the CGIs using the emerging epigenetic data. In summary, these three problems and their solutions are not isolated; they are linked to modern techniques in such diverse areas as signal processing, information-coding theory, artificial intelligence and cloud computing. These novel methods are expected to improve the efficiency and accuracy of computational tools and bridge the gap between biology and scientific computing.

Orientador: Flavia Lombardi Lopes / Banca: Silvia Helena Venturoli Perri / Banca: José Fernando Garcia / Banca: Ricardo da Fonseca / Banca: José bento Sterman Ferraz / Resumo: O objetivo desse estudo foi identificar polimorfismos de nucleotídeo único (SNPs) potencialmente sujeitos a controle epigenético exercido por metilação do DNA via seus envolvimentos na criação, remoção ou deslocamento de sítios CpG (meSNPs) e a partir de tal identificação criar um banco de dados para meSNPs, bem como determinar a possível associação desses marcadores com ilhas CpG (CGIs) e com o perfil metilacional de tecidos submetidos ao ensaio de recuperação de ilhas CpG metiladas combinado com plataformas de sequenciamento de nova geração (MIRA-seq) em bovinos. Usando as variantes anotadas para os SNPs identificados no Run5 do projeto 1000 Bull Genomes e a sequência genômica bovina de referência UMD3.1.1, identificamos e anotamos 12.836.763 meSNPs de acordo com o padrão de variação criado por cada SNP em um sítio CpG. Também analisamos a distribuição genômica desses meSNPs, sendo a maioria deles localizados em regiões intergênicas (68,00%) e intrônicas (26,32%). Globalmente, os meSNPs representam 22,53% dos 56.969.697 SNPs descritos na base de dados e 12,35% deles estão localizados em CGIs. Comparando o número observado com o número esperado de meSNPs nas CGIs e nos tecidos submetidos ao MIRA-seq, verificamos um enriquecimento médio (P<0,01) para meSNPs de 2,47 vezes em CGIs relaxadas e 1,90 vezes em CGIs rigorosas. Nos tecidos, o enriquecimento foi de 1,52 vezes em longissimus dorsi e 2,09 vezes em intestino delgado. Dez meSNPs com metilação diferencial, sendo 1 em longi... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The aim of this study was to identify single nucleotide polymorphisms (SNPs) potentially subject to epigenetic control exerted by DNA methylation via their involvement in creating, removing or displacement CpG sites (meSNPs) and from this identification create a database for meSNPs, as well as to determine its possible association with CpG islands (CGIs) and the methylation profile of tissues submitted to the methylated-CpG island recovery assay combined with next generation sequencing platforms (MIRA-seq) in cattle. Using the variant annotations for SNPs identified in Run5 of the 1000 bull genomes project and the UMD3.1.1 bovine reference genome sequence assembly, we identified and classified 12,836,763 meSNPs according to the pattern of variation caused at the CpG site. We have also analyzed the genomic distribution of the meSNPs, with the majority being located in intergenic regions (68.00%) and then in introns (26.32%) and the remainder distributed among proximal promoters (3.93%), coding regions (1.27%), untranslated regions (UTRs) (0.29%), non-coding RNAs (0.11%) and splice regions (0.08%). Overall, meSNPs represent 22.53% of 56,969,697 SNPs described in the database of which 12.35% are located in CGIs. Comparing the observed number with the expected number of meSNPs in the CGIs and tissues submitted to the MIRAseq we found a mean enrichment (P<0.01) for meSNPs of 2.47 times in the relaxed CGIs and 1.90 times in the strict CGIs. In the tissues the enrichment was of 1.52... (Complete abstract click electronic access below) / Doutor

Le cerveau et la moelle épinière adultes sont câblés pour traiter l’information sensorielle et la transformer en patrons d’activité cohérents qui forment la base de la perception du monde extérieur et de la motricité. Ces connexions très précises, loin d’être complètement établies à la naissance, sont affinées par l’activité électrique. Des activités spontanées (AS) peuvent être générées en l’absence de stimulation extrinsèque au système considéré. Au niveau de la moelle épinière, les AS sont responsables des mouvements spontanés et des tremblements myocloniques observés chez tous les mammifères immatures y compris chez l’humain. Elles constituent en quelque sorte les premiers pas du réseau locomoteur, ce qui signifie d’une part, qu’elles apparaissent avant l’émergence d’une activité de type locomotrice, et d’autre part, qu’elles participent à la mise en place et au raffinement d’un réseau moteur fonctionnel. La locomotion est générée par des réseaux de neurones localisés au niveau de la moelle épinière lombaire. Ces neurones constituent le générateur de rythme locomoteur (ou CPG, pour « central pattern generator »). Contrairement aux AS, l’activité locomotrice est déclenchée par une stimulation du réseau. Le patron locomoteur de décharges rythmiques est alterné de chaque côté de la moelle spinale, et entre les racines lombaires qui innervent des muscles extenseurs et fléchisseurs. / The mature brain and spinal cord are precisely wired to process sensory information into coherent patterns of activity that form the basis of our perception and motor behaviors. This precise wiring is not fully developed at birth. The pattern of connections that emerges during prenatal development only roughly approximates the final wiring. This initially coarse pattern of connections is subsequently refined by activity-dependent mechanisms that match precisely the presynaptic neurons to their appropriate target cells. In spinal cord, spontaneous activity (SA) is responsible for spontaneous limb movements and myoclonic twitching observed in all immature mammals, including human babies. SA can be seen as the first steps of the locomotor network since its participates in the development of the locomotor system.Locomotion is produced by neural networks located in the spinal cord (Central Pattern Generators (CPGs). Activation of CPGs, evokes a fictive locomotor pattern consisting of alternation between the motor bursts on the left and right sides of the spinal cord, as well as alternation between flexor and extensor bursts on the same side. Operation of neural networks depends on the balance between excitation and inhibition. At early stages of development, neuronal assemblies are hyperexcitable mainly because of GABA and glycine, the major inhibitory neurotransmitters in adults,that are depolarizing. GABA and glycine action depends on the intracellular concentration of chloride ([Cl-]i) which is finely regulated by specific cation-chloride co-transporters, called KCC2 and NKCC1.

Title: NMR Study of Oligonucleotide Structures Author: Václav Římal Department: Department of Low-Temperature Physics Supervisor: Prof. RNDr. Helena Štěpánková, CSc., Department of Low- Temperature Physics Abstract: The dynamics of nucleic acids plays a fundamental role in the interactions with proteins. Some processes are governed by changes in DNA stability and flexibility caused by sequence alterations or chemical modifications without substantial structural impact. We employed line-shape analysis of variable-temperature 1H NMR spectra to deeply investigate the melting transition of DNA structures. We observed a significant influence of the sequence beyond the nearest neighbours on chemical shifts and thermodynamics of the double helix. The CpG motif is especially sensitive to its distant surroundings and for a particular oligonucleotide it was even found to undergo an unexpected local conformational transition. A strong cooperativity in the duplex dis- ruption was witnessed by both the equilibrium and kinetic aspects as well as by distant effects of cytosine methylation. We propose a two-state melt- ing scheme also for a fragment of a transcription element proved here to fold as a hairpin with a six-membered loop. In addition, we describe the properties of self-assemblies of riboguanosine and...

RÉSUMÉ: Le charançon rouge des palmiers, Rhynchophorus ferrugineus (Oliv.), est l'insecte ravageur le plus destructif des palmiers cultivés en Asie, Moyen-Orient et bassin méditerranéen. Le piégeage olfactif peut jouer un rôle important en lutte intégrée contre ce ravageur mais l'usage indispensable de matériel végétal comme synergiste de sa phéromone d'agrégation limite son efficacité et son essor en zone désertique. Afin de disposer d'un synergiste synthéti-que de la phéromone, nous avons criblé par piégeage de terrain en Iran l'effet synergique avec la phéromone de molé-cules identifiées dans: 1- l'odeur de stipe de dattier, la plante-hôte de R. ferrugineus au Moyen-Orient lors de ce travail et 2- d'autres plantes-hôtes de Rhynchophorus spp. et décrites dans la bibliographie ou de structure apparentée. Nous avons utilisé la chromatographie en phase gazeuse (CPG) couplée à la spectrométrie de masse pour identifier la compo-sition d'odeurs de stipe de dattier isolées par collecte d'effluves. Les constituants potentiellement actifs sur le compor-tement de l'insecte ont été criblés par couplage CPG-électroantennographie (CPG-EAG) après une étude morphofonc-tionnelle de l'antenne par microscopie électronique à balayage et électroantennographie visant à vérifier s'il existait une régionalisation olfactive de la massue antennaire. Quatre types majoritaires de soies ont été identifiés, similaires à ceux décrits chez Rhynchophorus palmarum. Deux correspondent à des sensilles olfactives trichoïdes et basiconiques. La densité de sensilles basiconiques est plus élevée chez les femelles que chez les mâles et, chez les deux sexes, sur la face interne de la massue que sur la face externe. Les amplitudes des électroantennogrammes enregistrés à six molécules odorantes sont plus fortes à partir de la face interne de l'antenne que de la face externe mais le même profil de réponse a été obtenu que le branchement ait été réalisé en position, dorsale, centrale ou ventrale sur la massue. Au total, 109 molécules ont été identifiées dans les extraits d'odeur de dattier. Dix produits ont induit des électroantennogrammes reproductibles dans les analyses CPG-EAG dont un, l'anisole, a été identifié. L'effet phérosynergique de deux mélanges élaborés à partir des analyses d'odeurs de dattier, d'un mélange de molécules déclenchant des EAG de fortes amplitudes et de 21 mélanges bâtis à partir de 52 molécules issues de la bibliographie ont été étudiés. Parmi tous les mélanges testés sur le terrain, un mélange d'éthanol et d'acétate d'éthyle (50:50 v/v) à une dose de 300 mg/j a présenté un effet phérosynergique compétitif avec 0,35 kg de stipe de dattier et parfois avec 1,2 kg. Une variabilité importante de l'effet relatif entre ce mélange et le tissu de dattier a été observé au cours des trois saisons de travail. Aucun des mélanges plus complexes évalués n'a présenté d'effet notablement supérieur. L'activité antennaire de produits non identifiés dans l'odeur naturelle de dattier et l'absence d'effet phérosynergique des molécules testées, compétitif avec l'odeur naturelle, mettent en évidence le rôle important de composants de l'odeur du dattier non identifiés et/ou l'existence d'effets de mélange non évalués lors du présent travail. Le mélange d'éthanol et d'acétate d'éthyle, simple et bon marché, offre néanmoins une alternative intéressante pour améliorer le piégeage olfactif de R. ferrugineus dans la pratique, compte tenu du statut actuel de ce ravageur et de l'absence de meilleure alternative à l'usage d'appâts naturels.