Organometallic chemotherapeutic agents, many of which target DNA, have been shown to be effective in the treatment of cancer. With that said though, these compounds have a number of side affects such as nephrotoxicity. Two novel compounds, Ferrocene [ferrocenoyltrichloroacetone] and Rhodium-ferrocene [(1.5 cyclooctadiene)(1-ferrocenyl- 4,4,4-trichloro-1,3-butanedionate], synthesised by the research group of J Swarts (University of the Free State) were evaluated to determine their mechanism of action and their potential use as novel therapeutic agents. It is hypothesized, by merit of their chemical structures, that these compounds’ anti-cancer activity is due to their interaction with DNA. Both drugs were evaluated from a cellular to a molecular level, in vitro, to validate this hypothesis. Linearised DNA was exposed to both drugs and digested with a variety of restriction enzymes. It was found that the compounds bind to the PstI restriction site; thereby inhibiting the enzyme’s restriction activity. From this point it was necessary to show that the compounds are able to interact with DNA in a cellular system. By exposing a transformed breast epithelial cell line (MCF-12A) and a cancerous breast epithelial cell line (MCF-7) to the compounds, for various times, followed by flow cytometric analyses, it was found that both affect progression through the cell cycle. Cells accumulated at various phases of the cell cycle, as a result of checkpoint gene activation. Further flow cytometric analyses showed that both drugs induce necrosis in MCF-7 cells. The “normal” cell line however did not show this response as it is believed that cell cycle arrest and repair mechanisms were initiated, which would delay cell death. Gene expression analyses were performed by reverse transcriptase real-time PCR in which panels of cell cycle related genes as well as DNA damage associated genes were probed in two separate array formats. These studies revealed that a number of DNA damage and repair genes are activated; specifically those associated with excision repair and free-radical induced DNA damage. Members of the RAD family as well as the genes GADD45A, XPC and OGG1 were found to be upregulated as a result of Ferrocene treatment. This could be expected as it was shown that ferrocene binds to DNA, and it logically then follows that this would lead to excision repair being attempted by the cell. Similar gene expression patterns were found following Rhodium-ferrocene treatment with the up-regulation of genes such as OGG1, ATM and GADD45G, albeit to a lesser extent. It is hypothesised that the larger molecule may not interact as effectively with DNA, due to steric hinderance. Arrest mechanisms, for both drugs, were more pronounced in the “normal” cell line and it is believed that this is due to the fact that many of these genes have been inactivated in the cancerous cell line. We have shown, on multiple levels, that both compounds’ therapeutic action is as a result of their interaction with the cell’s DNA. This interaction leads to cell death in both the transformed and the cancerous cell line. In order to clarify these mechanisms it is suggested that proteomic and metabolomic studies should be performed. / Dissertation (MSc)--University of Pretoria, 2012. / Genetics / unrestricted

No description available.

Submitted by Rosivalda Pereira (mrs.pereira@ufma.br) on 2017-10-02T20:17:04Z No. of bitstreams: 1 ThiagoSantos.pdf: 1103502 bytes, checksum: 16ad7405a0ab31d83423293c43110ee8 (MD5) / Made available in DSpace on 2017-10-02T20:17:04Z (GMT). No. of bitstreams: 1 ThiagoSantos.pdf: 1103502 bytes, checksum: 16ad7405a0ab31d83423293c43110ee8 (MD5) Previous issue date: 2017-09-11 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Fundação de Amparo à Pesquisa e ao Desenvolvimento Científico e Tecnológico do Maranhão / Instituto Nacional de Ciência e Tecnologia em Bioanalítica / A photoelectroanalytical sensor was developed, based on deoxyribonucleic acid (DNA) and anatase titanium dioxide (TiO2) nanoparticles sensitized with bis(ethylenedithio)tetrathiofulvalene (BEDT-TTF) for determination of the adrenaline, also denominated as epinephrine. The photosensor composite developed was denominated as BEDT-TTF/DNA/TiO2/ITO and shows a high photocurrent for the adrenaline under light emitting diode (LED) irradiation in comparison to each component of the composite material. Under optimized conditions, the BEDTTTF/DNA/TiO2/ITO sensor shows a linear response range from 10 nmol L-1 up to 100 μmol L-1 with a sensitivity of 8,1 nA L μmol-1 and limit of detection of 1 nmol L-1 for the adrenaline. The photoelectrochemical sensor showed high photocurrent to adrenaline in comparison to photocurrent response to ascorbic acid and uric acid. The BEDT-TTF/DNA/TiO2/ITO photoelectrochemical sensor was successfully applied to urine samples, with recovery values between 96 and 106%. / Um sensor fotoeletroanalítico foi desenvolvido, baseado em ácido desoxirribonucleico (DNA) e nanopartículas de dióxido de titânio anatase (TiO2) sensibilizadas com bis(etilenoditio)tetratiofulvaleno (BEDT-TTF) para a determinação de adrenalina, também denominada como epinefrina. O fotossensor compósito desenvolvido foi denominado como BEDT-TTF/DNA/TiO2/ITO e exibiu uma elevada fotocorrente para a adrenalina sob a irradiação do diodo emissor de luz (LED) em comparação com cada componente do material compósito. Sob condições otimizadas, o sensor BEDT-TTF/DNA/TiO2/ITO exibiu um intervalo de resposta linear de 10 nmol L-1 para 100 μmol L-1 com uma sensibilidade de 8,1 nA L μmol-1 e limite de detecção de 1 nmol L-1 para a adrenalina. O sensor fotoeletroquímico mostrou elevada fotocorrente para a adrenalina em comparação com a resposta de fotocorrente para ácido ascórbico e ácido úrico. O fotossensor BEDTTTF/DNA/TiO2/ITO foi aplicado com sucesso em amostras de urina, com valores de recuperação entre 96 e 106%.

Of the many existing eukaryotic gene finding software programs, none are able to guarantee accurate identification of genomic protein coding regions and other biological signals central to pathway from DNA to the protein. Eukaryotic gene finding is difficult mainly due to noncontiguous and non-continuous nature of genes. Existing approaches are heavily dependent on the compositional statistics of the sequences they learn from and are not equally suitable for all types of sequences. This thesis firstly develops efficient digital signal processing-based methods for the identification of genomic protein coding regions, and then combines the optimum signal processing-based non-data-driven technique with an existing data-driven statistical method in a novel system demonstrating improved identification of acceptor splice sites. Most existing well-known DNA symbolic-to-numeric representations map the DNA information into three or four numerical sequences, potentially increasing the computational requirement of the sequence analyzer. Proposed mapping schemes, to be used for signal processing-based gene and exon prediction, incorporate DNA structural properties in the representation, in addition to reducing complexity in subsequent processing. A detailed comparison of all DNA representations, in terms of computational complexity and relative accuracy for the gene and exon prediction problem, reveals the newly proposed ?paired numeric? to be the best DNA representation. Existing signal processing-based techniques rely mostly on the period-3 behaviour of exons to obtain one dimensional gene and exon prediction features, and are not well equipped to capture the complementary properties of exonic / intronic regions and deal with the background noise in detection of exons at their nucleotide levels. These issues have been addressed in this thesis, by proposing six one-dimensional and three multi-dimensional signal processing-based gene and exon prediction features. All one-dimensional and multi-dimensional features have been evaluated using standard datasets such as Burset/Guigo1996, HMR195, and the GENSCAN test set. This is the first time that different gene and exon prediction features have been compared using substantial databases and using nucleotide-level metrics. Furthermore, the first investigation of the suitability of different window sizes for period-3 exon detection is performed. Finally, the optimum signal processing-based gene and exon prediction scheme from our evaluations is combined with a data-driven statistical technique for the recognition of acceptor splice sites. The proposed DSP-statistical hybrid is shown to achieve 43% reduction in false positives over WWAM, as used in GENSCAN.

Aufgrund einer komplexen Wechselwirkung mit Proteinen ist das Genom in einer Zelle ständig mechanischer Spannung und Torsion ausgesetzt. Daher ist es wichtig die Mechanik und die Dynamik von verdrillter DNA unter Spannung zu verstehen. Diese Situation wurde experimentell mittels einer sog. magnetischen Pinzette nachgestellt, indem sowohl Kraft als auch Drehmoment auf ein einzelnes DNA Molekül ausgeübt und gleichzeitig die mechanische Antwort des Polymers aufgezeichnet wurde. Als erstes Beispiel wurde der Übergang von linearer zu sog. plectonemischer DNA untersucht, d.h. die Absorption eines Teils der induzierten Verdrillung in einer superhelikalen Struktur. Eine abrupte Längenänderung am Anfang dieses Übergangs wurde bereits im Vorfeld publiziert. In der vorliegenden Arbeit wird gezeigt, dass diese abrupte DNA Verkürzung insbesondere von der Länge der DNA und der Ionenkonzentration der Lösung abhängt. Dieses Verhalten kann mittels eines Modells verstanden werden, in dem die Energie pro Verwringung der ersten Schlinge innerhalb der Superhelix größer ist als die aller nachfolgenden. Des Weiteren wurden DNA-DNA Wechselwirkungen in der Umgebung monovalenter Ionen durch die Analyse des Superspiralisierungsverhaltens einzelner DNA Moleküle bei konstanter Kraft charakterisiert. Solche Wechselwirkungen sind für die Kompaktierung des Genoms und die Regulation der Transkription wichtig. Oft wird DNA als gleichmäßig geladener Zylinder modelliert und ihre elektrostatischen Wechselwirkungen im Rahmen der Poisson-Boltzmann-Gleichung mit einem Ladungsanpassungsfaktor berechnet. Trotz erheblicher Anstrengung ist eine präzise Bestimmung dieses Parameters bisher nicht gelungen. Ein theoretisches Modell dieses Prozesses zeigte nun eine erstaunlich kleine effektive DNA Ladung von ~40% der nominalen Ladungsdichte. Abgesehen von Gleichgewichtsprozessen wurde auch die Dynamik eines Faltungsvorgangs von DNA untersucht. Spontane Branch Migration einer homologen Holliday-Struktur wurde genutzt, um die intramolekulare Reibung der DNA zu erforschen. Mittels einer magnetischen Pinzette wurde eine torsionslimitierte Holliday-Struktur gestreckt während die Längenfluktuationen der Zweige mit schneller Videomikroskopie bei ~3 kHz aufgezeichnet wurden. Einzelne diffusive Schritte der Basenpaare sollten auf einer sub-Millisekunden Zeitskala auftreten und viel kleiner als die Gesamtfluktuationen der DNA sein. Eine Analyse der spektralen Leistungsdichte der Längenfluktuationen ermöglicht eine eindeutige Beschreibung der Dynamik der Branch Migration. Die Holliday-Struktur wurde außerdem als nanomechanischer Linearversteller eingesetzt, um einen einzelnen fluoreszierenden Quantenpunkt durch ein exponentiell abfallendes evaneszentes Feld zu bewegen. Durch die Aufzeichnung der Emission des Quantenpunkts sowohl in dem evaneszenten Feld als auch unter gleichmäßiger Beleuchtung kann die Intensitätsverteilung des Anregungsfelds ohne weitere Dekonvolution bestimmt werden. Diese neue Technik ist von besonderem wissenschaftlichen Interesse, weil die Beschreibung dreidimensionaler inhomogener Beleuchtungsfelder eine große Herausforderung in der modernen Mikroskopie darstellt. Die Ergebnisse dieser Arbeit werden dem besseren Verständnis einer Vielzahl biologischer Prozesse, die in Verbindung mit DNA Superspiralisierung stehen, dienen und weitere technische Anwendungen des DNA-basierten Linearverstellers hervorbringen. / The genome inside the cell is continuously subjected to tension and torsion primarily due to a complex interplay with a large variety of proteins. To gain insight into these processes it is crucial to understand the mechanics and dynamics of twisted DNA under tension. Here, this situation is mimicked experimentally by applying force and torque to a single DNA molecule with so called magnetic tweezers and measuring its mechanical response. As a first example a transition from a linear to a plectonemic DNA configuration is studied, i.e. the absorption of part of the applied twist in a superhelical structure. Recent experiments revealed the occurrence of an abrupt extension change at the onset of this transition. Here, it is found that this abrupt DNA shortening strongly depends on the length of the DNA molecule and the ionic strength of the solution. This behavior can be well understood in the framework of a model in which the energy per writhe for the initial plectonemic loop is larger than for subsequent turns of the superhelix. Furthermore DNA-DNA interactions in the presence of monovalent ions were comprehensively characterized by analyzing the supercoiling behavior of single DNA molecules held under constant tension. These interactions are important for genome compaction and transcription regulation. So far DNA is often modeled as a homogeneously charged cylinder and its electrostatic interactions are calculated within the framework of the Poisson-Boltzmann equation including a charge adaptation factor. Despite considerable efforts, until now a rigorous quantitative assessment of this parameter has been lacking. A theoretical model of this process revealed a surprisingly small effective DNA charge of ~40% of the nominal charge density. Besides describing equilibrium processes, also the dynamics during refolding of nucleic acids is investigated. Spontaneous branch migration of a homologous Holliday junction serves as an ideal system where the friction within the biomolecule can be studied. This is realized by stretching a torsionally constrained Holliday junction using magnetic tweezers and recording the length fluctuations of the arms with high-speed videomicroscopy at ~3 kHz. Single base pair diffusive steps are expected to occur on a sub-millisecond time scale and to be much smaller than the overall DNA length fluctuations. Power-spectral-density analysis of the length fluctuations is able to clearly resolve the overall dynamics of the branch migration process. Apart from studying intramolecular friction, the four-arm DNA junction was also used as a nanomechanical translation stage to move a single fluorescent quantum dot through an exponentially decaying evanescent field. Recording the emission of the quantum dot within the evanescent field as well as under homogeneous illumination allows to directly obtain the intensity distribution of the excitation field without additional deconvolution. This new technique is of particular scientific interest because the characterization of three-dimensional inhomogeneous illumination fields is a challenge in modern microscopy. The results presented in this work will help to better understand a large variety of biological processes related to DNA supercoiling and inspire further technical applications of the nanomechanical DNA gear.

Of the many existing eukaryotic gene finding software programs, none are able to guarantee accurate identification of genomic protein coding regions and other biological signals central to pathway from DNA to the protein. Eukaryotic gene finding is difficult mainly due to noncontiguous and non-continuous nature of genes. Existing approaches are heavily dependent on the compositional statistics of the sequences they learn from and are not equally suitable for all types of sequences. This thesis firstly develops efficient digital signal processing-based methods for the identification of genomic protein coding regions, and then combines the optimum signal processing-based non-data-driven technique with an existing data-driven statistical method in a novel system demonstrating improved identification of acceptor splice sites. Most existing well-known DNA symbolic-to-numeric representations map the DNA information into three or four numerical sequences, potentially increasing the computational requirement of the sequence analyzer. Proposed mapping schemes, to be used for signal processing-based gene and exon prediction, incorporate DNA structural properties in the representation, in addition to reducing complexity in subsequent processing. A detailed comparison of all DNA representations, in terms of computational complexity and relative accuracy for the gene and exon prediction problem, reveals the newly proposed ?paired numeric? to be the best DNA representation. Existing signal processing-based techniques rely mostly on the period-3 behaviour of exons to obtain one dimensional gene and exon prediction features, and are not well equipped to capture the complementary properties of exonic / intronic regions and deal with the background noise in detection of exons at their nucleotide levels. These issues have been addressed in this thesis, by proposing six one-dimensional and three multi-dimensional signal processing-based gene and exon prediction features. All one-dimensional and multi-dimensional features have been evaluated using standard datasets such as Burset/Guigo1996, HMR195, and the GENSCAN test set. This is the first time that different gene and exon prediction features have been compared using substantial databases and using nucleotide-level metrics. Furthermore, the first investigation of the suitability of different window sizes for period-3 exon detection is performed. Finally, the optimum signal processing-based gene and exon prediction scheme from our evaluations is combined with a data-driven statistical technique for the recognition of acceptor splice sites. The proposed DSP-statistical hybrid is shown to achieve 43% reduction in false positives over WWAM, as used in GENSCAN.

Les métaux sont indispensables à la vie cellulaire car ils sont constitutifs des protéines. Les ions Ni, font partie intégrante des hydrogénases, enzymes primordiales pour le métabolisme énergétique. Paradoxalement, en excès, les métaux deviennent toxiques pour la cellule. Les bactéries luttent contre cette toxicité en produisant des systèmes de résistance ou d’adaptation. Les cellules procaryotes peuvent équilibrer les teneurs en métaux en contrôlant leur entrée ou leur efflux grâce à la biogenèse de transporteurs spécifiques. L’objectif de ces travaux de thèse a consisté à comprendre les mécanismes principaux permettant à la bactérie modèle Escherichia coli de s’adapter à de fortes variations en ions métalliques, en prenant comme modèle un stress provoqué par un excès d’ions Ni. Afin d’appréhender l’ensemble de la réponse cellulaire, l’effet de ce stress a été évalué sur l’expression de l’ensemble des gènes d’E. coli par des approches de transcriptomique couplées à une validation fonctionnelle. L’excès d’ions Ni induit le système d’efflux RcnRAB. En plus de la pompe d’efflux RcnA, ce système comporte une protéine périplasmique, RcnB, qui module le trafic des ions Ni ou Co via RcnA. Ces travaux ont montré que RcnB n’interagit pas avec les ions Ni ou Co mais de façon inattendue avec les ions Cu, définissant une nouvelle classe de cupro-protéines. Nous montrons que si RcnB n’intervient pas dans le contrôle de l’homéostasie du Cu, l’interaction avec ces ions est essentielle à sa fonction dans la modulation de l’efflux des ions Ni et Co. Ces résultats suggèrent des connexions entre les différents systèmes de maintien des homéostasies métalliques. Les résultats d’analyse transcriptomique montrent une forte modulation de l’expression des gènes impliqués dans les homéostasies du Cu et du Fe en présence d’un excès d’ions Ni, corrélée à une augmentation cellulaire de leur teneur mesurée par spectrométrie plasma. Ces métaux sont responsables de la production d’espèces réactives oxygénées entraînant de sérieux dégâts cellulaires, une des cibles privilégiée étant l’ADN. Nous montrons que les ions Ni ne provoquent pas de cassures de l’ADN et n’ont pas d’effet mutagène, par contre ils provoquent une modification importante de l’état de repliement de l’ADN. Nous proposons que ce relâchement de l’ADN soit dû à l’induction indirecte d’un stress oxydant. Ces travaux ont aboutis à l’identification du premier système de transport spécifique des ions Ni à travers la membrane externe chez E. coli. En résumé, un excès d’ions Ni affecte les systèmes spécifiques d’entrée et d’efflux des ions métalliques troublant les teneurs intracellulaires des autres métaux comme le Cu et le Fe. Ces métaux sont en partie responsables de la production de ROS létaux pour les cellules bactériennes. L’excès de Ni va induire une profonde reprogrammation génétique entraînant des changements physiologiques multifactoriels importants pour la survie bactérienne dans ces conditions de stress. / Metals are necessary components of all living cells because they are constitutive of many essential proteins. Nickel, for example, is required for hydrogenase activity, which is essential for the energetic metabolism. However, metals become toxic when present in excess. Prokaryotes can overcome this toxicity by using several systems of resistance or adaptation. Import systems must be repressed whereas export pathways activated. This work consists in bringing out the principal strategies established by Escherichia coli for accommodating a stress caused by an excess of Ni ions. In order to understand the cellular response, the effect of nickel stress has been evaluated in E. coli by a transcriptomic approach coupled to functional validation. Excess Ni induces the biosynthesis of the efflux system RcnRAB. In addition to the RcnA efflux pump, this system contains a periplasmic protein called RcnB. This protein modulates Ni and Co traffic. RcnB displayed no Ni or Co binding capacity but was shown to bing Cu ions. RcnB was characterized as a new family of cupro-protein. We showed that RcnB is not involved in the control of Cu homeostasis but that Cu binding is essential for its Ni and Co efflux function. Our results suggest connections between different systems of metals homeostasis. Indeed, RNA-Seq data analysis revealed that exposure to Ni induces strong variations of the expression of genes involved in Cu and Fe homeostasis. Our results correlated with an increase of intracellular Cu and Fe pools as assayed by plasma spectrometry. Both metals are involved in reactive oxygen species (ROS) production and generate serious cell damages, targeting DNA for example. We showed that Ni ions do not trigger DNA breakage and are not mutagenic. On the other hand, Ni stress has a strong effect on DNA folding. We propose that excess Ni causes DNA relaxation by the indirect induction of oxidative stress. Furthermore, we identified the first transport system specific for Ni ions localized in the outer membrane. This system, composed of YddA and YddB, allows the transfer of Ni ions accross the two membranes. The genes encoding these proteins are expressed in conditions evocative of a biofilm lifestyle. Moreover, this work showed that Ni stress promotes biofilm growth instead of a planktonic one. Indeed, in the presence of an excess of Ni ions, genes encoding flagella are down regulated whereas genes encoding adherence structures are up regulated. To conclude, an excess of Ni ions affects specific metals import and efflux systems unbalancing intracellular Fe and Cu contents. These metals in turn generate ROS that are toxic for the bacterial cells. Ni stress induces large transcriptomic modifications causing major physiological changes important for the survival of the bacteria.

There is a strong interest in phenolic compounds and nitrocompounds in the function of their diverse and significant biological activities. Such compounds can participate in electron transfer reactions and produce metabolites that influence the redox state at the cellular level, with consequent effect on vital biochemical processes. This work aimed to determine the electrodic mechanism involved in the oxidation of bergenin and the reduction of nitrobenzyl derivatives and to evaluate possible supramolecular interactions of bergenin with β-cyclodextrin (β-CD) and deoxyribonucleic acid (DNA) in order to increase its solubility in aqueous medium and help in understanding the molecular mechanism of biological action. The electrochemical studies in protic and aprotic medium were performed in potentiostat PGSTAT302 (AUT 73222) from Autolab® using voltammetric techniques. The influence of the interaction of different cyclodextrins on the solubility of bergenin in aqueous medium was verified through the phase transfer study by cyclic voltammetry. The bergenin:β-CD complex was prepared in 1:1 and 1:2 proportions by the coevaporation technique and characterized by spectroscopic techniques. The theoretical studies were performed through the Gaussian program 09. Bergenin in the free and complexed form were evaluated against antioxidant capacity (lipid peroxidation assays), cytotoxicity (cell viability versus macrophages), and interaction with estimated DNA through the use of dsDNA (double strand) electrochemical sensor and with ssDNA (single strand) for UV-Vis spectrophotometry in solution. The electrochemical results obtained for bergenin demonstrated that its oxidation mechanism in aprotic and protic environments involves, respectively, loss of 1e-/1H+ and 2e-/2H+, when using a glassy carbon electrode. Theoretical data contributed to clarify that oxidative mechanisms involve phenolic hydroxyls. The antioxidant activity of bergenin in the lipoperoxidation inhibition assays was favored in its complexed form with β-CD 1:1. The cytotoxicity of bergenin evaluated in macrophages was also influenced by interaction with β-CD. The electrochemical studies involving ssDNA demonstrated interaction between bergenin and the constituent bases of DNA, suggesting a possible mechanism of biological action. However, the dsDNA biosensor studies showed no interaction. Investigation of the interaction with dsDNA through UV-Vis spectrophotometry resulted in a binding constant between DNA and bergenin in the free and complexed form on the order of 103 and 104 M-1, respectively. The electrochemical behavior of the nitrobenzyl derivatives obtained in aprotic medium presented a voltammetric profile of great complexity, involving patterns related to autoprotonation and dissociative electron transfer reactions. The order of ease of reduction, based on values of first wave reduction potential, was found: ANB > EANBEN > EANB > AANB > ANOH > ATN = ENF > ANBNa > ENM > ANF, obtaining a positive correlation between the compounds with (less negative) with more pronounced biological activity (leishmanicidal and antitumor activities), already described in the literature, which justifies the importance of the electrochemical investigation of bioactive compounds as a tool in medical chemistry, in processes related to the transfer of electrons. / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Há forte interesse a cerca de compostos fenólicos e nitrocompostos em função de suas diversas e significativas atividades biológicas. Tais compostos podem participar de reações de transferência de elétrons e produzir metabólitos que influenciam o estado redox em nível celular, com consequente efeito em processos bioquímicos vitais. Deste modo, este trabalho teve como objetivo determinar o mecanismo eletródico envolvido na oxidação da bergenina e na redução dos derivados nitrobenzílicos e avaliar possíveis interações supramoleculares da bergenina com β-ciclodextrina (β-CD) e ácido desoxirribonucléico (DNA), a fim de aumentar sua solubilidade em meio aquoso e auxiliar no entendimento do mecanismo molecular de ação biológica. Os estudos eletroquímicos em meio prótico e aprótico foram realizados em potenciostato PGSTAT302 (AUT 73222) da Autolab® através de técnicas voltamétricas. A influência da interação de diferentes ciclodextrinas sobre a solubilidade da bergenina em meio aquoso foi verificada através do estudo de transferência de fase por voltametria cíclica. O complexo bergenina:β-CD foi preparado nas proporções 1:1 e 1:2 através da técnica de co-evaporação e caracterizado através de técnicas espectroscópicas. Os estudos teóricos foram realizados através do programa Gaussian 09. A bergenina na forma livre e complexada foram avaliadas frente à capacidade antioxidante (ensaios de peroxidação lipídica), citotoxicidade (viabilidade celular frente a macrófagos), e a interação com DNA estimada através da utilização de sensor eletroquímico de dsDNA (fita dupla) e com ssDNA (fita simples) e espectrofotometria UV-Vis, em solução. Os resultados eletroquímicos obtidos para bergenina demonstraram que seu mecanismo de oxidação em meio aprótico e prótico envolve, respectivamente, a perda de 1e-/1H+ e de 2e-/2H+, ao utilizar eletrodo de carbono vítreo. Os dados teóricos contribuiram para esclarecer que os mecanismos oxidativos envolvem as hidroxilas fenólicas. A atividade antioxidante da bergenina verificada nos ensaios de inibição de lipoperoxidação foi favorecida em sua forma complexada com β-CD na proporção 1:1. A citotoxicidade da bergenina avaliada em macrófagos também sofreu influência da interação com β-CD. Os estudos eletroquímicos envolvendo ssDNA demonstraram interação entre bergenina e as bases constituintes do DNA, sugerindo um possível mecanismo de ação biológico. Já os estudos com biossensor de dsDNA, não demonstraram interação. A investigação da interação com dsDNA através da espectrofotometria UV-Vis resultou numa constante de ligação entre DNA e bergenina na forma livre e complexada na ordem de 103 e 104 M-1, respectivamente. O comportamento eletroquímico dos derivados nitrobezílicos obtidos em meio aprótico apresentaram um perfil voltamétrico de grande complexidade, envolvendo padrões relacionados a reações de autoprotonação e de transferência de elétrons dissociativa. A ordem de facilidade de redução, baseada em valores de potencial de primeira onda de redução, encontrada foi: ANB > EANBEN > EANB > AANB > ANOH > ATN = ENF > ANBNa > ENM > ANF, obtendose correlação positiva entre os compostos com maior potencial de redução (menos negativos) com atividade biológica mais pronunciada (atividades leishmanicida e antitumoral), já descritas na literatura, o que justifica a importância da investigação eletroquímica de compostos bioativos como ferramenta em química medicinal, em processos relacionados à transferência de elétrons.

This thesis brings a new approach to the prediction of the effect of nucleotide polymorphism on human genome. The main goal is to create a new meta-classifier, which combines predictions of several already implemented software classifiers. The novelty of developed tool lies in using machine learning methods to find consensus over those tools, that would enhance accuracy and versatility of prediction. Final experiments show, that compared to the best integrated tool, the meta-classifier increases the area under ROC curve by 3,4 in average and normalized accuracy is improved by up to 7\,\%. The new classifying service is available at http://ll06.sci.muni.cz:6232/snpeffect/.

Aufgrund einer komplexen Wechselwirkung mit Proteinen ist das Genom in einer Zelle ständig mechanischer Spannung und Torsion ausgesetzt. Daher ist es wichtig die Mechanik und die Dynamik von verdrillter DNA unter Spannung zu verstehen. Diese Situation wurde experimentell mittels einer sog. magnetischen Pinzette nachgestellt, indem sowohl Kraft als auch Drehmoment auf ein einzelnes DNA Molekül ausgeübt und gleichzeitig die mechanische Antwort des Polymers aufgezeichnet wurde. Als erstes Beispiel wurde der Übergang von linearer zu sog. plectonemischer DNA untersucht, d.h. die Absorption eines Teils der induzierten Verdrillung in einer superhelikalen Struktur. Eine abrupte Längenänderung am Anfang dieses Übergangs wurde bereits im Vorfeld publiziert. In der vorliegenden Arbeit wird gezeigt, dass diese abrupte DNA Verkürzung insbesondere von der Länge der DNA und der Ionenkonzentration der Lösung abhängt. Dieses Verhalten kann mittels eines Modells verstanden werden, in dem die Energie pro Verwringung der ersten Schlinge innerhalb der Superhelix größer ist als die aller nachfolgenden. Des Weiteren wurden DNA-DNA Wechselwirkungen in der Umgebung monovalenter Ionen durch die Analyse des Superspiralisierungsverhaltens einzelner DNA Moleküle bei konstanter Kraft charakterisiert. Solche Wechselwirkungen sind für die Kompaktierung des Genoms und die Regulation der Transkription wichtig. Oft wird DNA als gleichmäßig geladener Zylinder modelliert und ihre elektrostatischen Wechselwirkungen im Rahmen der Poisson-Boltzmann-Gleichung mit einem Ladungsanpassungsfaktor berechnet. Trotz erheblicher Anstrengung ist eine präzise Bestimmung dieses Parameters bisher nicht gelungen. Ein theoretisches Modell dieses Prozesses zeigte nun eine erstaunlich kleine effektive DNA Ladung von ~40% der nominalen Ladungsdichte. Abgesehen von Gleichgewichtsprozessen wurde auch die Dynamik eines Faltungsvorgangs von DNA untersucht. Spontane Branch Migration einer homologen Holliday-Struktur wurde genutzt, um die intramolekulare Reibung der DNA zu erforschen. Mittels einer magnetischen Pinzette wurde eine torsionslimitierte Holliday-Struktur gestreckt während die Längenfluktuationen der Zweige mit schneller Videomikroskopie bei ~3 kHz aufgezeichnet wurden. Einzelne diffusive Schritte der Basenpaare sollten auf einer sub-Millisekunden Zeitskala auftreten und viel kleiner als die Gesamtfluktuationen der DNA sein. Eine Analyse der spektralen Leistungsdichte der Längenfluktuationen ermöglicht eine eindeutige Beschreibung der Dynamik der Branch Migration. Die Holliday-Struktur wurde außerdem als nanomechanischer Linearversteller eingesetzt, um einen einzelnen fluoreszierenden Quantenpunkt durch ein exponentiell abfallendes evaneszentes Feld zu bewegen. Durch die Aufzeichnung der Emission des Quantenpunkts sowohl in dem evaneszenten Feld als auch unter gleichmäßiger Beleuchtung kann die Intensitätsverteilung des Anregungsfelds ohne weitere Dekonvolution bestimmt werden. Diese neue Technik ist von besonderem wissenschaftlichen Interesse, weil die Beschreibung dreidimensionaler inhomogener Beleuchtungsfelder eine große Herausforderung in der modernen Mikroskopie darstellt. Die Ergebnisse dieser Arbeit werden dem besseren Verständnis einer Vielzahl biologischer Prozesse, die in Verbindung mit DNA Superspiralisierung stehen, dienen und weitere technische Anwendungen des DNA-basierten Linearverstellers hervorbringen. / The genome inside the cell is continuously subjected to tension and torsion primarily due to a complex interplay with a large variety of proteins. To gain insight into these processes it is crucial to understand the mechanics and dynamics of twisted DNA under tension. Here, this situation is mimicked experimentally by applying force and torque to a single DNA molecule with so called magnetic tweezers and measuring its mechanical response. As a first example a transition from a linear to a plectonemic DNA configuration is studied, i.e. the absorption of part of the applied twist in a superhelical structure. Recent experiments revealed the occurrence of an abrupt extension change at the onset of this transition. Here, it is found that this abrupt DNA shortening strongly depends on the length of the DNA molecule and the ionic strength of the solution. This behavior can be well understood in the framework of a model in which the energy per writhe for the initial plectonemic loop is larger than for subsequent turns of the superhelix. Furthermore DNA-DNA interactions in the presence of monovalent ions were comprehensively characterized by analyzing the supercoiling behavior of single DNA molecules held under constant tension. These interactions are important for genome compaction and transcription regulation. So far DNA is often modeled as a homogeneously charged cylinder and its electrostatic interactions are calculated within the framework of the Poisson-Boltzmann equation including a charge adaptation factor. Despite considerable efforts, until now a rigorous quantitative assessment of this parameter has been lacking. A theoretical model of this process revealed a surprisingly small effective DNA charge of ~40% of the nominal charge density. Besides describing equilibrium processes, also the dynamics during refolding of nucleic acids is investigated. Spontaneous branch migration of a homologous Holliday junction serves as an ideal system where the friction within the biomolecule can be studied. This is realized by stretching a torsionally constrained Holliday junction using magnetic tweezers and recording the length fluctuations of the arms with high-speed videomicroscopy at ~3 kHz. Single base pair diffusive steps are expected to occur on a sub-millisecond time scale and to be much smaller than the overall DNA length fluctuations. Power-spectral-density analysis of the length fluctuations is able to clearly resolve the overall dynamics of the branch migration process. Apart from studying intramolecular friction, the four-arm DNA junction was also used as a nanomechanical translation stage to move a single fluorescent quantum dot through an exponentially decaying evanescent field. Recording the emission of the quantum dot within the evanescent field as well as under homogeneous illumination allows to directly obtain the intensity distribution of the excitation field without additional deconvolution. This new technique is of particular scientific interest because the characterization of three-dimensional inhomogeneous illumination fields is a challenge in modern microscopy. The results presented in this work will help to better understand a large variety of biological processes related to DNA supercoiling and inspire further technical applications of the nanomechanical DNA gear.