Orientador: Marinônio Lopes Cornélio / Banca: Marcelo Andres Fossey / Banca: Hamilton Cabral / Resumo: Este trabalho apresenta um estudo do processo de desenovelamento da papaína via temperatura. Para tanto, foi utilizada a técnica espectroscópica de fluorescência, em conjunto com a espectroscopia de correlação bidimensional, com as análises sample-sample e variável- variável aplicadas à região do infravermelho médio. Desta forma, foi possível determinar três temperaturas de pré-transição: 34, 54 e 61ºC; sendo que a temperatura de 54ºC foi evidenciada tanto por fluorescência quanto pela análise sample-sample. Já as temperaturas de 34 e 61ºC foram evidenciadas somente na análise sample-sample. Além disso, a análise variável-variável descreveu a dinâmica conformacional durante o processo de desenovelamento. Assim, pode-se relacionar a temperatura e a dinâmica conformacional. / Abstract: This work presents a study of the unfolding of papain via temperature. In order to do so, the fluorescence spectroscopy technique was used along with two- dimensional correlation spectroscopy by sample-sample and variable-variable analyses applied to the medium infrared region. Thus, it was possible to determine the three papain thermal pre-transition temperatures, 34, 54 and 61º C. The 54º C one was obtained by fluorescence spectroscopy and sample-sample analyses; whereas the 34 and 61ºC, ones, were detected by sample-sample alone. Additionally, through variable-variable analysis it was possible to describe the conformational dynamics during the unfolding process. Therefore, the relationship between temperature and conformational dynamics could be matched. / Mestre

Protein misfolding is considered to be the major pathogenic mechanism in homocystinuria due to cystathionine beta-synthase (CBS) deficiency. The aim of this work was to study molecular mechanisms underlying protein misfolding of CBS mutants. Firstly, we studied spatial arrangement of normal human CBS protein. Using data from differential covalent labeling of solvent-exposed aminoacid residues, we identified interdomain contact area between the catalytic core and the regulatory domain in human CBS, and we subsequently generated the structural model of the full-length CBS. In the next step, we studied evolutionary divergence of CBS protein structures. We performed phylogenetic analysis that revealed unique spatial arrangement of CBS enzyme in nematodes; the domain architecture of CBS in Caenorhabditis elegans was studied experimentally in more detail. Finally, we determined conformational properties of a representative set of human CBS mutants that exhibited in various extent affected formation of tetramers and decreased catalytic activity. Using thermolysin-based proteolytic techniques for analysis of nine mutants expressed in E.coli, we found that an unfolded structure is a common intermediate occurring in CBS misfolding. The importance of protein unfolding for pathogenesis of CBS deficiency was...

Simulações moleculares podem fornecer informações e detalhes mecanísticos que são difíceis de obter de experimentos. No entanto, fenômenos bioquímicos como formação de complexos proteína-ligante e desenovelamento de proteína são lentos e difíceis de amostrar na escala de tempo geralmente atingida por simulações de dinâmica molecular (MD) convencionais. Esses fenômenos moleculares foram estudados aqui pela combinação de simulações de MD com diversos métodos e aproximações para aumentar a amostragem configuracional: método de energia de interação linear (LIE), a aproximação de ensemble ponderado (WE) e dinâmica molecular dirigida (SMD). Uma equação foi parametrizada para prever afinidades entre pequenas moléculas e proteínas baseada na aproximação LIE, que foca a amostragem computacional nos estados complexado e não-complexado do ligante. A flexibilidade proteica foi introduzida usando ensembles de configurações obtidos de simulações de MD. Diferentes esquemas de média foram testados para obter afinidades totais de complexos proteína-ligante, revelando que muitas configurações de complexo contribuem para as afinidades de proteínas flexíveis, enquanto as afinidades de proteínas rígidas são dominadas por uma configuração de complexo. O mutante L99A da lisozima T4 (T4L) é provavelmente a proteína mais frequentemente usada para estudar complexação de ligantes. Estruturas cristalográficas mostram que a cavidade de ligação artificial criada pela mutação é pouco acessível, portanto movimentos proteicos ou uma respiração conformacional são necessários para permitir a entrada e saída de ligantes. Simulações de MD foram combinadas aqui com a aproximação de WE para aumentar a amostragem de eventos infrequentes de saída do benzeno de T4L. Quatro possíveis caminhos foram encontrados e movimentações de alfa-hélices e cadeias laterais envolvidas na saída do ligante foram caracterizadas. Os quatro caminhos correspondem a túneis da proteína previamente observados em simulações de MD longas de T4L apo, sugerindo que a heterogeneidade de caminhos ao longo de túneis intrínsecos é explorada por pequenas moléculas para sair de cavidades de ligação enterradas em proteínas. Experimentos de microscopia de força atômica revelaram informações detalhadas do desenovelamento forçado e da estabilidade mecânica da rubredoxina, uma proteína ferro-enxofre simples. O desenovelamento completo da rubredoxina envolve a ruptura de ligações covalentes. Portanto, o processo de desenovelamento foi simulado aqui por simulações de SMD acopladas a uma descrição clássica da dissociação de ligações. A amostragem de eventos de desenovelamento forçado foi aumentada pelo uso de velocidades rápidas de esticamento. Os resultados foram analisados usando um modelo teórico válido para regimes de desenovelamento forçado lentos e rápidos. As simulações revelaram que mudanças no ponto de aplicação de força ao longo da sequência da rubredoxina levam a diferentes mecanismos de desenovelamento, caracterizados por variáveis graus de rompimento de ligações de hidrogênio e estrutura secundária da proteína. / Molecular simulations may provide information and mechanistic insights that are difficult to obtain from experiments. However, biochemical phenomena such as ligand-protein binding and protein unfolding are slow and hard to sample on the timescales usually reached by conventional molecular dynamics (MD) simulations. These molecular phenomena were studied here by combining MD simulations with several methods or approximations to enhance configurational sampling: linear interaction energy (LIE) method, weighted ensemble (WE) approach and steered molecular dynamics (SMD). An equation was parametrized to predict affinities between small molecules and proteins based on the LIE approximation, which focus computational sampling in ligand bound and unbound states. Protein flexibility was introduced by using ensembles of configurations obtained from MD simulations. Different averaging schemes were tested to obtain overall affinities for ligand-protein complexes, revealing that many bound configurations contribute to affinities for flexible proteins, while affinities for rigid proteins are dominated by one bound configuration. T4 lysozyme (T4L) L99A mutant is probably the protein most often used to study ligand binding. Crystal structures show the artificial binding cavity created by the mutation has low accessibility, so protein movements or conformational breathing are necessary to allow the entry and egress of ligands. MD simulations were combined here with the WE approach to enhance sampling of infrequent benzene unbinding events from T4L. Four possible pathways were found and motions on alpha-helices and side chains involved in ligand egress were characterized. The four pathways correspond to protein tunnels previously observed in long MD simulations of apo T4L, suggesting that pathway heterogeneity along intrinsic tunnels is explored by small molecules to egress from binding cavities buried in proteins. Previous atomic force microscopy experiments revealed detailed information on the forced unfolding and mechanical stability of rubredoxin, a simple iron-sulfur protein. Complete unfolding of rubredoxin involves rupture of covalent bonds. Thus, the unfolding process was simulated here by SMD simulations coupled to a classical description of bond dissociation. Sampling of forced unfolding events was increased by using fast pulling velocities. Results were analyzed using a theoretical model valid for both slow and fast forced unfolding regimes. Simulations revealed that changing the points of force application along the rubredoxin sequence leads to different unfolding mechanisms, characterized by variable degrees of disruption of hydrogen bonds and secondary protein structure.

Estudamos singularidades de famílias de matrizes simétricas. O objetivo é classificar as singularidades simples de tais famílias e estudar a geometria de alguns objetos associados a elas / We study the singularities of families of symmetric matrices. The aim of this work is to classify simple singularities of such families and study the geometry of some objects associated to them

High Energy Stereoscopic System (H.E.S.S.) est un réseau de cinq télescopes à imagerie Cherenkov atmosphérique, localisé dans l’hémisphère sud, ayant pour but principal l’étude de rayons cosmiques couvrant une gamme d’énergie de 30 GeV à plusieurs dizaines de TeV. La technique de détection Cerenkov ainsi que les spécificités de la méthode de reconstruction employée par H.E.S.S. I (première phase de l’expérience H.E.S.S.), sont décrites dans cette thèse. Après plus de dix ans d’activité, l’expérience H.E.S.S. a enregistré une quantité de données importante. En plus des régions d’intérêts sondées par ces détecteurs, où des sources astrophysiques ont déjà été dévoilées, l’étude d’événements collectés permet d’améliorer la compréhension de leur environnement sous-jacent. En effet, des émissions diffuses encore non-comprises se superposent aux rayonnements provenant de sources actives. Elles sont pourtant d’un intérêt significatif en astrophysique, physique des particules, cosmologie et même dans certains domaines de physique au-delà des modèles standards, tel que la recherche de matière noire. Les émissions diffuses et leurs précédentes études sont présentées dans cette thèse, ainsi que leurs possibles origines, depuis les mécanismes d’accélération de rayons cosmiques jusqu’à la production de rayon gamma dans les sources actives ou encore par des processus secondaires impliquant les interactions de rayons cosmiques avec le milieu interstellaire. Dans ce travail, des outils pour étudier les émissions diffuses ont été développés. L’approche choisie permet de distinguer les différentes composantes dans les données étudiées et extraire une estimation de leur poids dans le spectre. Elle prend en compte deux aspects, expliqués séparément dans cette thèse. Dans un premier temps, la morphologie de la source active présente dans le champ de vue est utilisée pour la modéliser et obtenir son spectre. Ensuite, pour distinguer les différentes contributions du fond, la méthode se base sur des fonctions de densité de probabilité construites à partir de variables discriminantes. L’étude et manipulation préliminaires nécessaires des variables discriminantes sont également détaillées. Des sources astrophysiques connues sont utilisées comme référence pour l’analyse. Les spectres résultants pour la source active, les électrons et hadrons diffus sont présentés et discutés, ainsi qu’une limite supérieure sur le flux de l’émission diffuse gamma extragalactique. Les erreurs systématiques associées ont été estimées. Une technique de "unfolding" a été mise en place et utilisée pour vérifier les résultats pour les émissions diffuses. / The High Energy Stereoscopic System (H.E.S.S.) is an array of five Imaging Atmospheric \v{C}erenkov Telescopes (IACT) located in the Southern Hemisphere, whose primary goal is the study of cosmic gamma-rays in the 30 GeV - few tens of TeV energy range. The detection technique used by IACT as well as the specificities of the reconstruction method of H.E.S.S. I (first phase of the H.E.S.S. experiment) are fully described in this thesis. After more than ten years of activity the H.E.S.S. experiment has registered a large amount of data. In addition to the regions of interest that its detectors probe and where astrophysical sources were unveiled, many events collected provide useful information on their surrounding environment. Indeed, acting as a background to the active sources, one can find the diffuse emissions, which are not well understood and yet are of significant interest for astrophysics, particle physics, cosmology and even physics beyond standard models, such as the search for dark matter. The diffuse emissions and their previous studies are presented in this thesis, as well as their possible origin, starting from the acceleration of cosmic-rays mechanism and the gamma-ray production in the active sources or from secondary process involving cosmic-rays interactions in the interstellar medium.In this work, tools to investigate the diffuse emissions were developed. The approach aims at disentangling the different components of the studied data so as to extract an estimation of their weight in the spectrum. It takes into account two aspects, explained separately in this thesis. On the one hand, the morphology of the active source in the studied field of view is used to modelize it and obtain its spectrum. Then, to disentangle the different contributions in the background, the method is based on probability density functions (PDF) built with discriminant variables. The necessary preliminary study and manipulation of the discriminant variables is also detailed. Well known astrophysical sources are used as benchmarks for the analysis. The resulting spectra for the active source, diffuse electrons and hadrons are presented and discussed, in addition to an upper limit on the extragalactic diffuse gamma-ray emission flux. The associated systematic errors were estimated.

Protein misfolding is considered to be the major pathogenic mechanism in homocystinuria due to cystathionine beta-synthase (CBS) deficiency. The aim of this work was to study molecular mechanisms underlying protein misfolding of CBS mutants. Firstly, we studied spatial arrangement of normal human CBS protein. Using data from differential covalent labeling of solvent-exposed aminoacid residues, we identified interdomain contact area between the catalytic core and the regulatory domain in human CBS, and we subsequently generated the structural model of the full-length CBS. In the next step, we studied evolutionary divergence of CBS protein structures. We performed phylogenetic analysis that revealed unique spatial arrangement of CBS enzyme in nematodes; the domain architecture of CBS in Caenorhabditis elegans was studied experimentally in more detail. Finally, we determined conformational properties of a representative set of human CBS mutants that exhibited in various extent affected formation of tetramers and decreased catalytic activity. Using thermolysin-based proteolytic techniques for analysis of nine mutants expressed in E.coli, we found that an unfolded structure is a common intermediate occurring in CBS misfolding. The importance of protein unfolding for pathogenesis of CBS deficiency was...

The mathematical theory of homogenization deals with the rigorous derivation of effective models from partial differential equations with rapidly-oscillating coefficients. In this thesis we deal with modeling and homogenization of random heterogeneous media. Namely, we obtain stochastic homogenization results for certain evolutionary gradient systems. In particular, we derive continuum effective models from discrete networks consisting of elasto-plastic springs with random coefficients in the setting of evolutionary rate-independent systems. Also, we treat a discrete counterpart of gradient plasticity. The second type of problems that we consider are gradient flows. Specifically, we study continuum gradient flows driven by λ-convex energy functionals. In stochastic homogenization the derived deterministic effective equations are typically hardly-accessible for standard numerical methods. For this reason, we study approximation schemes for the effective equations that we obtain, which are well-suited for numerical analysis. For the sake of a simple treatment of these problems, we introduce a general procedure for stochastic homogenization – the stochastic unfolding method. This method presents a stochastic counterpart of the well-established periodic unfolding procedure which is well-suited for homogenization of media with periodic microstructure. The stochastic unfolding method is convenient for the treatment of equations driven by integral functionals with random integrands. The advantage of this strategy in regard to other methods in homogenization is its simplicity and the elementary analysis that mostly relies on basic functional analysis concepts, which makes it an easily accessible method for a wide audience. In particular, we develop this strategy in the setting that is suited for problems involving discrete-to-continuum transition as well as for equations defined on a continuum physical space. We believe that the stochastic unfolding method may also be useful for problems outside of the scope of this work.

Cellular senescence is thought to play an essential role in many biological functions including tumor suppression and organismal aging. Senescent cells, which are permanently removed from the cell cycle, can be found both in vivo in many different tissue types and in vitro within cultures of non-immortalized cells. Despite their inability to proliferate, these cells persist and remain metabolically active for indefinite periods of time. This physiologic process occurs in response to a variety of cellular insults including oxidative stress, shortened telomeres, constitutive oncogene expression, and DNA damage, and can be initiated by upregulation of one of the two known senescent pathways, involving p16/Rb or p53/p21. The senescent cell phenotype is also characterized by changes to cell and nuclear morphology and to the secretory profile of the cell. Related to changes in nuclear morphology, epigenetic modifications to the packaging of DNA are thought to be key to the initiation and maintenance of the senescence program. While a large number of earlier studies focused on the findings that senescent cells gain regions of condensed heterochromatin, often in the form of Senescent Associated Heterochromatin Foci (SAHF), this thesis work shows that there is a marked loss of heterochromatin in the peri/centromeric regions of the genome. In fact, both α-satellite and satellite II sequences across the genome distend in a striking and unanticipated fashion; this can be readily visualized by fluorescence in situ hybridization (FISH) as their structure changes from a condensed spot to highly elongated and fine thread-like signals. We have termed this exceptional decondensation of constitutive heterochromatin Senescence Associated Distension of Satellites (SADS). Importantly, a series of experiments shows that SADS is both a consistent and an early event in the cell senescence process, which occurs as a result of every senescence induction method examined. We also observed that this distension was characteristic of both human and murine cells and in vivo in human benign Prostatic Intraepithelial Neoplasia (PIN) tissue. Furthermore, unlike SAHF formation, SADS can occur due to the activation of either of the two senescence pathways, p16/Rb or p53/p21. Additionally, the cytological dimensions of the thread-like satellite signals indicates that SADS represents “unraveling” of DNA on an unprecedented scale. Thus, it was surprising that this event was not facilitated by changes to several canonical histone modifications associated with condensed heterochromatin, namely H3K9Me3, H3K27Me3, or H3K4Me3, nor is it caused by loss of DNA methylation. Consequently, we believe that this marked distension of satellite DNA is due to changes in higher-order folding of the chromatin fiber. This is important for understanding fundamental events in the cell senescence process, but also provides a unique system for study of chromatin packaging that may provide new insights into the organization of DNA well beyond nucleosome packaging and the ten nanometer fiber. In fact, initial super resolution images of SADS suggest that the satellite sequences may be organized into domains or “globules”. Hence, we suggest that the changes to satellite sequence packaging may be facilitated by changes to higher-order nuclear structural proteins, such as LaminB1, which is reduced in senescent cells. Finally, this work provides analysis of the literature and preliminary experiments to consider the possibility that there are increased levels of cell senescence in Down syndrome (trisomy 21) cells. As individuals with Down syndrome (DS) experience many manifestations of premature aging (including early-onset Alzheimer’s Disease), have a resistance to solid tumor formation, are more susceptible to oxidative stress, and are trisomic for several genes implicated in causing senescence, our analysis provides plausibility for the hypothesis that accelerated rates of senescence may play a significant role in DS physiology. We also provide results of preliminary studies and outline the next steps for experimentation, using DS fibroblasts and a unique genetically engineered DS iPS cell system. As a final note, the quantification of cell senescence in trisomic versus disomic cells for these experiments relies substantially on the new single-cell marker of senescence discovered and established by this theses work, the Senescence-Associated Distension of Satellites.

The modelling of single-molecule experiments is of vital interest to gain new insights into processes which were hitherto not accessible by measurements performed on bulk systems. In the first part of this thesis, the kinetics of a triple-branch DNA molecule with four conformational states is investigated by employing pulling experiments with optical tweezers and theoretical modelling. Probability distributions of first rupture forces, which are calculated by applying transition rate theory to a free energy model, show good agreement with experimental findings. Permanently frayed molecules could be identified by analysing the number of opening base pairs in force jumps. In the second part of the thesis, DNA hairpin molecules with periodic base sequences are studied. Their unfolding kinetics allows an analytical treatment, because they exhibit a regular coarse-grained free energy landscape as a function of the number of opened base pairs. A procedure is developed for determining all relevant parameters of the landscape, which relies on probabilities that can be easily sampled from the unfolding trajectories. By means of Monte Carlo simulations it is shown that already 300 trajectories, as typically measured in single-molecule experiments, provide faithful results for the energetic parameters. The approach in particular opens a new access to improve loop contributions in the free energy landscape. In the third part of the thesis, a simulation method is developed for modelling the unfolding kinetics of DNA molecules with arbitrary base sequences. The method is validated against experimental data for five DNA hairpin molecules with different length of the end-loop. Applications of the method enable one, among others, to improve the parameter determination in functional forms suggested for the tail behaviour of work distributions. Such work distributions enter detailed and integral fluctuation theorems, which are useful for estimating free energy differences between folded and unfolded states from nonequilibrium measurements.

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