FrpC protein produced by Neisseria meningitidis in a human host belongs to the family of bacterial RTX toxins due to the presence of RTX domain. FrpC possesses a calcium-dependent auto-catalytic cleavage activity which is localized within its 177 amino-acids long segment Self-Processing Module (SPM). As the SPM is naturally intrinsically disordered protein without bound Ca2+, the calcium binding is crucial for SPM folding which is followed by the auto-catalytic processing. The elucidation of the SPM structure may be the key step for understanding of enzymatic and biological function. The structure of folded SPM itself can be characterized only with difficulties due to the presence of flexible loop according to preliminary NMR data. The subject of this work is the description of SPM using fluorescence methods, characterization of ions binding to SPM and structural changes occurring during Ca2+ binding. In this work, the ion binding properties of SPM segment and its ion-induced folding was characterized. It was found that the dissociation constant kD of 17 μM coincided with the folding of SPM into the native calcium-bound state which occurs in the concentration range between 1 and 20 μM Ca2+. In the attempt to characterize the structure of ion binding site, the fully active single tryptophan mutants...

The distribution of substance P (SP) binding sites in guinea-pig airway was examined by in vitro autoradiography with tritium and iodine-labeled SP. Specific SP binding sites were most abundant in tracheobronchial smooth muscle but were also detected in the mucosa/submucosa. Binding within the mucosa/submucosa was especially high in the region of glands. Binding of iodine-labeled SP to cartilage was negligible. Tritium-labeled SP bound non-specifically to airway cartilage. These observations are consistent with the proposed effects of SP-containing afferent nerves on airway resistance and vascular permeability. The localization of specific SP binding sites suggests that SP may also affect exocrine glands in the respiratory tract.

A central problem in the bioinformatics is to find the binding sites for regulatory motifs. This is a challenging problem that leads us to a platform to apply a variety of data mining methods. In the efforts described here, a combined motif discovery method that uses mutual information and Gibbs sampling was developed. A new scoring schema was introduced with mutual information and joint information content involved. Simulated tempering was embedded into classic Gibbs sampling to avoid local optima. This method was applied to the 18 pieces DNA sequences containing CRP binding sites validated by Stormo and the results were compared with Bioprospector. Based on the results, the new scoring schema can get over the defect that the basic model PWM only contains single positioin information. Simulated tempering proved to be an adaptive adjustment of the search strategy and showed a much increased resistance to local optima.

La voie de signalisation Sonic Hedgehog (Shh) joue un rôle fondamental au cours de l’embryogenèse pour la mise en place de nombreux tissus. Elle persiste à l’âge adulte et régulerait notamment le contrôle de fonctions cérébrales. Son activation requiert la liaison d’un peptide Shh sur le récepteur Patched (Ptc) qui réprime l’activité constitutive de Smoothened (Smo), un récepteur apparenté à la famille des récepteurs couplés aux protéines G (RCPG). Récemment, des essais cliniques pour le traitement de médulloblastomes et de diverses tumeurs solides chez l’Homme ont été menés avec des antagonistes de Smo. Cependant, ces molécules ont révélé des limitations à leur utilisation puisque des résistances au traitement sont apparues. Le travail de cette thèse a conduit au développement d’un modèle pharmacophorique des antagonistes de Smo qui a ensuite permis le criblage virtuel d’une banque de molécules et l’identification de nouvelles familles d’antagonistes de Smo. L’acylthiourée MRT-10 et l’acylurée MRT-14 ont été les deux premiers composés caractérisés. Des études de relations structure-activité ont permis l’identification d’une nouvelle famille d’inhibiteurs du récepteur Smo de haute affinité à laquelle l’acylguanidine MRT-83 appartient. Ce composé s’adapte parfaitement au modèle pharmacophorique des antagonistes de Smo. Les modifications structurales que MRT-83 présentes en comparaison avec les deux têtes de séries précédemment caractérisées sont à l’origine du gain d’activité de MRT-83 sur de nombreux tests cellulaires mettant en jeu l’activation de la voie Shh. Le composé MRT-83 inhibe la liaison de la BODIPY-cyclopamine sur le récepteur Smo humain et bloque la prolifération des précurseurs des cellules granulaires de rat avec une affinité de l’ordre du nanomolaire, comparable à celle des antagonistes de référence de Smo tels que le GDC-0449 et le LDE-225. Malgré l’homologie de séquence entre Smo et la famille des récepteurs Frizzled impliqués dans la signalisation Wnt, le composé MRT-83 ne présente aucun effet sur la voie Wnt. MRT-83 bloque la translocation de Smo dans le cil primaire induite par l’activation de la voie Shh dans les cellules NT2, une lignée issue d’un tératocarcinome humain, contrairement à l’antagoniste de Smo de référence, la cyclopamine qui induit l’adressage du récepteur dans le cil primaire. L’injection stéréotaxique dans le ventricule latéral de cerveau de souris adulte de MRT-83, contrairement à celle d’un composé de structure analogue, dépourvu d’activité sur Smo, inhibe l’expression des transcrits de Ptc induite par l’injection de Shh dans la zone sous-ventriculaire, l’une des deux principales aires de neurogenèse adulte. Ces résultats démontrent que les dérivés MRT bloquent également la signalisation Shh in vivo. Ainsi, les composés MRT-10, MRT-14, MRT-83 et les molécules de structure analogues caractérisées sont de puissants antagonistes de Smo. Ces molécules constituent de nouveaux outils pharmacologiques qui pourraient permettre d’améliorer notre compréhension des mécanismes moléculaires et biochimiques régulant la signalisation Hh et permettre le développement de nouvelles molécules en clinique pour le traitement des tumeurs Hh-dépendantes. / The Sonic Hedgehog (Shh) signaling pathway is implicated in multiple physiological responses including the control of brain functions. In mammals, the Shh pathway is expressed at the primary cilium and its activation requires the binding of a Shh peptide to the Patched (Ptc) receptor which represses the constitutive activity of Smoothened (Smo), a proposed member of the G-protein-coupled receptor (GPCR) family. Recently, clinical trials for treating medulloblastoma and various solid tumors in human have been conducted with Smo antagonists such as GDC-0449 or LDE-225. Such molecules may have some limitations leading to treatment resistance. In the present work, the development of a pharmacophoric model of Smo antagonists allowed a virtual screening strategy to identify novel Smo inhibitors. The acylthiourea MRT-10 and the acylurea MRT-14 were the two first leads identified. Structure-activity relationship experiments led to the discovery of new series of Smo inhibitors with high potency and to which the acylguanidine MRT-83 belongs. This inhibitor perfectly fits with the proposed pharmacophoric model for Smo antagonists. The discrete structural differences between MRT-83 and the original leads may account for the increased potency of MRT-83 observed in various in vitro Shh-based assays. MRT-83 inhibits BODIPY-cyclopamine binding to human Smo and Shh-mediated proliferation of rat granule cell proliferation with nanomolar potency similar to GDC-0449 or LDE-225. Despite significant homology of Smo with the Frizzled family of receptors which are involved in the Wnt signaling pathway, MRT-83 displays no significant effect on this pathway. MRT-83 blocks Smo translocation induced by Shh pathway activation to the primary cilia of NT2 cells that derive from a pluripotent testicular carcinoma whereas cyclopamine, a reference Smo antagonist, induces Smo accumulation of Smo signals at the primary cilium. Therefore, it might be anticipated that MRT-83, like GDC-0449 and LDE-225, interacts with Smo in a manner different from that of cyclopamine, suggesting that while their binding sites are overlapping, they are not identical. Stereotaxic injection of MRT-83 into the lateral ventricle of adult mice but not of a structurally-related compound inactive at Smo, abolished upregulation of Ptc transcription induced by Shh in the neighboring subventricular zone, one of the two main neurogenic areas of the adult brain. These data demonstrate that MRT derivatives efficiently antagonize Shh signaling in vivo. Thus, MRT-10, MRT-14, MRT-83 and structurally-related molecules are potent Smo antagonists. These compounds should be useful for clarifying the molecular and biochemical mechanisms underlying the resistance of Smo inhibitors in brain cancer cells and may help develop new therapies against Shh pathway-related brain diseases.

A doença de Alzheimer (DA) é a forma mais comum de demência, representando cerca de 80% dos casos. A DA é caracterizada por um processo de declínio progressivo e irreversível das funções cognitivas e da memória, que se estende para a desorganização do comportamento. Atualmente, 46,8 milhões de pessoas em todo o mundo foram diagnosticadas com demência. Embora vários fatores tenham sido implicados na DA, sua etiologia ainda não é completamente conhecida. Do ponto de vista neuropatológico, é observado no cérebro de indivíduos com DA atrofia cortical difusa, presença de grande número de placas senis, emaranhados neurofibrilares, processo inflamatório e perda neuronal. A progressão dos sintomas está associada a mudanças estruturais nas sinapses colinérgicas em certas regiões do cérebro, que consequentemente, apresentam neurotransmissão colinérgica reduzida. Os vários eventos patológicos interligados contribuem para o avanço da doença e direcionam diversas pesquisas na busca por tratamentos multialvos com base no processo multifatorial de DA. Assim o presente trabalho descreve a síntese de derivados híbridos dual binding site de donepezila-tacrina (fármacos inibidores de acetilcolinesterase), com potencial para agir em dois alvos terapêuticos pela (i) inibição da acetilcolinesterase em ambos os sítios ativo e periférico, como demonstrado pelos estudos de modelagem molecular, e (ii) na agregação do peptídeo A? neurotóxico induzido pela acetilcolinesterase, na tentativa de interromper a progressão da doença. A estratégia sintética envolveu a condensação da 5,6-dimetóxiindanona com a unidade 4-piperidinil carbaldeído, a qual forneceu o intermediário 5,6- dimetóxindan-1-ona-4-piperidinil-metileno-1-[(4-cloroquinolin-2-il)metil], seguido de redução da dupla ligação, gerada na reação de condensação anterior, e substituição do átomo de cloro-quinolina por amino para obtenção do produto final, ou manutenção da função olefina, seguido de substituição do átomo de cloro-quinolina por azido ou amino, gerando cinco híbridos estruturalmente correlacionados. Os híbridos foram testados em ensaio de inibição de acetilcolinesterase e butirilcolinesterase pelo método de Ellman, e o híbrido insaturado, contendo a função amino-quinolina foi o mais ativo da série com IC50 na faixa de nanomolar (0,014 ?M). Futuramente, os intermediários da reação e produto final serão submetidos ao ensaio de inibição da agregação do peptídeo A? neurotóxico pelo método da tioflavina T. Neste trabalho, também são descritos os testes de predição in vitro para permeação pela barreira hematoencefálica, bem como sua absorção intestinal, pelo método PAMPA. / Alzheimer\'s disease (AD) is the most commom form of dementia in almost 80% of the cases. AD is a process that causes a progressive and irreversible decline of cognitive functions, memory and includes disorganized behavior. Currently, 46.8 million people live with dementia around the world. Although several factors have been implicated in AD, its etiology is not fully understood. In the point of neuropathologic view, diffuse cortical atrophy, presence of senile plaques, neurofibrillary tangles, inflammatory process and neuronal loss are observed in the brain of the patients with AD. The progression of the disease symptoms is associated with structural changes in cholinergic synapses in certain brain regions and consequent impairment of cholinergic neurotransmission. The various interconnected pathological events contribute for the progression of the disease and drive several studies in the search for multitarget treatments based on the multifactorial process of AD. Therefore, this work describes the synthesis of dual binding site tacrine-donepezil hybrids, with the potential to act in two therapeutics targets by: (i) inhibition of the acetylcholinesterase in both peripheral and active sites, as demonstrated in molecular modeling studies, and (ii) the peptide A? neurotoxic aggregation induced by acetylcholinesterase, in an attempt to stop the progression of the disease. The synthetic strategy was based on the condensation between 5,6- dimethoxy-indanone and the 4-piperidinyl carbaldehyde core, which afforded the 5,6- dimethoxy-indanone-4-piperidinyl-methylene-1-[(4-chloroquinolin-2-yl)methyl] intermediate, followed by the reduction of the olefinic bond, formed in the previous condensation, and substitution of the chloro-quinoline atom by amine group to give the final product, or keeping the olefin function, followed by replacement of the chloro-quinoline atom by azide or amine to produce five structural related hybrids. The hybrids were tested in acetylcholinesterase and butyrylcholinesterase inhibition assays using Ellman method, and the unsaturated hybrid containing the amino-quinoline function was the most active of the series with IC50 in a nanomolar range (0,014 ?M). The ability of the hybrids to inhibit protein A? neurotoxic aggregation will be assessed by thioflavin T method. In addition, the blood brain and intestinal barrier permeation by PAMPA methodology were also predicted.

This dissertation presents a detailed computational investigation into the vibrational properties of quinones involved in solar energy conversion processes in photosynthetic reaction centers. In particular, we focus on the vibrational properties of the ubiquinone molecule that occupies the QA binding site in purple bacterial photosynthetic reaction centers. To provide a foundation upon which to base computational studies of pigments in protein binding sites density functional theory based calculations of the vibrational properties of neutral ubiquinone in the gas phase and in solvent were undertaken. From single point energy calculations it was shown that at least eight ubiquinone conformers, each with slightly different FTIR spectra, could be present in solvent at room temperature. The calculated and experimental spectra for neutral ubiquinone in solution are very different from the spectra associated with ubiquinone in the QA binding in purple bacterial reaction centers. For this reason an ONIOM method was undertaken in which the pigment was treated using density functional theory based methods while the protein was treated using molecular mechanics. The ONIOM calculations not only modeled the experimental QA FTIR difference spectra but also resolved the long standing issue of whether a very strong hydrogen bond exists between the bound ubiquinone and the imidazole nitrogen of a histidine residue (HisM219). To further validate the usefulness of the ONIOM approach experimental isotope edited FTIR spectra obtained using purple bacterial reaction centers with a range of chainless symmetrical quinones incorporated were modeled. Again, the agreement between the calculated and experimental spectra is outstanding. We also modeled the vibrational properties of the ubisemiquinone anion radical both in solvent and in the QA binding site. Vibrational modes of ubisemiquinone display a greater degree of mixing of the various molecular groups of the molecule. Nonetheless the calculated FTIR spectra for ubisemiquinone in solution and in the QA site agree very well with that found experimentally. Vibrational frequencies of ubisemiquinone obtained from ONIOM calculated Raman spectra also agree very well with that found in experimental resonance Raman spectra associated with the ubisemiquinone anion radical in the QA binding site.

This dissertation aims at analyzing complex problems arising in the context of dynamical networks, proteomics, and disease prevention. First, a new graph-based method for proving global stability of synchronization in directed dynamical networks is developed. This method utilizes stability and graph theories to clarify the interplay between individual oscillator dynamics and network topology. Secondly, a graph-theoretical algorithm is proposed to predict Ca2+-binding site in proteins. The new algorithm enables us to identify previously-unknown Ca2+-binding sites, and deepens our understanding towards disease-related Ca2+-binding proteins at a molecular level. Finally, an optimization model and algorithm to solve a disease prevention problem are described at the population level. The new resource allocation model is designed to assist clinical managers to make decisions on identifying at-risk population groups, as well as selecting a screening and treatment strategy for chlamydia and gonorrhea patients under a fixed budget. The resource allocation model and algorithm can have a significant impact on real treatment strategy issues.

<p>Uncovering the DNA regulatory logic in complex organisms has been one of the important goals of modern biology in the post-genomic era. The sequencing of multiple genomes in combination with the advent of DNA microarrays and, more recently, of massively parallel high-throughput sequencing technologies has made possible the adoption of a global perspective to the inference of the regulatory rules governing the context-specific interpretation of the genetic code that complements the more focused classical experimental approaches. Extracting useful information and managing the complexity resulting from the sheer volume and the high-dimensionality of the data produced by these genomic assays has emerged as a major challenge which we attempt to address in this work by developing computational methods and tools, specifically designed for the study of the gene regulatory processes in this new global genomic context. </p><p>First, we focus on the genome-wide discovery of physical interactions between regulatory sequence regions and their cognate proteins at both the DNA and RNA level. We present a motif analysis framework that leverages the genome-wide</p><p>evidence for sequence-specific interactions between trans-acting factors and their preferred cis-acting regulatory regions. The utility of the proposed framework is demonstarted on DNA and RNA cross-linking high-throughput data.</p><p>A second goal of this thesis is the development of scalable approaches to dimension reduction based on spectral decomposition and their application to the study of population structure in massive high-dimensional genetic data sets. We have developed computational tools and have performed theoretical and empirical analyses of their statistical properties with particular emphasis on the analysis of the individual genetic variation measured by Single Nucleotide Polymorphism (SNP) microrarrays.</p> / Dissertation

A doença de Alzheimer (DA) é a forma mais comum de demência, representando cerca de 80% dos casos. A DA é caracterizada por um processo de declínio progressivo e irreversível das funções cognitivas e da memória, que se estende para a desorganização do comportamento. Atualmente, 46,8 milhões de pessoas em todo o mundo foram diagnosticadas com demência. Embora vários fatores tenham sido implicados na DA, sua etiologia ainda não é completamente conhecida. Do ponto de vista neuropatológico, é observado no cérebro de indivíduos com DA atrofia cortical difusa, presença de grande número de placas senis, emaranhados neurofibrilares, processo inflamatório e perda neuronal. A progressão dos sintomas está associada a mudanças estruturais nas sinapses colinérgicas em certas regiões do cérebro, que consequentemente, apresentam neurotransmissão colinérgica reduzida. Os vários eventos patológicos interligados contribuem para o avanço da doença e direcionam diversas pesquisas na busca por tratamentos multialvos com base no processo multifatorial de DA. Assim o presente trabalho descreve a síntese de derivados híbridos dual binding site de donepezila-tacrina (fármacos inibidores de acetilcolinesterase), com potencial para agir em dois alvos terapêuticos pela (i) inibição da acetilcolinesterase em ambos os sítios ativo e periférico, como demonstrado pelos estudos de modelagem molecular, e (ii) na agregação do peptídeo A? neurotóxico induzido pela acetilcolinesterase, na tentativa de interromper a progressão da doença. A estratégia sintética envolveu a condensação da 5,6-dimetóxiindanona com a unidade 4-piperidinil carbaldeído, a qual forneceu o intermediário 5,6- dimetóxindan-1-ona-4-piperidinil-metileno-1-[(4-cloroquinolin-2-il)metil], seguido de redução da dupla ligação, gerada na reação de condensação anterior, e substituição do átomo de cloro-quinolina por amino para obtenção do produto final, ou manutenção da função olefina, seguido de substituição do átomo de cloro-quinolina por azido ou amino, gerando cinco híbridos estruturalmente correlacionados. Os híbridos foram testados em ensaio de inibição de acetilcolinesterase e butirilcolinesterase pelo método de Ellman, e o híbrido insaturado, contendo a função amino-quinolina foi o mais ativo da série com IC50 na faixa de nanomolar (0,014 ?M). Futuramente, os intermediários da reação e produto final serão submetidos ao ensaio de inibição da agregação do peptídeo A? neurotóxico pelo método da tioflavina T. Neste trabalho, também são descritos os testes de predição in vitro para permeação pela barreira hematoencefálica, bem como sua absorção intestinal, pelo método PAMPA. / Alzheimer\'s disease (AD) is the most commom form of dementia in almost 80% of the cases. AD is a process that causes a progressive and irreversible decline of cognitive functions, memory and includes disorganized behavior. Currently, 46.8 million people live with dementia around the world. Although several factors have been implicated in AD, its etiology is not fully understood. In the point of neuropathologic view, diffuse cortical atrophy, presence of senile plaques, neurofibrillary tangles, inflammatory process and neuronal loss are observed in the brain of the patients with AD. The progression of the disease symptoms is associated with structural changes in cholinergic synapses in certain brain regions and consequent impairment of cholinergic neurotransmission. The various interconnected pathological events contribute for the progression of the disease and drive several studies in the search for multitarget treatments based on the multifactorial process of AD. Therefore, this work describes the synthesis of dual binding site tacrine-donepezil hybrids, with the potential to act in two therapeutics targets by: (i) inhibition of the acetylcholinesterase in both peripheral and active sites, as demonstrated in molecular modeling studies, and (ii) the peptide A? neurotoxic aggregation induced by acetylcholinesterase, in an attempt to stop the progression of the disease. The synthetic strategy was based on the condensation between 5,6- dimethoxy-indanone and the 4-piperidinyl carbaldehyde core, which afforded the 5,6- dimethoxy-indanone-4-piperidinyl-methylene-1-[(4-chloroquinolin-2-yl)methyl] intermediate, followed by the reduction of the olefinic bond, formed in the previous condensation, and substitution of the chloro-quinoline atom by amine group to give the final product, or keeping the olefin function, followed by replacement of the chloro-quinoline atom by azide or amine to produce five structural related hybrids. The hybrids were tested in acetylcholinesterase and butyrylcholinesterase inhibition assays using Ellman method, and the unsaturated hybrid containing the amino-quinoline function was the most active of the series with IC50 in a nanomolar range (0,014 ?M). The ability of the hybrids to inhibit protein A? neurotoxic aggregation will be assessed by thioflavin T method. In addition, the blood brain and intestinal barrier permeation by PAMPA methodology were also predicted.

The genomes of several organisms have been sequenced and the need for methods to analyse the data is growing. In this project a method is described that tries to identify co-regulated genes. The method identifies transcription factor binding sites, documented in TRANSFAC, in the non-coding regions of genes. The algorithm counts the number of common binding sites and the number of unique binding sites for each pair of genes and decides if the genes are co-regulated. The result of the method is compared with the correlation between the gene expression patterns of the genes. The method is tested on 21 gene pairs from the genome of Saccharomyces cerevisiae. The algorithm first identified binding sites from all organisms. The accuracy of the program was very low in this case. When the algorithm was modified to only identify binding sites found in plants the accuracy was much improved, from 52% to 76% correct predictions.