The field of molecular bioenergetics deals with the energy transduction in biological cells. In this project, respiration and more specifically proton and sodium pumping enzymes and their coupling to electron transfer have been in focus. First we have been interested in the Na+-pumping NADH:quinone reductase from Vibrio cholerae which is the entry site of electrons in the respiratory chain of several pathogens. The role of specific flavin cofactors and amino acids involved in Na+ transfer has been shown in a combined IR spectroscopic and electrochemical approach. The interaction between proteins, namely the cytochrome c552 and the CuA fragment from the terminal ba3 oxidase from the organism Thermus thermophilus was then investigated. Structural reorganization during electron transfer was revealed by IR spectroscopy. Finally, in the third part of the project the interaction within the bc1-aa3 supercomplex from the respiratory chain from Corynebacterium glutamicum was analyzed.

Pattern discovery in protein structures is a fundamental task in computational biology, with important applications in protein structure prediction, profiling and alignment. We propose a novel approach for pattern discovery in protein structures using Particle Swarm-based flying windows over potentially promising regions of the search space. Using a heuristic search, based on Particle Swarm Optimization (PSO) is, however, easily trapped in local optima due to the sparse nature of the problem search space. Thus, we introduce a novel fitness-based stagnation detection technique that effectively and efficiently restarts the search process to escape potential local optima. The proposed fitness-based method significantly outperforms the commonly-used distance-based method when tested on eight classical and advanced (shifted/rotated) benchmark functions, as well as on two other applications for proteomic pattern matching and discovery. The main idea is to make use of the already-calculated fitness values of swarm particles, instead of their pairwise distance values, to predict an imminent stagnation situation. That is, the proposed fitness-based method does not require any computational overhead of repeatedly calculating pairwise distances between all particles at each iteration. Moreover, the fitness-based method is less dependent on the problem search space, compared with the distance-based method. The proposed pattern discovery algorithms are first applied to protein contact maps, which are the 2D compact representation of protein structures. Then, they are extended to work on actual protein 3D structures and interaction networks, offering a novel and low-cost approach to protein structure classification and interaction prediction. Concerning protein structure classification, the proposed PSO-based approach correctly distinguishes between the positive and negative examples in two protein datasets over 50 trials. As for protein interaction prediction, the proposed approach works effectively on complex, mostly sparse protein interaction networks, and predicts high-confidence protein-protein interactions — validated by more than one computational and experimental source — through knowledge transfer between topologically-similar interaction patterns of close proximity. Such encouraging results demonstrate that pattern discovery in protein structures and interaction networks are promising new applications of the fast-growing and far-reaching PSO algorithms, which is the main argument of this thesis. / Thesis (Ph.D, Computing) -- Queen's University, 2014-04-21 12:54:03.37

Genomic DNA is the template of life - the entity which is characterized by a self-sustaining anatomical development, regulated signaling processes, the ability to reproduce and to respond to stimuli. Through what is classically known as the central dogma, the genome is transcribed into mRNA, which in turn is translated into proteins. The proteins take part in most, if not all, cellular processes, and it is by unraveling these processes that we can begin to understand life and disease-causing mechanisms. In vitro and in vivo assays are two levels at which protein communication may be studied, and which permit manipulation and control over the proteins under investigation. But in order to retrieve a representation of the processes as close to reality as possible, in situ analysis may instead be applied as a complement to the other two levels of study. In situ PLA offers the ability to survey protein activity in tissue samples and primary cell lines, at a single cell level, detecting single targets in their natural unperturbed environment.   In this thesis new developments of the in situ PLA are described, along with a new technique offering in situ enzyme-free detection of proximity between biomolecules. The dynamic range of in situ PLA has now been increased by several orders of magnitude to cover analogous ranges of protein expression; the output signals have been modified to offer a greater signal-to-noise ratio and to limit false-positive-rates while also extending the dynamic range further; simultaneous detection of multiple protein complexes is now possible; proximity-HCR is presented as a robust and inexpensive enzyme-free assay for protein complex detection. The thesis also covers descriptions on how the techniques may be simultaneously applied, also together with other techniques, for the multiple data-point acquisition required by the emerging realm of systems biology. A future perspective is presented for how much more information may be simultaneously acquired from tissue samples to describe biomolecular interactions in a new manner. This will allow new types of biomarkers and drugs to be discovered, and a new holistic understanding of life.

La multiplication des résistances aux antibiothérapies actuelles et l’utilisation potentielle de bactéries pathogènes dans le cadre d’attentats bioterroristes rendent nécessaire la recherche de nouvelles cibles biologiques et la découverte de nouvelles stratégies antibiotiques. Dans ce contexte, les mécanismes d’assimilation du fer chez les bactéries à Gram négatif sont des cibles particulièrement prometteuses. Le fer est en effet un élément essentiel à la vie, mais peu biodisponible. Les bactéries ont donc développé des mécanismes efficaces pour subvenir à leurs besoins en fer. Ces mécanismes de transport nécessitent un apport d’énergie fourni par une machinerie bactérienne complexe, la machinerie TonB. La protéine TonB, qui joue un rôle central dans le fonctionnement de cette machinerie, est la cible de notre approche. Nous souhaitons séquestrer cette protéine dans le périplasme grâce à des composés peptidiques fonctionnalisés par des hétérocycles de type isoindole ou 1,2,4-triazine. La conception et la synthèse de ces molécules sont présentées dans ce manuscrit, ainsi que leurs perspectives de vectorisation en utilisant une stratégie dite du "cheval de Troie". Notre contribution à la mise au point d’un test d’affinité in vitro est également abordée. / The increasing resistances to the current antibiotherapies, and the potential use of pathogenic bacteria as biological weapons led us to the absolute necessity of discovering new biological targets and new antibiotic strategies. In this context, iron uptake pathways of Gram negative bacteria are promising targets. Indeed, iron is an essential nutrient, but it has a low bioavailability. Bacteria have developed efficient iron uptake pathways in order to proliferate. Iron is transported in the bacterial cell by specific outer membrane transporters and thanks to the energy provided by a complex molecular machinery, called TonB. The TonB protein, which is the keystone of this machinery, is a key target for the development of new antibiotics. We would like to sequester this protein in the periplasm thanks to molecules constituted of a peptidic moiety and a heterocyclic moiety such as isoindole or 1,2,4-triazine. The conception and the synthesis of these compounds are presented in this document, as well as their possibilities to be vectorized using a “Trojan Horse” strategy. Our contribution to the development of an in vitro test of affinity is presented as well.

No description available.

A fosforilação reversível de proteínas em resíduos de serina e treonina, catalisada por quinases e fosfatases desempenha papel chave na regulação do crescimento e na diferenciação celular em eucariotos. As serina/treonina proteínas fosfatases (PSTPs) são atualmente divididas em três famílias denominadas PPP (PhosphoProtein Phosphatase), PPM (Phosphoprotein Phosphatase Magnesium-dependent) e FCP/SCP (RNA polymerase II CTD phosphatase), sendo que os membros da família PPP são, frequentemente, holoenzimas compostas de uma subunidade catalítica associada a uma ou mais subunidades reguladoras, as quais definem a função, localização e especificidade ao substrato da fosfatase. Neste trabalho, analisamos, através de RT-qPCR, o perfil de expressão dos genes codificadores de subunidades catalíticas de PPPs de Dictyostelium discoideum (PP1c, PP2Ac, PP4c, PP4c-like, PP6c e PP5c) e de 16 potenciais parceiros moleculares de algumas destas subunidades catalíticas, tais como DdI-2 e DdI-3, sabidamente inibidores da PP1c. Em resposta ao estresse térmico de células da fase de crescimento, detectamos o aumento dos níveis de transcritos de PP4c e PP6c e também de DdI-2, DdI-3 e DDB_G0292194, esta última, uma proteína de função desconhecida que interage com a PP1c em ensaios de duplo-híbrido em leveduras. Por outro lado, durante o estresse hiper-osmótico observamos a diminuição dos níveis de transcritos de quase todos os genes analisados com exceção de DdI-2 e DDB_G0292194. O nível de expressão de DdPP1c, DdI-2, DdI-3 e DDB_G0292194 também foi analisado em resposta ao estresse oxidativo e apenas o DDB_G0292194 foi induzido nesta condição. Os genes de PP1c, PP4, PP5c e PP6c são expressos durante todo o ciclo de vida de D. discoideum, mas a expressão de alguns dos genes analisados aumenta em uma fase definida do ciclo de desenvolvimento como é o caso de DDB_G0292194 que tem níveis de transcritos aumentados na fase de agregação. Este gene codifica uma proteína hipotética de 559 aminoácidos, que apresenta um domínio FHA (ForkHead-Associated) em sua região aminoterminal, além de uma sequência similar ao motivo consenso de ligação à PP1c. Ensaios no sistema de duplo-híbrido em leveduras confirmaram que a interação entre DDB_G0292194 e DdPP1c independe do domínio FHA. Verificamos, também, que o mutante nocaute de DDB_G0292194 apresenta uma morfologia alterada em condições padrões de cultivo, tanto na fase de crescimento como durante o desenvolvimento, além de uma maior sensibilidade ao estresse oxidativo causado pelo peróxido de hidrogênio quando comparado à linhagem selvagem. Em conjunto, nossos resultados evidenciam a importância das PPPs na resposta a diferentes tipos de estresse e para o crescimento e desenvolvimento de D. discoideum. / Reversible phosphorylation of proteins on serine and threonine residues, catalyzed by kinases and phosphatases plays a key role in growth and cell differentiation regulation in eukaryotes. Protein serine/threonine phosphatases (PSTPs) are currently divided into three families named PPP (Phosphoprotein Phosphatase), PPM (Phosphoprotein Phosphatase Magnesium-dependent) and FCP/SCP (RNA polymerase II CTD phosphatase). The PPP family members are often holoenzymes composed of a catalytic subunit associated with one or more regulatory subunits, which define function, localization and substrate specificity of the phosphatase. In this work, we have examined, by RT-qPCR, the expression profile of genes encoding PPP catalytic subunits of Dictyostelium discoideum (PP1c, PP2Ac, PP4c, PP4c-like, PP6c and PP5c) and 16 potential molecular partners for some of these catalytic subunits, such as DdI-2 and DdI-3, both known as PP1c inhibitors. In response to heat stress of growth phase cells, we detected increased levels of transcripts of PP4c and PP6c as well as of DdI-2, DdI-3, and DDB_G0292194, the latter a protein of unknown function that interacts with PP1c in yeast two-hybrid assays. Moreover, during the hyperosmotic stress we observed decreased transcript levels of nearly all genes examined except DdI-2 and DDB_G0292194. The expression level of DdPP1c, DdI-2, DdI-3 and DDB_G0292194 was also analyzed in response to oxidative stress and only DDB_G0292194 was induced in this condition. PP1c, PP4c, PP5c and PP6c genes are expressed throughout growth and development of D. discoideum while transcript levels of some the analysed genes were increased at a defined stage of the developmental cycle as in the case of DDB_G0292194, which increased during aggregation. This gene encodes a hypothetical protein of 559 amino acids bearing a FHA (ForkHead-Associated) domain in its aminoterminal region and a sequence matching the PP1c binding consensus motif. Yeast two-hybrid assays confirmed that DDB_G0292194 and DdPP1c interaction does not depend on FHA domain. We also found that DDB_G0292194 knockout mutant exibits an altered morphology on standard growth and developmental conditions and shows an increased sensitivity to oxidative stress induced by hydrogen peroxide in comparison to the wild type strain. Taken together, our results highlight the importance of PPPs in the response to different types of stress and for growth and development of D. discoideum.

A biologia da reprodução de plantas é um campo de grande interesse, já que a maioria dos alimentos consumidos pelo homem é composta de partes reprodutivas das plantas (frutos e sementes). O pistilo é o órgão reprodutivo feminino, composto de estigma, estilete e ovário. Devido à importância central do pistilo no sucesso da reprodução de plantas, faz-se necessário um melhor conhecimento dos genes e processos que regulam seu desenvolvimento e funcionamento. Estudos comparativos da expressão gênica nos órgãos vegetativos e reprodutivos de Nicotiana tabacum revelaram genes de expressão preferencial nos órgãos reprodutivos, entre eles alguns codificando proteínas de função ainda desconhecida. Um destes genes foi caracterizado e denominado SCI1 (Stigma/style Cell-cycle Inhibitor 1), por apresentar um papel importante no desenvolvimento do estigma/estilete, atuando como um inibidor de ciclo celular tecido-específico (DePaoli et al., 2011). O presente trabalho teve como objetivo estudar os mecanismos moleculares pelos quais NtSCI1 regula o ciclo celular, investigando seus parceiros de interação. Em um ensaio de pull-down, utilizando-se extrato proteico nuclear de estigmas/estiletes de N. tabacum, vários putativos reguladores de ciclo celular foram identificados, sendo a interação entre NtSCI1 e NtCDKG;2 confirmada por BiFC e localizada no nucléolo. Uma biblioteca de cDNAs de estigmas/estiletes de N. tabacum, no sistema de duplo-híbrido de levedura, foi construída com sucesso. O screening desta biblioteca, utilizando BD-NtSCI1 como \"isca\", permitiu a identificação de vários parceiros de interação com NtSCI1, entre eles: uma helicase de RNA DEAD-BOX, a proteína 14-3-3D2, dois fatores de transcrição (HOMEOBOX-22 e STOREKEEPER), um fator de splicing portador do domínio SWAP, uma quinase de adenosina e uma transposase. As interações entre NtSCI1 e os três primeiros parceiros citados já foram confirmadas por BiFC (observadas no núcleo e nucléolo) e a interação entre NtSCI1 e Nt14-3-3D2 foi confirmada também por co-imunoprecipitação. O envolvimento de NtSCI1 com a regulação do ciclo celular foi corroborado pela interação entre NtSCI1 e a proteína NtCICLINA-L1 (subunidade regulatória de CDKG;2), confirmada por duplo-híbrido e por BiFC, no nucléolo. A interação entre NtSCI1 e NtCICLINA-RELATED também foi confirmada por BiFC. Para entender a dinâmica de NtSCI1 no nucléolo, foi estudada a localização subcelular da proteína de fusão NtSCI1-GFP durante as fases do ciclo celular. NtSCI1-GFP foi observada no nucléolo de células BY-2 em interfase e prófase, desaparecendo na metáfase e anáfase e reaparecendo no nucléolo no final da telófase, mostrando que a presença de NtSCI1 na célula é controlada pelo ciclo celular. A construção de uma primeira versão do interactoma de NtSCI1 mostrou seu envolvimento direto e indireto com proteínas relacionadas ao metabolismo de RNAs, controle da transcrição e regulação do ciclo celular. Estes resultados sugerem que NtSCI1 possa atuar no controle do ciclo celular de forma não canônica, por meio de múltiplos processos paralelos que interconectam aspectos da regulação da transcrição e o processamento de RNAs com o controle do ciclo celular. / The biology of plant reproduction is a field of great interest, since most of the food consumed by humans is composed of reproductive parts of plants (fruits and seeds). The pistil is the female reproductive organ, composed of stigma, style and ovary. Due to the central importance of the pistil in the success of plant reproduction, a better knowledge of the genes and processes that regulate pistil development and function is necessary. Comparative studies of gene expression in vegetative and reproductive organs of Nicotiana tabacum have revealed genes preferentially expressed in the reproductive organs, among them some encoding proteins of unknown function. One of these genes was characterized and denominated SCI1 (Stigma/style Cell-cycle Inhibitor 1), since it has an important role in stigma/style development, acting as a tissue-specific cell-cycle inhibitor (DePaoli et al., 2011). The objective of the present work was to study the molecular mechanisms through which NtSCI1 regulates the cell cycle investigating its interaction partners. In a pull-down assay, using nuclear protein extracts from N. tabacum stigmas/styles, several putative cell cycle regulators were identified. Among them, the interaction between NtSCI1 and NtCDKG;2 was confirmed by BiFC and localized in the nucleolus. A N. tabacum stigma/style cDNA library in the yeast two-hybrid system was successfully constructed. The screening of this library, using BD-NtSCI1 as bait, allowed the identification of several NtSCI1 interaction partners, among them: a DEAD-BOX RNA helicase; the 14-3-3D2 protein; two transcription factors (HOMEOBOX-22 and STOREKEEPER); a splicing factor containing a SWAP domain; an adenosine kinase; and a transposase. The interactions between NtSCI1 and the first three mentioned partners have already been confirmed by BiFC (observed in the nucleus and nucleolus) and the interaction between NtSCI1 and Nt14-3-3D2 was also wconfirmed by co-immunoprecipitation. The NtSCI1 involvement in cell cycle regulation was corroborated by the interaction between NtSCI1 and the NtCYCLIN-L1 (a regulatory subunit of CDKG;2), which was confirmed by two-hybrid and BiFC in the nucleolus. The interaction between NtSCI1 and NtCYCLIN-RELATED was also confirmed by BiFC. To understand the dynamics of NtSCI1 in the nucleolus, the subcellular localization of the fusion protein NtSCI1-GFP was studied during the different cell cycle phases. NtSCI1-GFP was observed in the nucleolus of BY-2 cells at interphase and prophase, disappearing at metaphase and anaphase and reappearing in the nucleolus at the end of telophase, showing that NtSCI1 presence in the cell is controlled by the cell cycle. The construction of the first version of NtSCI1 interactome showed its direct and indirect involvement with proteins related to RNA metabolism, transcription control and cell cycle regulation. These results suggest that NtSCI1 may act in cell cycle control in a non-canonical way, through multiple parallel processes interconnecting aspects of transcription regulation, RNA processing and cell cycle control.

Human immunodeficiency virus (HIV-1) is the pathogenic agent of HIV infection thatprecedes the total breakdown of cellular immunity, a condition known as acquiredimmunodeficiency syndrome (AIDS). The pandemic nature of the disease has promptedintense research into its biology. Already, much is known about HIV-1 infection, lifecycle,and progression to aids. Systems biology enables the combination of complex data fromthese studies into a framework where their effect on the various levels of cellularorganization (i.e. Pathways, cells, tissues, organs and the whole body) could be studied insilico. In this thesis, first, we reviewed our knowledge of the HIV-1 Human InteractionDatabase. We examined its contents and identified processes that HIV-1 was not previouslyknown to interact with. Then, we attempted an in silico dynamic model of HIV-1 interaction. We built a model of HIV-1 interaction with the CD4 T cell activation pathway comprised of137 nodes (16 HIV-1, 121 human) and 336 interactions. The model reproduced expectedpatterns of T cell activation. Using interaction graph properties, we identified 26 host cellfactors, including MAPK1&3, Ikkb-Ikky-Ikka and PKA, which contribute to the net activationor inhibition of viral proteins. By following a logical Boolean formalism, we identified 9 hostcell factors essential to the functions of viral proteins in the activation pathway. This wasthe first attempt to model dynamic viral-host interaction relationships. Then, we organize HIV-1 interacting host genes into modules to represent cellular processesneeded by the virus. We combined HIV-1 interactions with host gene GO annotations toclassify host genes according to these needed cellular processes. We obtained 201 modulesand found the same set of viral proteins do not interact with host genes having similarmodules suggesting intelligence in its co-ordination of host processes. This work is one of agrowing list that explores coordination of HIV-1 interactions. But more importantly, it would bebeneficial to functionally downsize the large dynamic HIV-1 interaction network. Finally, in our discussion, we discuss our results and suggest possible ways in which our workon dynamic models could be improved. This work is opening up a new field of systems virologythat studies the effect of viruses on the host in terms of its temporal and spatial aspects.

L’élucidation des interactions non-covalentes des complexes biologiques revêt d’une importance majeure dans la compréhension du fonctionnement cellulaire. L’objectif de ce travail de thèse est d’approfondir les développements de la spectrométrie de masse (MS) pour l’étude de ces complexes, que ce soit par MALDI-MS (la désorption-ionisation laser assistée par matrice) ou par ESI-MS (l’ionisation électrospray). Ce travail s’est articulé autour de trois axes : i) étude de la stœchiométrie et de la topologie du complexe SAGA HAT (Spt-Ada-Gcn5 Acétyltransferase, module Histone Acétyl Transferase) par pontage chimique couplé à la MS ; ii) suivi de la dimérisation des complexes formés par RAR-RXR (récepteur de l’acide rétinoïque - récepteur X des rétinoïdes) avec différents ADNs ; iii) mesure de la constante de dissociation des complexes RXR-ligand. Les méthodologies développées ont permis de repousser le potentiel de la MS et d’obtenir des informations structurales des complexes biologiques. / Elucidation of non-covalent interactions of biological complexes takes on great importance for the understanding of cellular function. The purpose of this thesis is a further development of mass spectrometry (MS) for the study of these complexes, either by MALDI-MS (matrix-assisted laser desorption-ionization) or by ESI-MS (electrospray ionization). This work was focused on three main lines: i) study of the stoichiometry and the topology of SAGA HAT (Spt-Ada-Gcn5 Acetyltransferase, Histone Acetyl Transferase module) complex by chemical cross-linking coupled to MS; ii) monitoring the dimerization of the complexes formed by RAR-RXR (retinoic acid receptor - retinoid X receptor) with different DNAs; iii) measuring the dissociation constant of RXR-ligand complexes. The developed methodologies made it possible to expand the potential of MS and get insight into structure of biological complexes.

Dans les thérapies anticancéreuses, les traitements anti-angiogéniques agissant sur l’axe VEGF/VEGFR ont une place importante en clinique. Dans ce contexte, nous avons conçu et évalué l’activité de nouveaux inhibiteurs de l’interaction VEGF/VEGFR. Une première approche a été la conception de molécules antagonistes du VEGFR1. Différents analogues hétérocycliques dérivant d’un composé de type (3-carboxy-2-ureido) thiophène ont été synthétisés. Des réactivités chimiques intéressantes ont été mises en évidence, mais l’activité biochimique de ces molécules ne s’est pas révélée concluante. Une seconde approche reposant sur la conception de peptides ciblant le VEGF a alors été initiée. A partir d’un peptide cyclique connu de 19 résidus ayant une affinité submicromolaire pour le VEGF, de nouveaux peptides et peptidomimétiques ont été développés.L’objectif a été de concevoir des composés de structures chimiques potentiellement plus simples et plus stables en milieu biologique, tout en optimisant l’affinité pour le VEGF. L’interaction de ces peptides avec le VEGF a été étudiée in vitro par ELISA et ITC, ainsi que par cristallographie pour le composé le plus affin. En parallèle, nous avons étudié l’effet du cuivre et d’autres métaux divalents sur l’interaction VEGF/VEGFR1. Au travers d’expériences réalisées au laboratoire ainsi qu’en collaboration, nous avons montré que certains métaux étaient capables non seulement d’inhiber l’interaction VEGF/VEGFR1 mais également d’induire une dimérisation non classique du domaine 2du récepteur. Sachant que les métaux, et en particulier le cuivre, sont connus pour jouer un rôle important dans l’angiogenèse, cette découverte apporte de nouveaux éléments de réponse sur leur mécanisme d’action. Ce travail de thèse s’inscrit donc non seulement dans une démarche de développement de nouveaux composés anti-angiogéniques mais également de compréhension du mécanisme de régulation de l’angiogenèse. / Inhibiting angiogenesis is an effective strategy of targeting therapy against cancer. In thiscontext, we develop an antiangiogenic strategy consisting in the design and evaluation of compoundsblocking the VEGF/VEGFR interaction. The first approach was the conception of antagonists of theVEGFR1. Starting from a (3-carboxy-2-ureido) thiophene hit, a variety of heterocyclic analogs wasdeveloped. Interesting chemical observations were made during the synthesis, but no optimization ofthe biochemical activity was achieved. The second approach was the design of peptides that bind tothe receptor-recognition surface of the VEGF. Starting from a cyclic peptide known to bind to theVEGF with a sub-micromolar affinity, new peptides and peptidomimetics were developed. Thestrategy was to design simplified and potentially more stable compounds, and to improve at thesame time the VEGF affinity. The interaction of VEGF with these ligands was studied in vitro by ELISAand ITC experiments, as well as X-ray diffraction for the best compound. Moreover, the investigationof the effects of copper and other divalent metals on the VEGF/VEGFR1 interaction was undertaken.Experiments realized in the laboratory and in collaboration showed that metals were able to displacethe VEGF/VEGFR1 interaction and to induce the dimerisation of the domain 2 of the receptor. Metalsare well known to play an important role in angiogenic phenomena, but their specific targets are stilla matter of debate. In this context, this discovery brings new response elements regarding theirmechanisms of action. Therefore, the objectives of this PhD thesis were the development of newantiangiogenic compounds, as well as the understanding of some aspects of the regulation of angiogenesis.