Cellular response to environmental changes involves a wide repertoire of complex signalling systems often resulting in up and down regulation of various genes. These mechanisms are generally conserved in a variety of organisms. These pathways are also constantly rewired in various organisms, which aid them in maintaining homeostasis and result in species-specific adaptation mechanisms. Protein kinases are central to these mechanisms and orchestrate a multitude of these pathways. This thesis aims to understand the selective forces behind evolution of signalling pathways. More specifically, this thesis focuses on structural and domain architecture differences of protein kinases. Protein kinases are one of the most populated families of proteins in many organisms and it constitutes about 2-3% of proteomes of most of the eukaryotic organisms. These kinases have evolved over ~400 million years and regulate nearly all major signalling pathways. Classification of kinases enables convenient association of kinases to the function and signalling pathway in which they participate. The current scheme of classification is based on the amino acid sequence of the catalytic region, which consists of about 200-300 residues. This scheme proposes division into 7 groups which show gross level similarities in function such as the TK group, which constitutes all tyrosine kinases, or AGC group which constitutes kinases regulated by second messengers. These groups are further divided into ~280 subfamilies providing us insights into function and regulation at a much finer level. This enables ascertaining information about signalling pathways, protein-protein interactions or substrates the kinase phosphorylates. Chapter 1 provides an elaborate introduction to the various types of protein kinases and their roles in signalling processes. This chapter discusses how protein kinases work in a concerted manner with several other players of a signalling pathway to generate a regulated response to external stimuli. Furthermore, it highlights both the evolutionary aspects and dynamical nature of such pathways. The subsequent part of this chapter deals with protein kinases, their evolution, regulation and structural features crucial to catalysis. Protein kinases are regulated in many ways ¬regulation is achieved from within the catalytic domain and also by means of additional domains tethered to the catalytic domain. The regulatory switch is triggered by various cellular and molecular events such as phosphorylation of specific residues, changes in spatial-temporal localization and altered redox states to name a few. The effects of regulatory domains on the overall function have also been discussed. The chapter concludes by highlighting structural analysis carried out to understand the regulatory aspect of kinases and uses this information in rational drug discovery. Chapters 2 and 3 report identification and analysis of a repertoire of protein kinases encoded in the genomes of two of the organisms which are frequently used in comparative genomics. Chapter 2 focuses on the distribution of kinases in Takifugu rubripes, a teleost fish which is a widely used model system for studying human genes. Use of remote homology detection methods identified 519 kinases in fugu. Although the group-wise distribution of kinases shows high similarity to that of human kinases, subfamily distribution shows considerable differences in 22 subfamilies. They are either under or over-represented in fugu. Most noticeable difference is seen for the DYRK subfamily, which is eight times higher in fugu than human. Detailed analysis of the DYRKs revealed interesting insights into and explained partially their high representation in fugu. Only about ten of these kinases classified into these subfamilies showed high sequence similarity and conserved localization signals to the human kinases and kinases commonly found in other eukaryotes such as C.elegans, S.cereviseae and D.melanogaster. Disparity at the level of genome may be attributed to the observation of unique domain architectures characteristic of this genome. A comparison of domain architectures of kinases documented in Pfam with that of the kinases in Takifugu also revealed two kinases with unique domain architectures in fugu; they are associated with Galectin domain and YkyA domains. Despite inconsistencies in the distribution, human and Takifugu kinases subfamilies remarkable similarity is observed in the MAP kinase pathway, which is ubiquitously found across eukaryotic organisms. Nearly 83% of the proteins in this pathway show more than 30% sequence identity between the two organisms thus, validating the use of Takifugu as a model system to study human signalling pathways. While addressing the possibilities of similar expansions of kinases in other teleosts, it was noticed that the Danio rerio genome (zebrafish) had a massively expanded kinome with ~1200 kinases. Chapter 3 explores the possible reasons for the expansion of kinome with kinases specific to Zebrafish. For e.g., the number of kinases from one subfamily (CAMK) is roughly similar to the total number of protein kinases encoded in the human genome. Further, the PIM kinase subfamily is the sole subfamily, which is massively over-represented (~30 times) in this genome. A detailed analysis of PIM kinases of zebrafish revealed that the sequences are divergent from the canonical PIM kinases. Despite this difference, the specific residues, which dictate the functional properties specific to PIM kinases, are highly conserved. These PIM kinases are usually constitutively active, features of which are conserved in PIM kinases of zebrafish as well. Unlike canonical PIM kinases in other eukaryotes, the post-transcriptional regulation of these PIM kinases might be different due to the absence of regulatory regions in the 3'UTR regions of the PIM gene. However, conservation of a S261 phosphorylation site highlights regulation by phosphorylation, which compensates for the constitutively active nature. A massive expansion of the substrate pool of PIM kinases in this genome seems to correlate well with the expansion. Since PIM kinases regulate large number of growth related pathways, we believe that, this might be associated with high regenerative capacity of organs observed in this fish, which makes it an ideal model to study most cancers. While the earlier two chapters primarily focused on the kinase catalytic domain and organism specific changes; the next two chapters address the contribution of domains tethered to the catalytic domain in the overall function of the kinase. Deviations from canonical kinase domain architectures indicate expansion in the functional repertoire of kinases. Chapter 4 is a study on human kinases from the latest revised version of the human genome sequence data. The initial part of the chapter focuses on the differences in the kinase repertoire upon revision of the human genomic data. Seven sequences gleaned from the earlier genomic data are absent and 16 new sequences are added to the kinome dataset according to the latest human genome sequence data. In addition, differences in transcripts for 23 kinases have led to differences in overall length and sub-family classification of these kinases. The identification of the kinome data from this latest version was a mandatory step prior to the study of outlier kinases due to variations in gene transcripts. The domain architectures of the human kinases have been compared with known subfamily-specific domain architectures, in order to identify outliers. Based on the type of domain architecture these outliers have been classified as “rogue” or “hybrid” kinases. Hybrid architecture represent kinases showing high sequence similarity within the kinase domain to a known sub¬family of kinases with the acquisition of non-kinase domains typically found in one of the other subfamilies of kinases. On the other hand rogue architectures belong to kinases with domain architectures not observed in any of the kinase sub-families. A total of 23 outliers have been identified in the human genome-13 hybrids and 10 rogues. The presence of such "hybrid" and "rogue" kinases makes classification of kinases into subfamilies a daunting task and hence necessitates a new method for classification using the full-length sequences. The use of one such alignment-free method, ClaP (Appendix), using full length sequences has been validated for classification of kinases. A similarity metric obtained from full protein sequence comparison further improved the existing methods of classification for 29 kinases, which utilize only the catalytic domain of kinases. Classification based on catalytic domain is incomplete without the knowledge of associated domains, which also have an important role in function. This necessitates a new approach in classification of kinases for function annotation-an integrated one that uses information from the full-length sequence of each kinase. Chapter 5 extends the learning from chapter 4 and aids in identification of 74 "Hybrid" and 18 "Rogue" kinases in other model eukaryotes, Mus musculus, C.elegans, S. Cerevisiae, D. melanogaster and Takifugu rubripes which show significant variations in the overall functions. These sequences due to their hybrid nature might facilitate cross-talk between signalling pathways. Thus annotating the function of each of these 92 outliers has highlighted the use of domain recombination in wiring new pathways and re-wiring existing pathways. Also, these sequences because of their hybrid nature cannot be classified under any of the existing sub-families. Therefore, it has been proposed in this chapter that they be classified as separate sub-family containing sequences with hybrid properties. To validate this, the ClaP method has been extended where the pair-wise distances between two sequences (using full length sequence) has been used to generate phylogenetic trees which have then been subjected to hierarchical clustering to generate sub-family based clusters. Further, a Shannon entropy based score has been used to identify clusters that contain sequences from diverse sub-families grouped together. Upon analysis of these clusters, it was observed that the hybrid and rogue kinases specifically cluster within four clusters with high entropy (constitute large number of sub-families) validating their status as emergent sub-families. In addition, more hybrids and rogues have been identified in these clusters, which have long regions without any domain assignments. Such sequences may contain domain families deviant from those that are currently known and information on their function can be obtained from further genomic studies in future. Lastly, the prevalence of such hybrid and rogue kinases in the genome of a protozoan parasite, P. falciparum has been studied in detail. The role of hybrids and rogues in host-pathogen interaction has been explored. Chapter 6 presents an in-depth analysis of the possible role of charge-neutralization around phosphosites in protein kinases and its substrates. This analysis was a follow up of a study and in collaboration with Dr.Warwicker's group in Manchester, which identified positively charged residues around phosphosites in kinase substrates. The current study not only aims to address the importance of charge neutralization around phosphosites, but also uses this feature for prediction of phosphosites in known structures of kinase substrates. A dataset of phosphosites mapped on a 3-D structure has been used to calculate peak electrostatic potentials around phosphosites based on the solution of a non-linear Poisson-Boltzmann equation. A comparison of peak potentials around phosphosites with that of non-phosphosites reveals a higher positive peak potential at ~10.0 Å radius around the phosphosite. This variation is significantly higher around tyrosine residues in comparison to Ser/Thr residues phosphosites. Further, this distinction in peak potential around the phosphosite is attributed to only certain families like protein kinases and pyruvate kinases. The concept of charge neutralization will therefore show greater success in prediction of phosphosites in such families in comparison to other families with phosphosites. The functional importance of such charge neutralizations has been studied in great detail in the protein kinase domain family due to prior knowledge that certain phosphorylation events contribute to conformational change, which may be correlated to the changes in peak potentials upon phosphorylation. Phosphorylation at certain sites within the kinase catalytic domain often mediates onset of certain signalling events including regulating activity levels of kinases, mediating protein-protein interactions and altering their localization. Therefore, by means of studying conservation patterns of such phosphosites or neutralizing residues, the variations in signalling pathways in homologues with differences in conservation patterns, have been highlighted. Among domain families which do not show clear differences in peak potentials between phosphosites and non-phosphosites, it was noted, in a few cases, that negatively charged ligands bind to the protein in the vicinity of phosphosites, in the un-phosphorylated forms of the protein. Structural studies on a few cases in ligand bound forms indicate a competitive mechanism between phosphorylation and ligand binding which helps in switching between different functional forms. Therefore, the role of phosphorylation as a regulatory mechanism for modulating ligand binding in such domain families has been highlighted. Chapter 7 of the thesis reports a study on disease causing mutations in kinases. So far 180 kinases have been reported to contain disease causing mutations. This chapter particularly focuses on understanding the deleterious effects of non-synonymous missense mutations in kinases. Mutations at certain sites are enriched as seen by the concentration of disease phenotypes upon mutations at these sites in comparison to others. Interactions involving Arginines in sub-domains VIB, VIII, IX and XI are perturbed which affect catalysis. Structural explanation of 10 such mutations, which occur in important sub-domains and not directly implicated in catalysis has been provided. Apart from analyzing the various evolutionary and structural aspects of protein kinases in this thesis an attempt has been made to provide a deeper structural understanding of Msh (MutS Homologues) proteins involved in eukaryotic chromosomal segregation. Chapter 8 deals with Msh4-Msh5 complex, which are eukaryotic homologues of the MutS family of proteins in bacteria. MutS proteins form homodimeric complexes in bacteria that aid in mismatch repair process. There are six MutS homologues in eukaryotes, which form hetero-dimers. Two of the homologues are Msh4 and Msh5, which form hetero-dimeric complexes which is a pre-requisite for its function. They are involved in chromosomal segregation during meiosis-I and aid in resolving Holliday junction DNA. Till date no structure of this complex is available and the exact mode of binding is unclear. In addition, Msh4 and Msh5 display asymmetry in DNA and ATP binding sites. These insights are derived from the severity in phenotypes upon mutation of various residues in these proteins. This work is in collaboration with Dr. Nishant from IISER, Trivandrum. The questions addressed in chapter 8 of the thesis are: What are the structural features that contribute to the asymmetry in function between Msh4 and Msh5 in DNA and ATP binding? Can a structural explanation be provided for each of the 27 mutations causing severe phenotypes (cross-over defects/viability) to predict their role in function of the Msh4-Msh5 complex? Can a prediction be provided for the mode of binding of the Holliday junction DNA? Can residues occurring at interface regions of Msh4 and Msh5 be identified on the basis of the structure which affects the complexation of Msh4 and Msh5? These questions are addressed by homology modelling of the Msh4-Msh5 complex using the Msh2-Msh6 complex as template. Structural explanations have been provided for 23 out of 27 mutations with severe phenotypes. Certain residues in Msh5 are shown to form tighter network of interactions than their counterparts in Msh4 and therefore likely to have a more prominent role in DNA and ATP binding which corroborate with the observed asymmetry in mutant functions. A volume based calculation has been used to suggest a possible mode of binding of the Holliday junction within the cavity of the complex. Finally, the model has been used to predict interface residues that play a crucial role in complexation and function. Experiments are being carried out in Dr. Nishant's laboratory to mutate these residues to validate the model. Chapter 9 summarizes the entire thesis work and also clearly states the chief conclusions from various chapters. Apart from studies embodied in the thesis, the author has been involved in one other study, which is provided as appendix.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Duguetia furfuracea is a common shrub from Brazilian cerrado areas, known as "ata brava", often used as a medicinal plant especially in treatment of renal colic and rheumatism. However, pharmacological studies with extracts obtained from different parts of this plant have shown cytotoxic activity, bactericidal and anti-tumor. Thus, this study aimed to identify and quantify the phenolic compounds of the hydroalcoholic extract of leaves D. furfuracea (HEDP), methanol (Mt-OH) and ethyl acetate (Ac-OEt) fractions by HPLC, and carry out phytochemical screening for different classes of compounds. In addition the antioxidant activity by the DPPH and FRAP assays, toxicity of the crude extract in the Drosophila melanogaster model, and the antifungal/modulatory activity were evaluated. The phytochemical screening revealed the presence of alkaloids, tannins, xanthones, chalcones, flavonoids, aurones and phenolic acids. HPLC analysis revealed that major components of HEDF were caffeic acid (33.17 ± 0.03 mg/g) and rutin (20.56 ± 0.01 mg/g), for the Mt-OH fraction were, caffeic acid (32.47 ± 0.03 mg/g) and quercitrin (31.96 ± 0.03 mg/g), and for Ac-OEt fraction were, quercitrin (32.97 ± 0.03 mg/g) and isoquercitrin (31.56 ± 0.01 mg/g). Although highest levels of phenols and total flavonoids were found in Ac-OEt fraction, the crude extract showed the highest antioxidant (in vitro) potential. The toxicity of the extract was confirmed (in vivo) associating the extract with standard diet of D. melanogaster at different concentrations. The extract caused a significant increase in mortality on the third day of exposure of flies at higher concentrations (100 and 200 mg/ml). The 50mg/ml concentration promoted decrease in motility of flies. Interestingly, the activity of acetylcholinesterase (AchE) was increased in flies exposed to concentrations of 1 and 10mg/ml and inhibited by the concentration of 50 mg/ml of HEDF. The cell viability was significantly compromised flies at all HEDF concentrations (1, 10 and 50 mg/ml) although it has been observed a significant increase in production of reactive oxygen species flies exposed only in the concentration of 50mg/ml. In this context it was evaluated the influence of extract (ex vivo) in the activity of antioxidant defenses of D. melanogaster exposed to concentrations of 1 and 10 mg/ml of HEDF resulted in a significant increase in the activity of enzymes Glutathione s-transferase (GST), Superoxide dismutase (SOD) and catalase (CAT), however, for the concentration of 50 mg/ml of extract, there was a dramatic decrease in the activity of GST with no change in SOD activity and CAT. The cleavage of PARP investigated as a general index of cell death by apoptosis was confirmed in flies exposed to all HEDF concentrations. Yet exposure of the flies to HEDF 10 mg/mL significantly increased the phosphorylation of kinase ERK. The extract and fractions tested against Candida albicans, Candida tropicalis and Candida krusei showed no fungicidal activity since the minimum inhibitory concentration (MIC) was ≥ 1.024 μg/ml for all fungal strains tested. However, the HEDF and the fractions Ac-OEt and Mt-OH had a synergistic effect when combined with fluconazole, indicating modulatory action against fungi when associated with clinically relevant medicine. The HEDF and the fraction Mt-OH potentiated the effect of fluconazole when tested against C. kruzei, and the fraction Mt-OH also showed synergy with fluconazol against C. tropicalis. The fraction Ac-OEt potentiated the effect of fluconazol against C. albicans. The set of results suggests that oxidative stress may be an important mechanism underlying the toxicity induced by extract of D. furfuracea in Drosophila melanogaster. Additionally modulatory activity of the extract and fractions towards fluconazol improve the biomedical applications D. furfuracea. / Duguetia furfuracea é um arbusto comum em áreas do cerrado brasileiro, conhecida como ata brava , costuma ser usada como planta medicinal especialmente no combate a cólica renal e reumatismo. Entretanto, estudos farmacológicos com extratos obtidos de diferentes partes dessa planta têm evidenciado atividades citotóxica, bactericida e antitumoral. Assim, esse trabalho teve como objetivos identificar e quantificar os compostos fenólicos do extrato hidroalcoólico das folhas de Duguetia furfuracea (EHDF) e frações metanólica (Mt-OH) e acetato de etila (Ac-OEt) por meio de HPLC, e realizar triagem fitoquímica para diferentes classes de compostos. Ainda avaliar a atividade antioxidante por meio dos ensaios DPPH e FRAP, a toxicidade do extrato bruto no modelo de Drosophila melanogaster, a atividade antifúngica e modulatória do extrato e frações. A triagem fitoquímica revelou presença de alcalóides, taninos, xantonas, chalconas, flavonoides, auronas e ácidos fenólicos. A análise por HPLC no EHDF revelou como principais componentes ácido caféico (33,17 ± 0,03 mg/g) e rutina (20,56 ± 0,01 mg/g), para a fração Mt-OH, o ácido caféico (32,47 ± 0,03 mg/g) e quercitrina (31,96 ± 0,03 mg/g), e para Ac-OEt, quercitrina (32,97 ± 0,03 mg/g) e isoquercitrina (31,56 ± 0,01 mg/g). Os mais altos níveis de fenois e flavonoides totais foram encontrados na fração Ac-OEt, entretanto, o extrato bruto apresentou maior poder antioxidante (in vitro) do que as frações. A toxicidade do extrato foi confirmada no modelo in vivo Drosophila melanogaster. O extrato promoveu aumento significativo da mortalidade no terceiro dia de exposição das moscas nas concentrações mais altas (100 e 200mg/ml). A concentração de 50mg/ml promoveu redução na motilidade das moscas. A atividade da acetilcolinesterase (AchE) foi aumentada nas moscas expostas as concentrações de 1 e 10 mg/ml e inibida na concentração de 50 mg/ml do EHDF. A viabilidade celular das moscas foi significativamente comprometida em todas as concentrações do EHDF (1; 10 e 50mg/ml) embora tenha sido observado um aumento significativo na produção de espécies reativas de oxigênio apenas nas moscas expostas a concentração de 50mg/ml. Nesse contexto foram avaliadas as defesas antioxidantes de D. melanogaster A exposição das moscas as concentrações de 1 e 10 mg/ml do HEDF resultou em aumento significativo na atividade das enzimas Glutationa s-transferase (GST), Superóxido dismutase (SOD) e Catalase (CAT), entretanto, na concentração de 50 mg/ml do extrato, houve diminuição na atividade da GST sem alterações na atividade da SOD e CAT. A clivagem da PARP, investigada como índice geral de morte celular por apoptose, foi confirmada nas moscas expostas a todas as concentrações do EHDF. Ainda a exposição das moscas a 10 mg/mL do EHDF aumentou significativamente a fosforilação de ERK. O extrato e frações testados contra Candida albicans, Candida tropicalis e Candida krusei não apresentaram atividade fungicida considerando que a concentração inibitória mínima (CIM) foi ≥ 1,024 μg/ml para todas as estirpes de fungos testadas. Entretanto, o extrato e as frações Ac-OEt e Mt-OH apresentaram efeito sinérgico quando associadas ao fluconazol, indicando ação moduladora contra fungos mediante associação a medicamento clinicamente relevantes. O EHDF e a fração Mt-OH potencializaram o efeito do fluconazol quando testados contra a C. kruzei, e a fração Mt-OH também apresentou sinergismo com fluconazol contra C. tropicalis. A fração Ac-OEt potencializou o efeito do fluconazol contra C. albicans. O conjunto dos resultados sugere que o estresse oxidativo pode ser um mecanismo importante subjacente a toxicidade induzida por Duguetia furfuracea em Drosophila melanogaster, e atividade modulatória do extrato e frações junto ao fluconazole amplia as aplicações biomédicas de Duguetia furfuracea.

Streptococcus pneumoniae (ou pneumocoque) est un agent pathogène humain responsable de maladies invasives et potentiellement mortelles. Les mécanismes impliqués dans le processus d'invasion restent largement inconnus, mais plusieurs observations suggèrent que les processus de signalisation impliquant la phosphorylation des protéines participeraient au caractère invasif du pneumocoque. Le génome de S. pneumoniae code pour une seule tyrosine-kinase (CpsD) et une seule sérine/thréonine-kinase (StkP). Cette dernière serait notamment impliquée dans la virulence, la compétence et la division cellulaire. Elle représente donc une cible thérapeutique potentielle intéressante pour lutter contre les infections liées au pneumocoque. L'objectif de cette thèse a donc été de caractériser le rôle de cette sérine/thréonine-kinase StkP dans la division cellulaire du pneumocoque. StkP est une protéine transmembranaire qui se caractérise par la présence de motifs structuraux conservés dans son domaine catalytique appelés motifs de Hanks. De plus, StkP possède un domaine extracellulaire composé de la répétition de quatre domaines PASTA (Penicillin-binding protein And Serine/Threonine kinase Associated). Le modèle actuel suggère que ces domaines PASTA seraient capables de fixer des fragments de la paroi cellulaire afin de permettre l'activation de StkP qui se comporterait donc comme un récepteur membranaire permettant de réguler la division cellulaire du pneumocoque. Mes travaux de thèse ont permis de revisiter ce modèle en démontrant que les domaines PASTA ne servent pas uniquement à contrôler l'activité protéine-kinase de StkP mais également à contrôler l'épaisseur de la paroi cellulaire et ainsi permettre la constriction de la cellule. Plus précisément, j'ai démontré que le domaine PASTA distal est spécialisé dans l'interaction avec une hydrolase de la paroi cellulaire alors que les trois autres domaines PASTA sont nécessaires à l'activité kinase de StkP mais également au positionnement du domaine PASTA distal. Ainsi, le domaine extracellulaire de StkP se comporterait comme une règle permettant de définir l'épaisseur de la paroi cellulaire de la bactérie. Ces travaux permettent donc de proposer un nouveau modèle d'activation et de régulation de la division cellulaire par la sérine/thréonine-kinase StkP / Streptococcus pneumoniae (the pneumococcus) is one of the most important human pathogens that causes potentially fatal invasive diseases. Mechanisms required for the pneumococcal invasion process remain largely unknown, but several observations suggest that phosphorylation-based signaling processes will be at play in the invasiveness of the pneumococcus. S. pneumoniae encodes only one tyrosine-kinase (CpsD) and one serine/threonine-kinase (StkP). The latter would be involved in virulence, competence, and cell division. StkP represent therefore a promising target to combat pneumococcal infections. My aims were to better understand the role of StkP in pneumococcal cell division. StkP is a transmembrane protein characterized by the presence of a series of conserved structural motifs called Hanks motifs in its catalytic domain. In addition, StkP possesses an extracellular domain composed of the repetition of four PASTA domains (Penicillin-binding protein And Serine/Threonine kinase Associated). The current model proposes that PASTA domains are able to bind cell wall fragments resulting in StkP kinase activation. StkP would thus behave as an authentic kinase receptor regulating cell division. My thesis works has allowed to revisit this model by showing that PASTA domains do not only serve StkP kinase activation. Rather, they contribute to determine the cell wall thickness and govern cell constriction. More precisely, I demonstrated that the distal PASTA domain possesses unique features for the binding of a cell wall hydrolase whereas the other three contributes to StkP kinase activation and the positioning of the distal PASTA domain. Thus, the extracellular domain of StkP acts as a ruler determining the cell wall thickness. This work allows to propose an alternative model of activation and regulation of cell division by the serine/threonine-kinase StkP

Legionella pneumophila est une bactérie opportuniste qui émerge de l'environnement après multiplication dans des amibes et peut infecter accidentellement les macrophages alvéolaires humains, provoquant une pneumonie sévère, la légionellose. La capacité de L. pneumophila à survivre dans ses cellules hôtes est strictement dépendante du système de sécrétion de type 4 Dot/Icm, qui sécrète un large répertoire d'effecteurs dans le cytosol de l'hôte. Identifier la contribution individuelle de chaque protéine bactérienne sécrétée par le système Dot/Icm, dans le cycle infectieux de L. pneumophila reste un enjeu majeur pour comprendre les bases moléculaires de la virulence des légionelles. Mes travaux de thèse participent à cet objectif en caractérisant la voie cellulaire ciblée par la protéine kinase LegK2. Des tests d'interaction et de phosphorylation ont identifié le complexe nucléateur d'actine ARP2/3 comme cible de LegK2. Suite à l'adressage de LegK2 à la surface de la vacuole après sa translocation dans le cytosol de l'hôte, l'interaction LegK2-ARP2/3 inhibe la polymérisation d'actine sur le phagosome. Cette inhibition permet à Legionella de diminuer le trafic des endosomes tardifs et/ou des lysosomes vers le phagosome et favorise ainsi l'évasion du phagosome à la voie de dégradation endocytique. L'interaction LegK2-ARP2/3 met en évidence un mécanisme original de virulence dans lequel le remodelage local du cytosquelette d'actine de la cellule hôte permet à la bactérie de manipuler le trafic vésiculaire pour échapper aux défenses de l'hôte / Legionella pneumophila is an opportunistic bacterium that emerges from the environment after multiplication in protozoans and can accidentally infect human alveolar macrophages leading to a severe pneumonia, the legionellosis. The L. pneumophila ability to survive within host-cells is strictly dependent on the Dot/Icm Type 4 Secretion System that translocates a large repertoire of effectors into the host cell cytosol. Deciphering the individual contribution of each bacterial protein translocated by the Dot/Icm system in the L. pneumophila infectious cycle remains a major challenge to understand the molecular basis of Legionella virulence. My works contribute to this objective by characterizing the cellular pathway targeted by the protein kinase LegK2. Interaction and phosphorylation assays identified the actin nucleator ARP2/3 complex as the target of LegK2. Following the LegK2 addressing to the vacuole surface after its translocation into host cytosol, LegK2- ARP2/3 interplay inhibits the actin polymerization on the phagosome. This inhibition allows Legionella to decrease the late endosome/lysosome trafficking towards the phagosome and promotes the phagosome evasion from endocytic degradation pathway. LegK2-ARP2/3 interplay highlights an original mechanism of virulence wherein the local actin cytoskeleton remodeling of host cell allows bacteria to hijack the vesicles trafficking in order to escape host-cell defenses

Malgré les modèles établis pour certaines bactéries, la morphogenèse de bactéries de formes atypiques est peu comprise. C'est le cas de la bactérie pathogène pour l'homme Streptococcus pneumoniae, ou pneumocoque, qui possède une forme ovo-diplococcale. Cependant, à mon arrivé au laboratoire, il avait été démontré qu'une sérine/thréonine protéine-kinase membranaire appelée StkP était indispensable à la division cellulaire et à la morphogenèse du pneumocoque. L'objectif de ma thèse a ainsi été de caractériser certains substrats de StkP et d'étudier leur rôle, ainsi que l'impact de leur phosphorylation, au cours du processus de division cellulaire. Dans ce contexte, j'ai montré que le substrat DivIVA et son paralogue GpsB coordonnent l'élongation et la division cellulaire du pneumocoque. Ces travaux permettent de proposer un nouveau modèle de morphogenèse du pneumocoque dans lequel la triade StkP/DivIVA/GpsB organise la synthèse de la paroi cellulaire nécessaire à l'élongation et à la division de la cellule. J'ai également mis en évidence que la protéine MapZ interagit avec la paroi cellulaire lors de l'élongation cellulaire afin de marquer de manière permanente le site de division, où elle recrute la protéine FtsZ. Ces travaux ont ainsi permis d'identifier un système inédit de régulation positive du positionnement du site de division chez les bactéries. Enfin, j'ai caractérisé les déterminants moléculaires du positionnement de MapZ au centre de la cellule. S. pneumoniae étant un pathogène humain important, nous pouvons anticiper que nos données pourraient servir de base fondamentale à des projets plus appliqués de lutte contre les infections bactériennes / Despite the established models for some bacteria, the morphogenesis of bacteria with atypical shapes is poorly understood. This is the case of the human pathogen Streptococcus pneumoniae, or pneumococcus, that displays an ovo-diplococcal shape. However, when I joined the lab, it had just been shown that a membrane serine/threonine kinase named StkP was crucial for the cell division and the morphogenesis of the pneumococcus. The goal of my thesis was to characterize the substrates of StkP and to study their function as well as the impact of their phosphorylation in the cell division process. First, I have shown that the substrate DivIVA together with its paralog GpsB coordinate cell elongation and division of the pneumococcus. Based on these observations, we propose a new model of pneumococcal morphogenesis in which the triad StkP/DivIVA/GpsB organizes cell wall synthesis involved in cell elongation and division. In a second part of my work, I have studied another substrate of StkP that was of unknown function and that we named MapZ. I have shown that MapZ interacts with the cell wall during the cell elongation to position at midcell. Then MapZ recruits the cell division protein FtsZ and controls the closure of the Z-ring. This work has uncovered a new mechanism of positive regulation for the positioning of the division site in bacteria. Finally, I characterized the molecular determinants of MapZ positioning at the division site. S. pneumoniae is an important human pathogen, we can thus anticipate that our work will represent a fundamental base for applied projects in order to develop new strategies against bacterial infections

No description available.

AMP-activated protein kinase (AMPK) is a key regulator of energy homeostasis in cells. It has been implicated as a therapeutic target for various metabolic diseases like type II diabetes and obesity. However, its role in cancer is context-dependent and therefore warrants further studies to explore its possible use as a therapeutic target. AMPK can either promote or retard the growth of cancer cells depending on other cues and stresses in the milieu of the cancer cells. This study aims to understand AMPK signalling in response to extracellular cues of matrix deprivation and matrix stiffness that are important determinants of metastasis. 1) Calcium-Oxidant Signalling Network Regulates AMPK Activation upon Matrix Deprivation. Recent work from our lab, as well as others, has identified a novel role for the cellular energy sensor AMP-activated protein kinase in epithelial cancer cell survival under matrix deprivation. However, the molecular mechanisms that activate AMPK upon matrix-detachment remain unexplored. In this study, we show that AMPK activation is a rapid and sustained phenomenon upon matrix deprivation, while re-attachment to the matrix leads to its dephosphorylating and inactivation. Since matrix-detachment leads to loss of integrin signalling, we investigate whether integrin signalling negatively regulates AMPK activation. However, modulation of FAK or Src, the major downstream components of integrin signalling, fails to cause a corresponding change in AMPK signalling. Further investigations reveal that the upstream AMPK kinases, LKB1 and CaMKKβ, contribute to AMPK activation upon detachment. Additionally, we show LKB1 phosphorylation and cytosolic translocation upon matrix deprivation, which might also contribute to AMPK activation. In LKB1-deficient cells, we find AMPK activation to be predominantly dependent on Caskβ. We observe no change in ATP levels under detached conditions at early time points suggesting that rapid AMPK activation upon detachment is not triggered by energy stress. We demonstrate that matrix deprivation leads to a spike in intracellular calcium as well as oxidant signalling and both these intracellular messengers contribute to rapid AMPK activation upon detachment. We further show that ER calcium release induced store-operated calcium entry (SOCE) contributes to intracellular calcium increase, leading to ROS production, and AMPK activation. We additionally show that the LKB1/CaMKK-AMPK axis and intracellular calcium levels play a critical role in anchorage-independent cancer sphere formation. We find a significant increase in LKB1 as well as pACC levels in breast tumour tissues in comparison to normal tissues. Further, we observe a significant correlation between LKB1 and pACC levels in breast tumour tissues suggesting that LKB1-AMPK signaling pathway is active in vivo in breast cancers. Thus, the Ca2+/ROS triggered LKB1/CaMKK-AMPK signalling cascade may provide a quick, adaptable switch to promote survival of metastasising cancer cells. 2) Extracellular Matrix Stiffness Regulates Stemless through AMPK. Cancer cells experience changes in extracellular matrix stiffness during cancer progression. However, the signalling pathways utilised in sensing matrix stiffness are poorly understood. In this study, we identify AMPK pathway as a possible mechanosensory pathway in response to matrix stiffness. AMPK activity, as measured by downstream target phosphorylation, is found to be higher in soft matrix conditions. We additionally show that compared to stiff matrices, soft matrices increase stemless properties, as evidenced by the increased expression of stemless markers, which is dependent on AMPK activity. Thus, we elucidate a novel mechanotransduction pathway triggered by matrix stiffness that contributes to stemness of cancer cells by regulating AMPK activity. Taken together, our study identifies a novel calcium-oxidant signaling network in the rapid modulation of AMPK signaling in the context of matrix detachment. This pathway is especially relevant in the context of metastasising cancer cells that may not face energy stress in the blood stream but are matrix-deprived. Inhibition of AMPK might compromise the viability of these circulating cells thereby reducing the metastatic spread of cancer. Our study further suggests that varying stiffnesses experienced by cancer cells can modulate AMPK activity and this, in turn, regulates stem-like properties. Thus our study provides novel insights into various extracellular cues that regulate this kinase and contribute to cell survival and metastasis. This knowledge can be utilised in the stage-specific use of AMPK inhibitors in the treatment of breast cancer patients.

Pyrazoloanthrone and its analogues form the central core of the thesis and the work is focused on the evaluation of chemical and biological applications of pyrazoloanthrones. Selective and sensitive detection of biologically, environmentally and industrially important molecular species such as fluoride, cyanide and picric acid by using pyrazoloanthrones as sensors form the first part while the second part deals with selective and specific kinase inhibition by pyrazoloanthrones to moderate inflammation associated disorders like septic shock. All the investigations are based on extensive crystallographic studies of the participating molecules. Chapter 1 provides a brief review on the history and biological importance of 1,9-pyrazoloanthrones. The potential of these molecules as probes in sensor chemistry and protein kinase inhibition is envisaged. A short account of the techniques employed for the investigations along with a preamble is presented. Chapter 2 is divided into two parts. Part A deals with the design of a colorimetric and “turn-on” fluorescent chemosensor based on 1,9-pyrazoloanthrone specifically for cyanide and fluoride ion detection. A remarkable solid state reaction indicated by the development of intense red color occurs when crystals of tetrabutylammonium cyanide/fluoride are brought in physical contact with 1,9¬pyrazoloanthrone resulting in corresponding molecular complexes (Figure 1). X-ray crystal structures of these complexes and also of 1,9-pyrazoloanthrone have been determined and the ion sensing activity has been substantiated on the basis of spectroscopic (absorption, fluorescence and NMR) and structural analyses. The crystal structure of the parent compound exhibits a disorder as a consequence of tautomerism and the disorder gets carried on to the complexes as well with even the cyanide and the fluoride ions showing partial occupancy sites. The presence of the –NH group and associated intramolecular charge transfer upon complex formation is attributed to the extreme sensitivity of 1,9-pyrazoloanthrone for cyanide and fluoride (detection limits of 0.2 ppb and 2 ppb) ions respectively. Figure 1. Development of intense red color during the solid state reaction (shown on left) and the turn on fluorescence behavior (shown to the right) Part B demonstrates the utilization of electron rich N-alkyl substituted pyrazoloanthrones to design sensors for detecting explosive and electron deficient nitro aromatics such as picric acid (PA). The N-alkyl derivative of 1,9-pyrazoloanthrone has been synthesized, characterized by single crystal X-ray diffraction studies and evaluated as a potent sensor for picric acid. NMR and fluorescence lifetime measurements validate that the fluorescence quenching of sensor compound by PA (Figure 2) as due to the formation of excited state charge-transfer complex resulting in dynamic quenching. Figure 2. Fluorescence quenching measurements demonstrating the dynamic quenching in the charge transfer complex. Chapter 3 deals with the biological evaluation of 1,9-pyrazoloanthrone and its alkyl derivatives towards the inhibition of a decisive protein kinase called c-Jun N-terminal Kinase (JNK), an important member of MAP kinase family. JNK controls crucial cellular processes like apoptosis and cell proliferation and is implicated in disorders associated with inflammation such as septic shock, arthritis, inflammatory bowel disease, etc. Therapeutic inhibition of JNK activity by small molecules has proven to be advantageous in the treatment of diseases coupled with derailed inflammation. In this context, it is already established that 1,9-pyrazoloanthrone (SP600125) effectively and selectively inhibits JNK at concentrations beyond 10 M. A series of alkyl isomers of pyrazoloanthrone derivatives have been synthesized to evaluate the structural implications of inhibition and to elevate both selectivity and sensitivity at lower concentrations. The crystal structures of these isomers have been characterized and their utility as inhibitors has been tested for their in vitro inhibitory activity over c-Jun N-terminal kinase (JNK). The minimum inhibitory concentrations required by these molecules to inhibit JNK was found to be lesser as compared to 1,9-pyrazoloanthrone (<5 µM; Figure 3). Critically, it turns out that among the various inhibitors synthesized, the lead candidates SPP1 and SPB1 display specific inhibition of JNK among other LPS activated MAP kinases like ERK1/2 and p38. These results suggest that N-alkyl (propyl and butyl) bearing pyrazoloanthrone scaffolds provide promising therapeutic inhibitors for JNK in regulating inflammation associated disorders. Figure 3. Inhibition of JNK in macrophages by the SPP1 and SPB1 compared to the known SP600125. Inspired by the results reported in the previous chapter, Chapter 4 is devoted to the generation of a library of compounds based on SPP1 and SPB1 with a purpose to design inhibitors of JNK which perform at the lowest possible concentrations and the consequent evaluation of their potential on endotoxin induced septic shock. Severe sepsis or septic shock is one of the rising causes for mortality worldwide representing nearly 10% of intensive care unit admissions. Susceptibility to sepsis is identified to be mediated by innate pattern recognition receptors and responsive signaling pathways of the host. The c-Jun N-terminal Kinase (JNK)-mediated signaling events play critical role in bacterial infection triggered multi-organ failure, cardiac dysfunction and mortality. Figure 4. Two selected molecules for specific inhibition studies of JNK at lower concentrations. It is demonstrated that alkyl and halogen substitution on the periphery of anthrapyrazolone increases the binding potency of the inhibitors specifically towards JNK. Based on the results from both in vitro with macrophages and in vivo with the mouse model of septicemia, the potential role of two selected molecules D1 and D2 (Figure 4) in regulating endotoxin induced inflammation is firmly established. Further, it is demonstrated that hydrophobic and hydrophilic interactions generated by these small molecules effectively block endotoxin-induced inflammatory genes expression in in vitro and septic shock in vivo, in a mouse model, with remarkable efficacies. Altogether, the in vitro as well as the in vivo data clearly potentiates the selective inhibitory capacity of small molecule inhibitors like D1 and D2 which can facilitate the treatment of current inflammatory disorders when used in combination with the available drugs having varied efficacies. The results rationalize the significance of the diversity oriented synthesis of small molecules for selective inhibition of JNK and their potential in the treatment of severe sepsis.

Cellular senescence is a permanent cell cycle arrest that is induced as a response to cellular stress. Replicative senescence is a well-described mechanism that limits the replicative capacity of cells and must be overcome by cancer cells. Oncogene-induced senescence (OIS) is a form of premature senescence and a potent tumor suppressor mechanism. OIS is induced in normal cells as a result of deregulated oncogene or tumor suppressor gene expression. An exciting area of research is the identification of novel targets that induce senescence in cancer cells as a therapeutic approach. In this study, a novel mechanism is described where the inhibition of Hsp90 in small cell lung cancer (SCLC) cells induced premature senescence rather than cell death. The senescence induced following Hsp90 inhibition was p21-dependent and the loss of p21 allowed SCLC cells to bypass the induction of senescence. Additionally, we identified a novel mechanism where the depletion of PKCι induced senescence in glioblastoma multiforme (GBM) cells. PKCι depletion-induced senescence did not activate the DNA-damage response pathway and was p21-dependent. Further perturbations of mitosis, using an aurora kinase inhibitor, increased the number of senescent cells when combined with PKCι depletion. This suggests that PKCι depletion-induced senescence involves defects in mitotic progression. Senescent glioblastoma cells at a basal level of senescence in culture, induced by p21 overexpression, and induced after PKCι depletion had aberrant centrosomes. Mitotic slippage is an early exit from mitosis without cell division that occurs when the spindle assembly checkpoint (SAC) is not satisfied. Senescent glioblastoma cells had multiple markers of mitotic slippage. Therefore, PKCι depletion-induced senescence involves mitotic slippage and results in aberrant centrosomes. A U87MG cell line with a doxycycline-inducible shRNA targeting PKCι was developed to deplete PKCι in established xenografts. PKCι was depleted in established glioblastoma xenografts in mice and resulted in decreased cell proliferation, delayed tumor growth and improved survival. This study has demonstrated that both Hsp90 and PKCι are novel targets to induce senescence in cancer cells as a potential therapeutic approach.

Synaptic long-term modifications following neuronal activation are believed to be at the origin of learning and long-term memory. Recent experiments suggest that these long-term synaptic changes are all-or-none switch-like events between discrete states of a single synapse. The biochemical network involving calcium/calmodulin-dependent protein kinase II (CaMKII) and its regulating protein signaling cascade has been hypothesized to durably maintain the synaptic state in form of a bistable switch. Furthermore, it has been shown experimentally that CaMKII and associated proteins such as protein kinase A and calcineurin are necessary for the induction of long-lasting increases (long-term potentiation, LTP) and/or long-lasting decreases (long-term depression, LTD) of synaptic efficacy. However, the biochemical mechanisms by which experimental LTP/LTD protocols lead to corresponding transitions between the two states in realistic models of such networks are still unknown. We present a detailed biochemical model of the calcium/calmodulin-dependent autophosphorylation of CaMKII and the protein signaling cascade governing the dephosphorylation of CaMKII. As previously shown, two stable states of the CaMKII phosphorylation level exist at resting intracellular calcium concentrations. Repetitive high calcium levels switch the system from a weakly- to a highly phosphorylated state (LTP). We show that the reverse transition (LTD) can be mediated by elevated phosphatase activity at intermediate calcium levels. It is shown that the CaMKII kinase-phosphatase system can qualitatively reproduce plasticity results in response to spike-timing dependent plasticity (STDP) and presynaptic stimulation protocols. A reduced model based on the CaMKII system is used to elucidate which parameters control the synaptic plasticity outcomes in response to STDP protocols, and in particular how the plasticity results depend on the differential activation of phosphatase and kinase pathways and the level of noise in the calcium transients. Our results show that the protein network including CaMKII can account for (i) induction - through LTP/LTD-like transitions - and (ii) storage - due to its bistability - of synaptic changes. The model allows to link biochemical properties of the synapse with phenomenological 'learning rules' used by theoreticians in neural network studies.