L'amibe Dictyostelium discoideum est un eucaryote unicellulaire capable de se déplacer et de se nourrir par phagocytose. Cet organisme est très utilisé pour décrypter les mécanismes moléculaires du chimiotactisme et de la motilité cellulaire. Les travaux de S.Fache au laboratoire ont notamment montré que la motilité de Dictyostelium est stimulée par une contrainte mécanique, et que la vitesse atteinte dépend du calcium extracellulaire. <br />Dans ce travail, nous avons étudié l'étalement de Dictyostelium sur un substrat, processus qui peut être apparenté à certaines étapes de la motilité cellulaire. Nous avons montré que l'étalement de Dictyostelium est un processus quasi-linéaire et anisotrope. De plus, nous avons mis en évidence des variations périodiques de l'aire gagnée par les cellules dont nous n'avons pu identifier l'origine moléculaire. <br />Ces travaux sur l'étalement cellulaire nous ont permis de caractériser le rôle de la protéine Phg2 dans la motilité cellulaire. Phg2 est une kinase connue pour être impliquée dans la phagocytose et la motilité. Nous avons établi que Phg2 contrôle la polarisation cellulaire via son domaine de liaison aux protéines de type Ras, et joue également un rôle dans la polymérisation locale de l'actine via son domaine kinase. <br />Enfin, nous avons inactivé deux gènes codant pour des canaux calciques chez Dictyostelium, et les études préliminaires menées semblent indiquer qu'ils ne participent pas à la réponse calcique de la motilité induite par une contrainte.

Les matériaux inorganiques ont des propriétés uniques à l'échelle nanométrique. Ces propriétés ont généré beaucoup d'intérêt pour fabriquer des nouveaux matériaux utilisant des nano-objets comme unité de construction. Nous avons suivi une approche biomimétique pour la fabrication de dispositifs à base de nanoparticules afin d'améliorer les méthodes actuelles de fabrication top-down et bottom-up. Certaines protéines naturelles se lient en effet spécifiquement à des matériaux inorganiques, et déclenchent notamment la croissance de cristaux inorganiques. Une première étape dans cette approche biomimétique est de comprendre comment des protéines se lient spécifiquement à des nanomatériaux inorganiques. Nous avons exploré ce mécanisme de reconnaissance en sélectionnant des anticorps (les protéines de notre système immunitaire spécialisées dans les interactions avec de nombreuses cibles) contre des matériaux inorganiques par la méthode combinatoire biotechnologique appelée "phage display". Cette technique permet d'obtenir la séquence génétique codante des anticorps sélectionnés se liant à leur cible à partir d'une banque aléatoire d'anticorps. L'analyse statistique des séquences des anticorps sélectionnés fournit de nouvelles informations sur les interactions protéines/matériaux inorganiques. Notre principale conclusion est l'identification de l'acide aminé arginine en tant que contributeur majeur dans les interactions protéine/or. L'ingénierie génétique des anticorps permet de fonctionnaliser ces nouvelles sondes de matériaux inorganiques en vue de leur utilisation pour des applications dans le domaine des nanomatériaux. Les anticorps recombinants sélectionnés et leurs dérivés fonctionnalisés peuvent être exprimés par sécrétion à l'aide d'un hôte eucaryote (Dictyostelium discoideum) mis au point au cours de cette thèse.

Thesis (Ph. D.)--University of Missouri-Columbia, 1997. / Typescript. Vita. Includes bibliographical references (leaves 109-130). Also available on the Internet.

Thesis (Ph. D.)--University of Missouri-Columbia, 1997. / Typescript. Vita. Includes bibliographical references (leaves 109-130). Also available on the Internet.

Argonaute proteins play substantial roles in post-transcriptional regulation of gene expression within RNA interference (RNAi) pathways, making them crucial subjects for research, aimed at understanding their interactions with small non-coding RNAs (ncRNAs) and other RNAi components. This study focuses on investigating these properties of Argonaute proteins, particularly Argonaute protein A (AgnA), in the social amoeba Dictyostelium discoideum that is renowned for its broad genetic toolbox and unique life cycle. While previous studies have examined the disruption of three Argonaute genes (agnB, agnC, agnE) and their effect on mRNA levels and small ncRNA expression, this study extends to agnA gene, which remains less studied. Key questions surrounding the influence of AgnA on the cellular processes such as the cell growth rate, development, gene expression, as well as potential targets and small ncRNA binding, remain unanswered. A well-established approach that could provide the necessary answers is the disruption of the gene through traditional homologous recombination, by insertion of a drug-resistance cassette flanked by homology arms complementary to the target locus. However, the emerging CRISPR/Cas9 gene editing tool on contrary offers straightforward protocols for disruption of gene expression through efficient induction of genomic knockouts, point mutations and deletions. In this study, both approaches were applied in parallel to knockout the agnA gene, enabling comparison of knockout efficiency and further study of the growth rate, development and gene expression in the knockout strains. Moreover, important information regarding the growth patterns of both wild-type and agnE knockout strains were also elucidated, complementing the previous growth rate analyses. The obtained data from this research could provide valuable insights for future studies ofthe RNAi machinery components and particularly the function of Argonaute proteins in D. discoideum.

Glycogen phosphorylase 2 (gp-2) is a key enzyme during the development of Dictyostelium discoideum. The gp-2 enzyme breaks down glycogen into glucose monomers that are subsequently used to synthesize the terminal end products of cellular differentiation. This gene is an ideal candidate for studying the process of selective gene expression because its product figures so prominently in the development of this organism, implying a dependable control mechanism responsible for its developmentally regulated expression. I present in this thesis the identification of several putative cis-acting elements of gp-2 as revealed through footprint analysis. Due to the extreme AT-bias characteristic of Dictyostelium promoters, footprinting conditions required intensive optimization with respect to template, nonspecific competitor, source of protein extract and DNase I digestion. Using an endlabeled fragment containing seven repeated sequences (3 TA boxes [TAATTATA], 2 TAG boxes [TAAAAATGGT] and 2 C boxes [ACCCACT]), purified replication protein A and several developmental nuclear extracts were tested for DNA binding activity. Small footprints were observed on the TAG and C boxes of the promoter for both protein sources. However, using a more sensitive footprinting strategy involving multiple rounds of primer extension, larger footprints spanning the same promoter regions were detected. In both cases, the appearance of the footprints coincided with the documented transcriptional activity of the gene. It can be concluded from the data obtained that the TAG and C boxes are very likely cis-acting elements involved in the regulation of gp-2 expression. / Master of Science

Amoebae are commonly studied to understand embryogenesis, and the best-characterized amoebozoan species is Dictyostelium discoideum (Dd). Dd has a very simple life cycle with a range of developmental stages, among which we are most interested in the stage of a migrating slug. It has been observed that different sizes of Dd slugs maintain a proportional distribution of prestalk cells and prespore cells: prestalk cells occupy the anterior 20% of the slug, while prespore cells occupy the posterior 80%. However, it remains unknown how the migrating slug forms and preserves this anterior-posterior proportional pattern under so many different dynamics including cell movement, signaling, and cell differentiation. Therefore, we constructed a mathematical model to simulate the cell movement and chemical distribution during slug migration, and we conducted numerical experiments to explore possible factors for this pattern. In particular, we divided the problem of interest into the following three parts to be investigated. (1) differential motion: the ability of prestalk cells to move through all the prespore cells and stay in the anterior region of the slug; (2) signaling: how cells of different types produce, receive, and respond to the signals in the environment; (3) cell differentiation: how prestalk and prespore cells differentiate into each other under the regulation of signaling. We finally combined and balanced these mechanisms appropriately to achieve the desired patterns observed in migrating slugs.

Molecular chaperones are proteins that interact with and aid in stabilization and activation of other proteins. Chaperones help proteins attain their three dimensional conformation, without forming a part of the final structure. Many of the chaperones are stress proteins known as Heat shock proteins (Hsps). Their expression is upregulated in response to various kinds of stress such as heat stress, oxidative stress etc., which threaten the protein homeostasis, by structurally destabilizing cellular proteins, and increasing the concentration of aggregation-prone folding intermediates. The Hsps are classified according to their molecular weight into Hsp40, Hsp60, Hsp70, Hsp90, Hsp100, and the small Hsp families. Some of them are constitutively expressed and play a fundamental role in de novo protein folding. They further aid in proteome maintenance by assisting in oligomeric assembly, protein trafficking, refolding of stress denatured protein, preventing protein aggregation and protein degradation. Heat shock protein 90 (Hsp90) are one of the important representatives of this class of proteins. Hsp90 are highly conserved class of molecular chaperones. They are found in bacteria, eukaryotes, but not in archaea. In contrast to the eukaryotes which require a functional cytoplasmic Hsp90 for viability, the bacterial counterpart (HtpG) is typically nonessential. Hsp90 is an ATP dependent chaperone. Hsp90 form dimers, with each protomer consisting of three functional domains: N- terminal, ATP binding domain, Middle domain and C-terminal domain. Hsp90 is a dynamic protein, and undergoes an elaborate conformational cycle during its ATPase cycle, which is essential for its chaperoning activity. The Hsp90 chaperone cycle is regulated by interaction with diverse cochaperones. Hsp90 interacts with specific set of substrate proteins. Many of these substrate proteins function at the heart of several cellular processes like signalling, cell cycle, apoptosis. Studies from protozoans like Leishmania, Plasmodium, Trypanosoma etc. have also implicated the role of Hsp90 in their growth and stage transitions. Thus, selective inhibition of Hsp90 has been explored as an intervention strategy against important human diseases such as cancer, malaria and other protozoan diseases. The ATP binding N-terminal domain (NTD), has been explored as the target domain for inhibition of Hsp90 using competitive inhibitors of ATP. Several chemical classes of Hsp90 inhibitors are known, including ansamycins, macrolides, purines, pyrazoles, and coumarin antibiotics. However, many inhibitors are observed to be toxic, less soluble and unstable. Hence, there is a requirement for new approach to design inhibitors which are more soluble and less toxic and serve as effective therapeutic drugs.inhibitors are observed to be toxic, less soluble and unstable. Hence, there is a requirement for new approach to design inhibitors which are more soluble and less toxic and serve as effective therapeutic drugs. The work presented in this thesis mainly concerns with the structural studies and biochemical and biophysical characterization of Hsp90 from two different sources viz. Dictyostelium discoideum, a cellular slime mould and a plant source Oryza sativa (rice). The structural analyses of these two proteins have been carried out by X-ray crystallography. Though yeast has been explored extensively as a model system to understand the different roles of Hsp90, it lacks the various signalling pathways essential for growth and development present in case of higher eukaryotes. D. discoideum has been employed as a model system to understand multicellular development, which occurs in response to starvation induced stress. D. discoideum has the advantages due to its ease of manipulation. The organism's genome also shows many signalling pathway for growth and differentiation that are conserved between D. discoideum and mammals. With this motivation, we have studied several structural aspects of the cytosolic isoform of Hsp90 from D. discoideum called HspD. HspD was also observed to play a role in the multicellular development of D. discoideum. It has been demonstrated that the treatment of D. discoideum with inhibitors like Geldanamycin or Radicicol causes an arrest in the multicellular development at the mound stage, and the few which escaped this arrest gave rise to abnormal fruiting bodies. A subset of the proteins involved in this mound arrest phenotype, were observed to have homologs in humans, which are clients of Hsp90. Therefore, a structural perspective of HspD can aid in better understanding of the role of this protein in the organism, as well as, elucidate any structural differences observed as compared to other species, which may have an impact on its activity. Studies on the physiological role of Hsp90 in plants began much later as compared to fungi and humans. In plants Hsp90 are involved in various abiotic stress responses. In addition, their roles have also been implicated in plant growth and development, innate immune response and buffering genetic variations. However, the molecular mechanisms of these various actions are not clearly understood. Also, the structural aspects of plant Hsp90 are yet to be explored. The structure of the NTD of Hsp90 from barley is the only one available from a plant source till now. We have initiated the studies on rice Hsp90 with the objective to understand the mechanism of Hsp90 in plants, which may aid in improving stress tolerance in plants. The thesis has been divided into five chapters. The first chapter introduces the various aspects of Hsp90 protein. The chapter starts with a general overview of concept of molecular chaperones and describes briefly the different classes of molecular chaperones. This is followed by a detailed description of different aspects of Hsp90 with main emphasis on the structure and its conformational flexibility. The chapter describes the association of Hsp90 with other accessory proteins like cochaperones and its interaction with its substrate proteins and explains the functional significance of Hsp90 as a drug target and the need for the development of new class of inhibitors, followed by the significance of the study of Hsp90 in the two model systems (D. discoideum and rice) chosen to be studied. The second chapter gives a brief overview of the principles behind the different experimental methods employed during the course of this research, which includes the tools of X-ray crystallography and other biochemical and biophysical techniques employed for the characterization of the protein. Chapter 3 describes the crystal structure of NTD of Hsp90 from D. discoideum. The structure of NTD was solved in two different native (ligand-free) forms viz. monoclinic and hexagonal. The two forms differed in local structural rearrangement of a segment of NTD known as the lid region. The lid region in the hexagonal form showed a shift in its position as compared to the other solved structures of NTD. The structure of NTD was also solved in complex with various ligands which include ADP, substrate analogs and an inhibitor molecule. A comparison of all the structures showed that the overall structure is well-conserved. One of the crystal structures of NTD showed a heptapeptide (part of the vector) bound at the active site. The peptide was observed to make several complementary interactions with the residues of the ATP binding pocket and retain several interactions which the nucleotide makes with the NTD. The NTD showed subtle conformational differences when compared with the NTD of Hsp90 from yeast. Chapter 4 details the structural and functional characteristics of full length Hsp90 protein from D. discoideum. Due to the large size and flexibility, the full length protein did not crystallize in spite of several attempts. Hence, HspD was studied using different solution studies like Small Angle X-ray Scattering (SAXS) and Dynamic Light Scattering (DLS). Both the studies showed the presence of higher oligomers. The SAXS data showed the presence of tetramers and hexamers while, the addition of the ligand shifts the protein from a dimer to a higher oligomer as observed from DLS studies. The chapter also describes the study of interaction of HspD with a cochaperone protein p23. The interactions were studied using ITC, which showed a strong binding. The ATPase activity was also evaluated in the presence of increasing concentrations of p23, which was observed to decline with increasing concentrations of p23. In chapter 5, we describe the biochemical characterization of Hsp90 from Oryza sativa (rice) and the crystallographic analysis of its NTD. Binding of the rice Hsp90 to ATP and an inhibitor were studied by fluorescence. The ATPase activity of rice Hsp90 was checked by radioactive assay and the protein was observed to be active. The NTD of rice Hsp90 crystallized as a monomer in complex with a substrate analog AMPPCP and the structure was determined.

No description available.

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