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. / Inorganic materials have unique properties at the nanometer scale. These properties have generated a lot of interest among researchers to fabricate novel materials using nano objects as building units. In this PhD thesis, we have attempted to mimick nature in the fabrication of nanoparticle based devices in order to improve upon current top-down and bottom-up nanomaterial fabrication methods. Proteins can specifically bind inorganic materials and trigger crystal growth and thus are considered as the main building units for a biomimetic approach of fabrication. The first step towards mimicking nature is to explore how proteins bind specifically to nanomaterials. We have explored this recognition mechanism by selecting antibodies (the protein binders of our immune system) against inorganic nanomaterials using the combinatorial biotechnology method of phage display. This technique provides us with the genetic sequence of selected antibodies from a random antibody library exposed against a target. Statistical analysis of selected antibody sequences provides new information on proteins/inorganics interactions. Our main finding in this regard is the identification of the amino acid arginine as a major contributor to protein/gold interactions. Additional functionality to these new binders of inorganic materials is obtained by antibody engineering, allowing for their value added use in nanomaterial science applications. Selected recombinant antibodies and their engineered derivatives along with other recombinant protein can be expressed and secreted using a eukaryotic expression platform (Dictyostelium discoideum) developed during this thesis.

As serina/treonina fosfatases (PPs) são enzimas responsáveis pela desfosforilação de resíduos de fosfoserina e/ou fosfotreonina e estão subdivididas em duas famílias gênicas designadas PPP e PPM. A família PPP está dividida em cinco subfamílias, que compreendem as PPs do tipo 1 (PP1), 2A (PP2A), 2B (PP2B), 5 (PP5) e 7 (PP7), de acordo com a similaridade entre as sequências de aminoácidos das subunidades ou domínios destas enzimas. Novas PPs estão sendo descobertas em diferentes organismos e classificadas nestas famílias ou subfamílias com base na análise comparada de suas sequências. Uma delas é a proteína fosfatase do tipo 4 (PP4), descoberta originalmente em coelhos e cujas funções biológicas vêm sendo progressivamente elucidadas. Neste estudo tivemos como objetivos a identificação e caracterização de uma nova serina/treonina fosfatase de Dictyostelium discoideum. Para isto, rastreamos uma biblioteca de cDNA e um cDNA completo que codifica a subunidade catalítica da fosfatase do tipo 4 (PP4c) foi isolado e seqüenciado. Verificamos que o gene da PP4c é essencial e está presente em cópia única localizada no cromossomo 2, originando um mRNA expresso ao longo de todo o ciclo de vida de D. discoideum. Imunodetecção da PP4c realizada com anticorpo específico indicou que os níveis desta proteína também são constitutivos. Observamos que a superexpressão da PP4c sob o controle de um promotor constitutivo não causa alterações fenotípicas detectáveis. A análise da localização celular da PP4c expressa em células de D. discoideum como proteína de fusão com GFP (Green Fluorescent Protein) revelou que a enzima está localizada no citossol, contrastando com outros organismos, onde ela se encontra enriquecida no centrossomo. Por fim, descrevemos neste trabalho, além da organização genômica da PP4, a estrutura dos genes de todas as serina/treonina fosfatases da família PPP encontradas no cromossomo 2 e apresentamos dados das relações entre estas enzimas de D. discoideum. / The serine/threonine protein phosphatases (PPs) are enzymes responsible for dephosphorylation of phosphoserine and/or phosphothreonine residues and are divided in two gene families designated as PPP and PPM. The PPP family is divided in five subfamilies, which comprise type 1 (PP1), 2A (PP2A), 2B (PP2B), 5 (PP5) and 7 (PP7) phosphatases. This subdivision is based on aminoacid sequence similarity or enzyme domains. Novel PPs have been discovered in different organisms and classified in these families or subfamilies based on comparative sequence analysis. One of these is type 4 protein phosphatase (PP4), originally discovered in rabbit, which functions have been progressively uncovered. In this study our goal is to identify and characterize a novel serine/threonine phosphatase in Dictyostelium discoideum. We first screened a cDNA library and isolated a complete cDNA encoding the catalytic subunit of protein phosphatase 4 (PP4c), confirmed by manual DNA sequencing. The PP4c is an essential, single-copy gene located in chromosome 2, which encondes a mRNA constitutively expressed throughout D. discoideum life cycle. Immunodetection of PP4c performed with specific antibodies indicated corresponding protein levels. Overexpression of PP4c under a strong promoter caused no detectable phenotype. Subcellular localization of PP4c expressed as a GFP-fusion protein revealed its cytosolic location, in contrast to other organisms, where it has been reported to be enriched in centrosomes. We also describe here the genetic organization of PP4c, the genetic structure of all serine/threonine protein phosphatases belonging to the PPP family found in chromosome 2, and a phylogenetic analysis indicating relationships among these enzymes in D. discoideum and other selected organisms.

Cells invest a lot of energy in order to get their proteins to fold correctly and attain functionality. It is the functional proteome of a cell that defines the ‘life of a cell’. Cells have therefore employed dedicated machinery called chaperones to enable protein folding. One class of these chaperones is heat shock proteins named so because they were initially discovered to be heat inducible and particularly important during heat stress. However the role of heat shock proteins has now been extended from merely being important for stress tolerance. Heat shock proteins are prominently involved in maintaining the correct folding and conformation of proteins and are vital in regulating the stability between protein synthesis and degradation. One of the heat shock proteins, Hsp90, is an evolutionarily conserved molecular chaperone essential in all known eukaryotes examined so far. Unlike other chaperones, Hsp90 is unique in binding to substrate proteins, which are at a late stage of folding, poised for activation by either ligand binding or interaction with other cellular factors. The most common clients of Hsp90 are signaling proteins, the classic example being steroid hormone receptors and signaling kinases. Several other proteins including transcription factors, proteins involved in cell division and development have also been shown to rely on Hsp90 functioning for their maturation. Hsp90 has emerged as an important molecular chaperone due to the large number of proteins that depend on the activity of Hsp90 for their functionality. Hsp90 plays a central role in multiple cellular processes. Since knock-out of hsp90 is lethal to most eukaryotes, inhibitors of Hsp90 have been widely used to study its function. The most widely used inhibitor is geldanamycin (GA). GA binds to the N-terminal/ATP binding site of Hsp90 which results in the degradation of client proteins. Hsp90 clients have been shown to be proteins important for diverse cellular processes such as protein trafficking, signal transduction, cell-cycle, cellular motility and development in eukaryotes. Exploring new Hsp90 clients gives an insight into more pathways that Hsp90 regulates. Intriguingly, many proteins interact with Hsp90 in a context dependent manner, i.e., under certain environmental cue, or in a particular tissue, or only under certain diseased states. It is therefore essential to study Hsp90 functioning and examine Hsp90-client interactions in more than one model organism. Dictyostelium discoideum: a model organism to study the role of Hsp90 in development The eukaryote, Saccharomyces cerevisiae that has been explored extensively for studying the diverse clientele of Hsp90, lacks various signaling pathways important for growth and differentiation as prevalent in higher eukaryotes. It is desirable to develop a model system that would combine the advantages of a lower eukaryote, in terms of its ease of manipulation and retain the complexities of higher eukaryotes. With this motivation, the social slime mold D. discoideum was explored to examine potential roles of cytoplasmic Hsp90 in growth and development. D. discoideum is ideal for studying signaling pathways important for growth and differentiation and to understand how these pathways control cellular responses to external stimuli. Multicellular development in D. discoideum occurs in response to starvation induced stress. As in case of many other protozoans, we conjectured that Hsp90 may participate in regulating developmental transition from unicellular to multicellular stages in Dictyostelium as well. My initial study attempts, to address the role of Hsp90 (HspD), in development of D. discoideum. Towards this two approaches were taken: through genetic interference of HspD, and the other, through its pharmacological inhibition. An antisense HspD plasmid was designed which upon transfection in D. discoideum, showed a very slow growth phenotype, and the cells did not survive beyond few generations. Therefore to further study the functions of HspD, I resorted to pharmacological inhibition by using the specific, well characterized inhibitor, GA. As a first step towards this I examined whether GA was capable of binding to HspD from D. discoideum cell lysate. Towards this, GA was immobilized to NHS-sepharose beads, and bound proteins were examined. Western blot of the bound fraction, using antibody specific to HspD, identified it as a predominant protein being pulled down. This was further confirmed by mass spectrometry. To be able to compare Hsp90 from D. discoideum with Hsp90s from other model organisms, HspD was cloned, purified and biochemically characterized. Comparison of ATPase activities of HspD with Hsp90’s from other systems indicates HspD to possess a relatively low ATPase activity with a Kcat of 1.6 x 10-3 min-1. The dissociation constant of GA for HspD was found to be 0.8 µM, which was in the range similar to Hsp90s from other systems. In addition, we have now obtained structural data on HspD in collaboration with crystallography groups. The N-terminal domain of HspD has been crystallized, both in -free and ligand-bound forms. Crystal structure comparison of HspD with Hsp90 from S. cerevisiae shows overall fold similarity yet some important differences in side chain orientations of specific residues in the ATP binding domain. Interestingly, on treating D. discoideum cells with GA or another Hsp90 N-terminal inhibitor, Radicicol, it was found that, while control cells progressed to develop into fruiting bodies, GA/Radicicol treated cells resulted in delayed development, and were finally arrested at the ‘mound’ stage. This suggested potential involvement of HspD in developmental progression beyond the mound stage. In order to identify the pathways that are probably affected by HspD in D. discoideum development, cells were treated with/without GA and subjected to comparative proteomics using mass spectrometric analysis. Amongst other differences, there was an obvious absence of peptides corresponding to the protein paxillin in GA treated cells. The results were verified by Western blot analysis, using a specific antibody against paxillin, wherein a drastic decrease in paxillin levels were observed in cells treated with GA. Paxillin is a key player in focal adhesion sites that functions as an adaptor protein to recruit diverse cytoskeletal and signaling proteins into a complex, and is essential for cellular proliferation and cell-substrate adhesion. My studies suggest that one of the pathways through which HspD regulates development is through cellular motility as Hsp90 was involved in regulating proteins necessary for motility and cytoskeletal organization at focal adhesion points during development in D. discoideum. Hsp90 as a target for Trypanosoma evansi infections In addition to examining the role of Hsp90 in differentiation in D. discoideum, I have also looked at the potential of Hsp90 under diseased conditions. Towards this, I explored the protozoan parasite, T. evansi, which causes a fatal disease ‘surra’. Surra is a neglected disease that mainly affects domestic and wild animals including equines, camels, cattle and buffaloes. The parasite causes significant economic losses to livestock industry. While this infection is mainly restricted to domestic (camels, equines, cattle, buffaloes, goats, sheep, pigs, dogs etc.) and wild animals, recent reports indicate their ability to infect humans. There are no reliable sensitive and specific diagnostic tests or vaccines available against this disease and the available drugs show significant toxicity. There is an urgent need to develop improved methods of diagnosis and control measures for this disease. Unlike its related human parasites T. brucei and T. cruzi whose genomes have been fully sequenced T. evansi genome sequence remains unavailable. With a view to identifying potential diagnostic markers and drug targets I have studied the clinical proteome of T. evansi infection using mass spectrometry. I have been able to identify almost 166 proteins of T. evansi, which also included potential drug and vaccine targets. Due to absence of any genome sequence information from T. evansi, most of the peptides obtained matched to its related species, T. brucei, T. cruzi and also few from Leishmania major. Importantly, I was also able to identify peptides from Hsp90. Hsp90 from T. evansi was cloned and its sequence was also obtained. To investigate the possibility of exploring Hsp90 as a target against Surra infections, TeHsp90 protein was purified by expressing it in bacterial cells, and its drug (GA) binding ability was examined in-vitro. The dissociation constant of GA for HspD was found to be 1.4 µM, which was in the range similar to Hsp90s from other systems. The ability of 17AAG (a derivative of GA) was examined in inhibiting T. evansi infection at pre-clinical level. Towards this, swiss female mice were infected with purified parasites and then the drug was injected either immediately, in one group of mice, and in another group of mice the parasites were challenged with the drug only after the onset of infection. Interestingly, both groups of mice were found to get cured using Hsp90 inhibitor. The pre-clinical results suggested that Hsp90 was an interesting drug target and its inhibitor could indeed be used against ‘surra’ infections. Hsp90 from Giardia lamblia: An unusual case Hsp90 was also examined from another pathogenic protozoan, Giardia lamblia, one of the leading causes of diarrhea in the world. Previous studies from our lab have shown Gardial Hsp90 to be coded by two different ORFs, spliced together in trans. This is indeed the only example of trans-splicing in Hsp90 known so far. My study further characterizes this finding through analysis of transcription levels of the individual ORFs, using Northern blot analysis. Importantly, I was able to detect transcripts of all three forms of Hsp90; full-length, N terminus as well as C terminus, suggesting that these are expressed and may have biological significance. To understand the significance of these independent transcripts, I have examined relative levels of expression of all three forms by Real-time PCR analysis wherein there was almost 90 fold and 5 fold lesser transcript level of N terminus and C terminus Hsp90 observed, respectively as compared to the full-length GlHsp90 expression. Previous reports have shown Hsp90 from all known organisms, to get up regulated during heat shock. Thus it was important to examine the effect of heat stress on the expression of these independent transcripts. Interestingly, different domains were found to get independently induced during heat stress. The transcript level of HspC was seen to be almost similar to that of full-length upon heat shock. There was also a significant up regulation observed in HspN transcript upon heat shock. Taking together all these observations, these results suggest a possible role for the independent domains, HspN and HspC during heat stress in G. lamblia. Furthermore, I have cloned and purified one of the individually expressed domains, HspN and characterized it biochemically. HspN was found to be able to bind to ATP, however lacked ATPase activity. Taking together all these observations, it suggests a possible role for the independent domains, HspN and HspC which needs to be investigated further. Summary Altogether, my studies establish the importance of alternate model systems in understanding the biology of Hsp90. The importance of Hsp90 was first established in growth and development of a nonpathogenic protozoan D. discoideum. My results provide significant insights into the additional pathways that Hsp90 regulates during D. discoideum development. One such important pathway was delineated to be cellular locomotion and motility. Further, I have also studied the importance of Hsp90 in neglected infectious diseases. In addition to providing a glimpse into the pathways operational during disease manifestation in T. evansi, we have shown Hsp90 to be effective in pre-clinical trials against T. evansi infections. Hsp90 from another pathogenic protozoan, G. lamblia, has also been studied. This is by far the only organism, in which there is an independent expression of the N-and C-terminal domain of Hsp90. The rare gene organization, coupled with independent expression of domains of Hsp90, makes this organism important to examine novel functions of this chaperone.

Doctor of Philosophy / Department of Physics / Bret N. Flanders / Directed electrochemical nanowire assembly (DENA) is a method for fabricating nano-structured materials via electrochemical dendritic solidification. This thesis presents two new applications of nano-structured materials that are fabricated via the DENA methodology: cellular force sensors to probe adhesive sites on living cells and single-crystalline metallic dendrites as surface coating materials. Fast migrating cells like D. discoideum, leukocytes, and breast cancer cells migrate by attachment and detachment of discrete adhesive contacts, known as actin foci, to the substrate where the cell transmits traction forces. Despite their importance in migration, the physics by which actin foci bind and release substrates is poorly understood. This gap is largely due to the compositional complexity of actin foci in living cells and to a lack of technique for directly probing these sub-cellular structures. Recent theoretical work predicts these adhesive structures to depend on the density of adhesion receptors in the contact sites, the receptor-substrate potential, and cell-medium surface tension. This thesis describes the fabrication of sub-microscopic force sensors composed of poly(3,4-ethylene dioxythiophene) fibers that can interface directly with sub-cellular targets such as actin foci. The spring constants of these fibers are in the range of 0.07-430 nN m-1. These fibers were used to characterize the strength and lifetime of adhesion between the single adhesive contacts of D. discoideum cells and the fibers, finding an average force of 3.1 ± 2.7 nN and lifetime of 23.4 ± 18.5 s. This capability is significant because direct measurement of these properties will be necessary to measure the cell-medium surface tension and to characterize the receptor-substrate potential in the next (future) stage of this project. The fabrication of smart materials that are capable of the high dynamic range structural reconfiguration would lead to their use to confer hydrophobic, lipophobic, and anti-corrosive character to substrates in a regenerative manner. As a step towards this goal, we have extended the DENA method to enable repetitive growth and dissolution of metallic dendrites to substrates. The experimental parameters that control this process are the frequency and duty cycle of the alternating voltage signal that initiates the dendritic growth.

Legionella pneumophila wurde erstmals 1977 beschrieben, nachdem der Erreger aus dem Lungengewebe eines Patienten isoliert wurde, der an einer schweren atypischen Pneumonie erkrankt war. Das Bakterium zeichnet sich durch ein duales Wirtssystem aus und kann sich sowohl in Protozoen als auch in humanen Zellen vermehren. Ein Ziel dieser Arbeit war es, wichtige Faktoren einer Legionelleninfektion seitens der Wirtszelle zu betrachten. Als Wirtsmodell diente die soziale Amöbe Dictyostelium discoideum. Mit Hilfe eines von Patrick Farbrother (Universität Köln) etablierten Dictyostelium DNA-Microarrays mit 5906 genspezifischen Sonden, wurde die Genexpression von D. discoideum in Reaktion auf eine Infektion durch L. pneumophila untersucht. Zur Kontrolle dienten uninfizierte Zellen, und Zellen, die mit L. hackeliae bzw. einer dotA-Mutante von L. pneumophila koinkubiert wurden. Diese beiden Stämme weisen eine verminderte Pathogenität bzw. ein deletiertes Pathogenitätsgen auf. Für den Zeitpunkt 24 h nach Infektionsbeginn wurden 140 Gene gefunden, die in D. discoideum in Reaktion auf eine Infektion mit L. pneumophila differentiell exprimiert werden. Einige Gene codieren bereits bekannte Proteine von D. discoideum. Dazu gehören das RtoA (ratioA, Fusion von Vesikeln), Discoidin I, CotB (spore coat Protein SP70) und die lysosomale α-Mannosidase. Mit Hilfe von Homologie-Suchen konnte weiteren unbekannten Proteinen eine Funktion zugeteilt werden. Hierzu zählen die Chaperone ClpB (heat shock protein Hsp104), β’-COP (coat protein) und drei calciumbindende Proteine. Nach Einteilung in funktionelle Kategorien, konnte gezeigt werden, dass viele Gene reguliert werden, deren Produkte am Aminosäure-Metabolismus beteiligt sind oder bei denen es sich um ribosomale Proteine handelt. Des Weiteren wurde in dieser Arbeit das Nramp-Protein von D. discoideum näher untersucht. Nramp transportiert zweiwertige Kationen über die phagosomale Membran. Es konnte festgestellt werden, dass die Aufnahme von L. pneumophila und M. avium in eine nramp-Mutante deutlich reduziert ist. Allerdings ist eine vermehrte Replikation von Legionellen und Mykobakterien in der Wirtsmutante zu beobachten. In Zusammenarbeit mit Salvatore Bozzaro (Turin, Italien) konnte gezeigt werden, dass während einer Infektion mit L. pneumophila die nramp-Expression sinkt und bereits nach 48 h annähernd keine nramp-RNA im Northern-Blot nachweisbar ist. Im Gegensatz dazu bleibt die nramp-Expression während einer Infektion mit M. avium relativ konstant. In Infektionsstudien konnte nachgewiesen werden, dass sich die Endozytobionten TUME1, UWE25 und UWC6 in D. discoideum vermehren können. Mit Hilfe von spezifischen Cy3-markierten 16S-rRNA Sonden wurde die intrazelluläre Zunahme der Bakterien über 48 h beobachtet. Zum Zeitpunkt 48 h nach Inokulation konnte eine erhöhte Anzahl der drei Endozytobionten in D. discoideum festgestellt werden. Anhand von elektronenmikro-skopischen Aufnahmen konnte gezeigt werden, dass die Stämme TUME1 und UWE25 im Zytoplasma des Wirtes von membranösen Strukturen eng umschlossen sind. UWC6 konnte sowohl in Vakuolen als auch frei im Zytoplasma nachgewiesen werden. Die Lokalisierung der Endozytobionten entspricht ihrer Lokalisierung in ihren natürlichen Wirten. Ein weiteres Ziel dieser Arbeit war die Funktionsanalyse des Mip-Proteins aus L. pneumophila. Das Mip-Protein von Legionella gehört in die Klasse der FK506-Bindeproteine. Es besitzt Peptidyl-Prolyl cis/trans Isomeraseaktivität und bildet Homodimere. Mip kann an die extrazelluläre Matrix von Lungenepithelzellen binden, speziell an das Collagen IV. Mit Hilfe von Transwell-Versuchen konnte festgestellt werden, dass Mip für die Penetration von Legionella durch eine Barriere aus Lungenepithelzellen verantwortlich ist. Die Penetrationsfähigkeit konnte nach Hemmung der PPIase-Aktivität durch FK506 bzw. Rapamycin gehemmt werden. Ebenso waren Legionellen nach Hemmung der Serinproteaseaktivitäten im Transwell-System nicht mehr in der Lage die Barriere aus Epithelzellen mit extrazellulärer Matrix zu durchwandern. Mit Hilfe von Degradationsassays mit S35-markierter extrazellulärer Matrix konnte gezeigt werden, dass mip-positive Legionellen extrazelluläre Matrix degradieren können. Nach Hemmung der PPIase-Aktivität bzw. Serinproteaseaktivität konnten mip-positive Legionellen extrazelluläre Matrix nicht mehr degradieren. / The facultative intracellular bacterium Legionella pneumophila is the etiological agent of Legionnaire’s disease. It was first described in the year 1977 after isolation of the lung of a patient who had a severe atypical pneumonia. The bacterium possesses a dual host system and can replicate within protozoa and human cells. The main focus of this work was to identify host cell factors which are important during Legionella infection. As a host model system we used the social amoeba Dictyostelium discoideum. In cooperation with Patrick Farbrother (University of Cologne) who established a Dictyostelium DNA-microarray we have been able to investigate the gene expression of Dictyostelium during Legionella infection. The microarray consists of 5906 gene-specific probes representing about half the genome of D. discoideum. As controls we isolated RNA from uninfected cells and cells after coincubation with L. hackeliae as well as a dotA-mutant of L. pneumophila. Both are Legionella strains with reduced pathogenicity and a deleted pathogenicity gene respectively. Approximately 24 h after infection we identified 140 differentially expressed D. discoideum genes. Some of these genes encode well characterised D. discoideum proteins like RtoA (ratioA, vesicle fusion protein), Discoidin I, CotB (spore coat protein SP70) and the lysosomal α-Mannosidase. By homology searches the functions of others could be assigned, like the chaperones ClpB (heat shock protein Hsp104), β’-COP (coat protein) and three calcium-binding proteins. When characterising the probes by cellular processes the alteration of gene expression was analysed on functional level. A lot of genes were regulated whose products are involved in nucleotid metabolism or that are ribosomal proteins. Furthermore we investigated the role of the Nramp-protein of D. discoideum. L. pneumophila as well as M. avium were more efficiently phagocytosized from wildtype Dictyostelium than from nramp-mutants. In contrast to phagocytosis, the replication rate of both bacteria was much higher in the mutant than in the wildtype. In cooperation with Salvatore Bozzaro (Turin, Italy) we have been able to show a decrease of nramp-expression during infection with Legionella. Approximately 48 h after infection virtually no nramp-RNA was detectable by northern blots. In contrast to these results, the nramp-expression was nearly constant during an infection by Mycobacteria. By infection studies it was shown that the endocytobionts TUME1 UWE25 and UWC6 were able to replicate within D. discoideum. The intracellular increase of the bacteria within 48 h was detected by fluorescent in situ hybridisation with specific Cy3-labeled 16S-rRNA probes. By means of electron micrographs we were able to show that the strains TUME1 and UWE25 resided within the host cytoplasm closely surrounded by membranous structures. UWC6 was found in vacuoles as well as in the cytoplasm. Localisation of the endocytobionts corresponds to their localisation in their natural hosts. A further aim of this work was to investigate the function of the L. pneumophila Mip-protein. The Mip-protein of Legionella belongs to the FK506 binding proteins. Mip exhibits peptidyl-prolyl cis/trans isomerase activity and creates homodimers. We showed that mip binds to the extracellular matrix of lung epithelial cells especially to collagen IV. By using the transwell-system we demonstrated that Mip is responsible for Legionella penetrating a barrier of lung epithelial cells and their extracellular matrix. After blocking the PPIase-activity by FK506 or Rapamycin, the ability of Legionella-penetration was dramatically reduced. Also after inhibition of serinprotease-activities in the transwell-system, Legionella was not able to penetrate through the barrier. Degradation-assays with S35-labeled extracellular matrix indicated that mip-positive Legionella are able to degrade extracellular matrix. After inhibiting the PPIase-activity or serinprotease-activities mip-positive Legionella were no longer able to degrade extracellular matrix.

Die haploide Amöbe Dictyostelium discoideum hat sich als geeinet erwiesen für die Untersuchung der zellulären Aspekte der Legionella Infektion. Nach der Aufnahme befindet sich L. pneumophila innerhalb eines unreifen Phagosoms das weder angesäuert wird noch mit Lysosomen fusioniert. In dieser Studie wurden die Wirtzellfaktoren untersucht, die Legionella eine erfolgreiche Kolonizierung des Wirt ermöglichen. Phagozytoseversuche mit spezifischen zellulären Inhibitoren und die Analyse der Aufnahme in definierten Wirtzell-Mutanten haben gezeigt, daß das zytoplasmatische Kalziumniveau, Zytoskelettproteine und die Kalzium-bindenden Proteine des ERs, Calreticulin und Calnexin, spezifisch die Aufnahme und das intrazelluläre Wachstum von L. pneumophila beeinflussen. Mikroskopisches Untersuchungen mit GFP-markierten Calnexin und Calreticulin haben gezeigt, dass beide Proteine spezifisch in den "phagocytic cups" der L. pneumophila-infizierten Wirtszellen akkumulieren. Beide Proteine umhüllten die replikative Vakuole von L. pneumophila während der gesamten Replikation des Bakteriums. Die kumulativen Effekte intrazellulären Kalziumniveaus, die räumliche Verteilung von Calnexin und Calreticulin und die Defekte Aufnahme und intrazelluläre Vermehrung von L. pneumophila im Calnexin- und Calreticulin-minus der Zellen deuten darauf hin, daß diese Faktoren ein Teil des Regulationssystems sind, der zu der Bildung der spezifische Vakuole von L. pneumophila führt. / The haploid amoeba Dictyostelium discoideum is a versatile host system for studying cellular aspects of Legionella pathogenicity. Previous studies have shown that the internalization of L. pneumophila leads to an endoplasmic reticulum (ER)-derived organelle that supports intracellular replication of the bacteria. In this study a roadmap of host-cell factors involved in this process was developed. Phagocytosis assays with specific cellular inhibitors and the effects of well defined host-cell mutants revealed that cytoplasmic calcium levels, cytoskeleton-associated proteins and the calcium-binding proteins of the ER, calreticulin and calnexin, specifically influence the uptake and intracellular growth of L. pneumophila. Confocal microscopic time series with green fluorescent protein (GFP)-tagged calnexin and calreticulin demonstrated the accumulation of both proteins in the phagocytic cup of L. pneumophila-infected host cells. In contrast to the control experiment with Escherichia coli-containing phagosomes, both proteins decorated the replicative vacuole of L. pneumophila during the entire growth phase of the bacteria. The cumulative effects of cytosolic calcium levels, the spatial distribution of calnexin and calreticulin, and the defective invasion and replication of L. pneumophila in calnexin- and calreticulin-minus cells suggest that these factors are part of a regulatory system that leads to the specific vacuole of L. pneumophila.

No description available.

Excitable media are an important class of systems, examples of which include epidemics, predator-prey interactions, nervous systems, and heart muscle. Aggregating cellular slime moulds are an example of an excitable medium. The species of cellular slime mould Dictyostelium discoideum is an important model organism that many science laboratories use. Studying the aggregation of slime moulds increases knowledge about excitable media generally. One method of studying the aggregation of slime mould is to simulate theft behaviour on a computer model. This thesis presents the author's computer model of cellular slime mould Dictyostelium discoideum and the results of experiments carried out using the computer model. The experiments investigate the relation between the aggregation patterns and the various parameters of the model. These parameters are the density of artificial slime moulds, the acrasin threshold, the acrasin degradation rate, and the rate of acrasin secretion. Randomness has an effect on the aggregation patterns produced. Results of experiments are presented that examine the effect of randomness. Two forms of randomness are investigated: random secretion of acrasin by the artificial slime moulds; random initial reactivity of the artificial slime moulds. The computer model describes an artificial environment in which artificial slime mould amoebae interact with each other and their environment. Out of these individual interactions the global patterns that characterize slime mould aggregations emerge. The model facilitates the study of these individual interactions and hence the global patterns that emerge. The model and the experimental results described in this thesis contribute to the study of the aggregation phase of the life cycle of Dictyosteliuni discoideum. The author proposes mechanism that could underlie certain classes of aggregation patterns. These patterns include net-like aggregations and loop aggregations. The computer model presented in this thesis is successful in emulating the behaviour of the cellular slime mould Dictyostelium discoideum. In its present form the model is a useful tool to biologists. The results of experiments conducted with the model suggest mechanisms that may underlie certain pattern produced by living slime moulds. A result of particular interest is the initiation of the spiral wave pattern from a loop wave, which produces a loop aggregation.

Thesis (M.Sc.)--York University, 20uu. Graduate Programme in Biology. / Typescript. Includes bibliographical references (leaves 105-113). Also available on the Internet. MODE OF ACCESS via web browser by entering the following URL:http://gateway.proquest.com/openurl?url%5Fver=Z39.88-2004&res%5Fdat=xri:pqdiss&rft%5Fval%5Ffmt=info:ofi/fmt:kev:mtx:dissertation&rft%5Fdat=xri:pqdiss:MQ99332

Thesis (M.S.)--The University of North Carolina at Greensboro, 2008. / Directed by Paul Steimle; submitted to the Dept. of Biology. Title from PDF t.p. (viewed Mar. 19, 2010). Includes bibliographical references (p. 30-31).