All cells maintain essential metal nutrients at optimal levels by metal homeostasis. P-type ATPases, a crucial superfamily of integral membrane proteins, are involved in the active transport of metal ions across biological membranes driven by the motive force of ATP- hydrolysis. The PIB-type ATPase subfamily, also called CPx-ATPases, fulfills a key role in heavy metal homoeostasis among the most widespread species from bacteria to human. In humans, the defect in copper transporters is the direct cause of severe neurological and hepatic disorders such as Wilson and Menkes diseases, therefore, understanding the molecular function of these pumps is of paramount importance in human health. Cu+-ATPases have two transmembrane metal binding sites (TM-MBS) and three cytosolic domains, namely the actuator (A-domain) and phosphorylation and nucleotide-binding domain (PN), and regulatory N-terminal heavy metal binding domain (HMBD). Here, we have studied the Legionella pneumophila CopA (LpCopA) and its isolated cytosolic domains to improve our understanding of the functional interaction of the protein domains during metal transport relate this to the known structure of this ATPase. To elucidate how cytosolic ligands (Cu+ and nucleotide) stimulate the interactions among the cytosolic domains and may transmit conformational changes to the TM-MBS, the interactions among recombinant isolated cytosolic domains were first examined biochemically by co-purification and spectroscopically by circular dichroism, time-resolved fluorescence and site-directed fluorescent labeling assays. The Cu+-dependent interaction between the A-domain and HMBD has been postulated as a mechanism for activating the ATPase cycle. This question was addressed here by studying copper-dependent interactions between the isolated expressed domains. Spectroscopic evidence is provided that an HMBD-A complex is formed in the presence of Cu+ which binds with 100-200 nM affinity to the recombinant HMBD. In contrast, the A-domain interacts with the PN domain in a nucleotide-dependent fashion. This molecular recognition is required for the dephosphorylation step in the catalytic cycle. The interaction was investigated in more detail by the use of a decameric peptide derived from the PN-binding interface of the A-domain and carrying the conserved TGE-motif involved in dephosphorylation. Its binding to the isolated PN domain in a weakly nucleotide-dependent manner, is demonstrated here by stopped-flow fluorescence spectroscopy. Several ATPase assays were modified to assess the functionality of the PN-domain and full length LpCopA. The peptide was found to reduce the catalytic turnover of full length LpCopA. This agrees with the expected slowing down of the reformation of the PN-A-domain interaction since the peptide occupies their binding interface. Thus, the synthetic peptide provides a means to study specifically the influence of PN-A-domain interactions on the structure and function of LpCopA. This was done by time-correlated single photon counting (TCSPC) method. The time-dependent Stokes shift of the environmentally sensitive fluorophore BADAN which was covalently attached to the conserved CPC-motif in the TM-MBS was measured. The data indicate that the interior of the ATPase is hydrated and the mobility of the intra-protein water varies from high to low at C382 at the “luminal side” and C384 at the “cytosolic side” of the TM-MBS, respectively. This finding is consistent with the recent MD simulation of LpCopA, bringing the first experimental evidence on a luminal-open conformation of E2~P state. The A-domain-derived decapeptide, although binding to the cytosolic head piece, induces structural changes also at the TM-MBS. The peptide-stabilized state (with a disrupted PN-A interface) renders the C384 environment more hydrophobic as evidenced by TCSPC. Taken together, the data from cytosolic domain interactions, ATPase assays and of time-dependent Stoke shift analyses of BADAN-labeled LpCopA reveal the presence of hydrated intramembraneous sites whose degree of hydration is regulated by the rearrangement of cytosolic domains, particularly during the association and dissociation of the PN-A domains. Copper affects this arrangement by inducing the linkage of the A-domain to the HMBD. The latter appears to play not only an autoinhibitory but also a chaperone-like role in transferring Cu+ to the TM-MBS during catalytic turnover.

Includes bibliographical references (leaves 115-120) Establishes a Bayesian Belief Network (BBN) to model uncertainty of aerosols released from cooling towers and Geographic Information Systems (GIS) to create a wind dispersal model and identify potential cooling towers as the source of infection. Demonstrates the use of GIS and BBN in environmental epidemiology and the power of spatial information in the area of health.

Les espèces bactériennes exhibent plusieurs états de structure de populations pouvant varier de clonale à panmictique selon l'importance des transferts horizontaux et la nature de leur écosystème. Dans mon travail de thèse, je me suis intéressé à trois espèces bactériennes, Staphylococcus aureus, Legionella pneumophila et Pseudomonas aeruginosa qui reflètent trois situations différentes. Afin de pouvoir décrire de façon rapide de grandes collections de souches, j'ai utilisé comme marqueurs de diversité le polymorphisme de séquences répétées en tandem appelées VNTRs, pour Variable Number Tandem Repeat. La méthode MLVA, ou Multiple Loci VNTR Analysis, est une méthode de typage moléculaire qui s'appuie sur l'étude concomitante du polymorphisme de plusieurs loci VNTRs. Dans un premier temps, j'ai conçu des protocoles de typage automatisés pour les trois espèces considérées, puis j'ai appliqué ces outils pour traiter de questions d'épidémiologie. S. aureus, espèce à structure clonale, est un pathogène majeur responsable notamment de toxi-infections alimentaires collectives (TIAC). Les travaux réalisés ont permis de démontrer la spécificité d'hôte de certains complexes clonaux et l'origine humaine des cas de TIAC. L. pneumophila est un pathogène de l'environnement dont la structure de population est atypique : présumée panmictique dans la nature, la bactérie semble connaitre une évolution clonale lorsque son écosystème est restreint, dans un milieu anthropique par exemple. L'étude épidémiologique menée sur la population de L. pneumophila dans la ville de Rennes a mis en évidence la présence d'un écotype, non impliqué dans les cas cliniques épidémiques, particulièrement adapté aux réseaux d'eau. P. aeruginosa, modèle de bactérie panmictique, colonise les bronches de patients atteints de mucoviscidose. Le suivi longitudinal de patients indique que les souches installées sont persistantes et quasi-exclusive de la niche qu'elles occupent. L'exploration de cette diversité du monde bactérien est un préalable à l'investigation épidémiologique des maladies infectieuses. Avec un même outil moléculaire de première intention, cette thèse retrace l'épidémiologie et la structure de trois espèces bactériennes très différentes. L'adaptation à un nouvel environnement (hôte animal, niche écologique, organe) est l'occasion d'expansions clonales.

Blood-sucking louse Polyplax serrata harbor two phylogenetically distinct symbiotic bacteria from the orders Legionellales (Gammaproteobacteria) and Neisseriales (Betaproteobacteria). Based on rigorous phylogenetic analyses, the first bacteria was confirmed as member of genus Legionella, and the name Candidatus Legionella polyplacis, sp. n. was proposed. Vertical transgeneration transmission was proved by visualisation of the symbiont in host tissues. Improved genome of latter symbiont is considerably larger and possesses more metabolic functions. In the genome, I found horizontally transferred operon, which encode synthesis of enzymes involved in urea cleavage.

<p>  </p> <p>The pandemic prompted buildings globally to transition to low or no occupancy as social distancing to reduce the spread of Coronavirus Disease (COVID-19). This consequence prompted concerns about the chemical and microbiological safety of building drinking water due to stagnation. At the same time, microplastic (MP) pollution received increasing global attention due to their presence in the environment and recent discoveries within water distribution systems and at building faucets. MP sources have primarily been targeted as originating within the drinking water sources, but plastic plumbing components are less discussed and known to deteriorate into fragments and smaller pieces that reach faucets. Literature at the time of this work as sparse on stagnation impacts to drinking water quality and the fate of MPs in plumbing. In particular, health officials and building owners issued and received many differed guidance documents telling building owners do different things and no standard guideline was available to reduce the health risks caused by stagnant building drinking water. This dissertation  examined three different types of buildings during closed to low water use conditions and conducted bench-scale testing to explore the phenomena observed in the field. Chapter 1 describes water quality impacts during a 7 year old ‘green’ middle school as it transitioned from Summer (low water use) to Fall (normal use). Field experiments revealed that more than half of first draw water samples exceeded the copper (acute) health-based action limit during low water use. Copper concentration within the school increased as distance from building entry point increased. Chapter 2 and 3 describe report on chemical and microbiological water quality in buildings at a university buildings (Chapter 2), and elementary school (Chapter 3). Chapters 2 and 3 revealed that stagnation negatively impacted chemical and microbiological building water quality (cold and hot) but flushing was effective at remediating high concentration of heavy metals and <em>Legionella pneumophila</em> at most locations. But in large buildings, where building plumbing system was more complicated, flushing did not always result in improved water quality. Also discovered was that water quality again deteriorated even after whole building water system was flushed. It is important to understand own building systems to maintain water quality as each building complexity requires specific knowledge and solutions. Chapter 4 describes current knowledge associated with MPs in drinking water and results of bench scale experiments on MP fate and transport in building plumbing. This work identified that while MPs have been reported at building faucets, sampling details lacking from available studies often resulted in study results not being comparable across others. Based on the review of the issue, it was found that MPs have likely reached building faucets for decades but have received no characterization until recently. Bench-scale testing using two MPs, of different density, in copper and crosslinked polyethylene (PEX) pipes revealed size influenced the amount of MPs retained in a pipe. Research needs were identified to determine the fundamental factors that control MP fate in plumbing and their presence at building faucets. </p>

The task of this diploma thesis is to prepare the design of sanitary and gas pipeline installations in the assigned hospital building. The building has one underground and four above-ground floors. On the underground floor there are technical premises including a boiler room, hygienic facilities of staff and rooms for storage and cleaning. On the first floor there are ambulances of various kinds. Patient rooms are located over the second and third floors. Each room has its own built-in bathroom. There are also doctor's examination rooms and daily room of staff. On the fourth floor there is an ICU. In the theoretical part I deal with the legionella bacterium. I explain what causes this bacterium, what are the conditions for its reproduction, where it occurs and what measures will prevent its multiplication in water pipes. In the next part, I compare two possible variants of the solution of the design of water pipe routes with the subsequent selection of a more suitable option. The calculation part contains all the calculations used to design sewerage, water supply and gas pipeline, including their connection to utilities.

No description available.