Thesis (M.S.)--University of Wisconsin--Madison, 1962. / Typescript. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 112-118).

Thesis (M.S.)--University of Wisconsin--Madison, 1962. / Typescript. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 76-82).

S. aureus est une bactérie pathogène opportuniste de l’homme qui pose un grave problème de santé publique. Le pouvoir pathogène de S. aureus est conféré par un très grand nombre de facteurs de virulence, dont l’expression est finement régulée à de multiples niveaux. Les effecteurs de cette régulation sont à la fois des protéines et des ARN non codants (ARNnc) aussi appelés ARN régulateurs. Je me suis concentré au cours de ma thèse sur la classe majoritaire qui sont les ARNnc qui régulent la traduction d’ARNm. Ils sont impliqués dans de complexes réseaux de régulation qui permettent de contrôler la physiologie de la cellule ainsi que sa virulence. Pour élargir nos connaissances de ces réseaux, j’ai développé deux approches méthodologiques, appelées MAPS et Grad-seq, que j’ai appliquées in vivo chez S. aureus en utilisant RsaA et RsaC comme modèles. L’application du MAPS a permis d’identifier de nouvelles cibles directes pour RsaA et des cibles potentielles pour RsaC. L’approche Grad-Seq est un outil puissant mais demande encore des ajustements. J’ai également pu déterminer un rôle probable pour l’ARNnc RsaC dans la régulation de l’homéostasie oxydo-réductive de S. aureus, en lien avec la résistance au stress oxydatif et avec la persistance lors de l’internalisation par les ostéoblastes. / S. aureus is an opportunistic pathogen of the human species which can express a large array of virulence factors whose expression is under tight regulation at multiple levels. The regulation can be done by proteins and by particular molecules of RNA called non-coding RNA (ncRNA). I focused during my thesis on the main category of ncRNA in S. aureus, which are regulating the translation of mRNA. These ARNs are involved in complex regulatory networks, impacting the physiology of the bacterial cell and its virulence. To understand further these networks, I developped two methodological approaches in vivo in S. aureus, called MAPS and Grad-seq, which were applied using RsaA and RsaC as models of studies. MAPS allowed to find new direct targets of RsaA and plausible targets for RsaC. The Grad-Seq method showed to be a powerful tool but still needs refinements. I also could determine a possible role for RsaC in the regulation of oxydo-reductive homeostasis, in direct link with oxydative stress resistance and persistance during internalisation by osteoblasts.

Pathogens in drinking water are the cause of over 1.5 million deaths around the world every year, mostly in developing countries. Practical, cheap, and effective tools for detection of these pathogens are critical to advance public health in many areas around the globe. Micro electro-mechanical systems (MEMS) are miniaturized structures that can be used for a variety of purposes, including, but not limited to, small scale sensors. Therefore, MEMS can be used in place of expensive laboratory equipment and offer a cheap and practical tool for pathogen detection. The presented work's research objective is to introduce a new technique called embedded passivated-electrode insulator-based dielectrophoresis (EπDEP) for preconcentration, separation, or enrichment of bioparticles, including living cells. This new method combines traditional electrode-based DEP and insulator-based DEP with the objective of enhancing the electric field strength and capture efficiency within the microfluidic channel while alleviating direct contact between the electrode and the fluid. The EπDEP chip contains embedded electrodes within the microfluidic channel covered by a thin passivation layer of only 4 μm. The channel was designed with two nonaligned vertical columns of insulated microposts (200 μm diameter, 50 μm spacing) located between the electrodes (600 μm wide, 600 μm horizontal spacing) to generate the nonuniform electric field lines to concentrate cells while maintaining steady flow in the channel. The performance of the chip was demonstrated using Gram-negative (Escherichia coli) and Gram-positive (Staphylococcus aureus) bacterial pathogens in aqueous media. Trapping efficiencies of 100% were obtained for both pathogens at an applied AC voltage of 50 V peak-to-peak and flow rates as high as 10 uL/min. / Master of Science

Bacteria are enclosed by a peptidoglycan sacculus, an exoskeleton-like polymer composed of glycan strands cross-linked by short peptides. The sacculus surrounds the cell in a closed bag-like structure and forms the main structural component of the bacterial cell wall. As bacteria grow and divide, cell wall remodelling by peptidoglycan hydrolases results in the release of peptidoglycan fragments from the sacculus. In Gram-negative bacteria, these fragments are efficiently trapped and recycled. Gram-positive bacteria however shed large quantities of peptidoglycan fragments into the environment. For nearly five decades, Gram-positive bacteria were thus assumed not to recycle peptidoglycan and this process has remained enigmatic until recently. In this thesis, the occurrence and physiological role of peptidoglycan recycling in the Gram-positive pathogen Staphylococcus aureus was investigated. S. aureus is an important pathogen, and is becoming increasingly resistant to many antibiotics. Through bioinformatic and experimental means it was determined that S. aureus may potentially recycle components of peptidoglycan and novel peptidoglycan recycling components were identified and characterised. Though disruption of putative peptidoglycan recycling in S. aureus appears not affect growth or gross morphology of this bacterium, potential roles for peptidoglycan recycling in cell wall homeostasis and in virulence were identified. This is to my knowledge the first demonstration of a potential role of peptidoglycan recycling in either of these aspects of bacterial physiology in any Gram-positive bacterium. This is an important step forward in understanding the basic biology of Gram-positive bacteria, and in understanding the mechanisms of virulence in S. aureus. Future study of this process in S. aureus and other Gram-positive bacteria promises to reveal yet further facets of this process and its functions, potentially leading to the identification of novel therapeutic approaches to combat infections.

Chapter I: This chapter briefly gives an introduction about microorganisms, their varieties, growth, reproduction etc. In particular, about bacterial function. A sincere attempt is made to review this briefly, including an account of the studies already reported in the literature. Chapter II deals with the antimicrobial activity of Ag/agar film on Escherichia coli (E. coli), Staphylococcus aureus (S. aureus), and Candida albicans (C. albicans). In particular, films were repeatedly cycled for its antimicrobial activity. The antimicrobial activity of Ag/agar film was found to be in the order, C. albicans > E. coli > S. aureus. Chapter III describes the synthesis of Ag@AgI NPs in agarose matrix. A detailed antibacterial studies including repetitive cycles are carried out on E. coli and S. aureus bacteria. EPR and TEM studies are carried out on the Ag@AgI/Agarose and the bacteria, respectively, to elucidate a possible mechanism for killing of the bacteria. The hybrid could be recycled for the antibacterial activity many times and is found to be non toxic towards human cervical cancer cell (HeLa cells). Chapter IV reports the antibacterial efficacy of silver nanoparticles (Ag NPs) deposited alternatively layer by layer (LBL) on chitosan polymer in the form of a thin film over a quartz plate and stainless steel strip against E. coli. AFM studies are carried out on the microbe to know the morphological changes affected by the hybrid film. The hybrid films on aging (3 months) are found to be as bioactive as before. Cytotoxicity experiments indicated good biocompatibility. Chapter V describes the fabrication of carbon foam porous electrode modified with reduced graphene oxide-Ag nanocomposites. The device can perform sterilization by killing pathogenic microbes with the aid of just one 1.5V battery with very little power consumption. Chapter VI Here we have studied in particular a property say the influence of surface defect in the production of ROS by ZnO NPs and in turn the bactericide activity. Secondly, a homogeneous ZnO and ZnO/Ag nanohybrid has been synthesized by employing chitosan as mediator. The synergistic antibacterial effect of ZnO/Ag nanohybrid on bacteria is found to be more effective, compared to the individual components (ZnO and Ag). A possible mechanism has been proposed for the death of bacteria by ZnO/Ag nanohybrid, based on EPR studies and TEM studies.