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).

Staphylococcus aureus é uma espécie de grande importância médica, pois está frequentemente associado com diversas infecções em seres humanos. Essa bactéria pode causar doenças que vão desde infecções simples, até infecções potencialmente letais como endocardite, pneumonia, meningite, síndrome do choque tóxico, septicemia, osteomielite, entre outras. O S. aureus é o agente mais comumente encontrado em infecções na pele e tecidos moles, infecções ósseas e de próteses ósseas. Tendo em vista a dificuldade de identificação destes focos infecciosos torna-se importante o desenvolvimento de novas técnicas diagnósticas. O diagnóstico por cintilografia apresenta vantagens sobre outros métodos, pois é capaz de identificar tecidos doentes sem a necessidade de procedimentos invasivos e é capaz de realizar um diagnóstico precoce antes mesmo de haver modificações anatômicas. Assim, a medicina nuclear poderia contribuir para um diagnóstico preciso de infecções bacterianas, desde que radiofármacos específicos fossem desenvolvidos. Aptâmeros são oligonucleotídeos que apresentam alta afinidade e especificidade para seus alvos moleculares e vem se apresentado como uma classe de moléculas promissoras para o desenvolvimento de radiofármacos. Aptâmeros radiomarcados, específicos para os agentes infecciosos, poderiam dar uma contribuição significativa para o diagnóstico de infecções através da cintilografia. Neste estudo, aptâmeros selecionados para S. aureus foram marcados com 99mTc e utilizados para identificação desta bactéria em ensaios in vitro e in vivo. Os aptâmeros foram inicialmente marcados com 32P e incubados com células de S. aureus em ensaio in vitro, mostrando alta afinidade pelas células de S. aureus quando comparados com a biblioteca de oligonucleotídeos com sequências aleatórias, utilizada como controle. Os aptâmeros foram marcados com 99mTc e incubados com células de S. aureus em ensaio in vitro e demostraram afinidade pelas células de S. aureus quando comparados com a biblioteca, porém ligações inespecíficas foram também verificadas. A estabilidade da marcação foi avaliada e os aptâmeros radiomarcados foram estáveis em solução salina 0,9%, em plasma de camundongos Swiss e em excesso de cisteína. Em ensaios in vivo em modelo experimental com camundongos Swiss, com infecção intramuscular na coxa direita, os aptâmeros marcados com 99mTc injetados por via intravenosa, foram capazes de identificar a infecção causada por S. aureus, apresentando uma relação alvo/não alvo de 4,035 0,46 após 4 h da injeção do radiomarcado. Foram utilizados como controle camundongos infectados com C. albicans, que apresentaram uma relação alvo/não alvo de 2,02 0,42 e camundongos com inflamação asséptica induzida por zimosan nos quais a relação alvo/não alvo foi de 1,146 0,226. Imagens cintilográficas mostraram uma elevada captação do radiofármaco pelos rins e eliminação pela bexiga em todos os grupos. O grupo infectado por S. aureus apresentou maior captação na coxa direita infectada sendo visível a diferença em relação a coxa esquerda (controle) no tempo de 1 h. Para os outros grupos (C. albicans e zimosan) não foram visualizadas diferenças. Os resultados obtidos neste estudo demonstram que aptâmeros marcados diretamente com 99mTc são promissores no diagnóstico de infecção pela cintilografia. / Staphylococcus aureus is specie of great medical importance because it is often associated with many infections in humans. This bacterium can cause diseases ranging from simple infections to life-threatening infections such as endocarditis, pneumonia, meningitis, toxic shock syndrome, septicemia, osteomyelitis, among others. S. aureus is the most commonly agent found in infections of the skin and soft tissues, bone infections and bone prostheses. The difficulty in early detection of specific foci caused by bacteria has raised the need to search for new techniques for this purpose. Diagnosis by scintigraphy has advantages over other methods because it is able to identify damage tissues without the need of invasive procedures and is able to perform an early diagnosis even before anatomic changes. Thus, nuclear medicine could contribute to an accurate diagnosis of bacterial infections, since specific radiopharmaceuticals were developed. Aptamers are oligonucleotides that have high affinity and specificity for their molecular targets and are emerging as a new class of molecules for radiopharmaceuticals development. Radiolabeled aptamers specific to the infectious agents, could give a significant contribution to the infection diagnosis by scintigraphy. In this study, aptamers selected to S. aureus were labeled with 99mTc and used for the bacteria identification in vitro and in vivo. The aptamers labeled with 32P and incubated in vitro with S. aureus cells showed high affinity for the bacterial cells when compared with the library of oligonucleotides with random sequences used as control. The aptamers labeled with 99mTc also showed affinity for S. aureus cells when compared with the library, but unespecific binding was also verified. The 99mTc labelled aptamers were stable in 0.9% saline, plasma of Swiss mice and in excess of cysteine. The in vivo biodistribution studies using Swiss mice with intramuscular infection in the right thigh showed that the aptamers labeled with 99mTc were able to identify the infection foci caused by S. aureus displaying a target/non-target ratio of 4,035 0.46 after 4 h. Mice infected with C. albicans presented a target/non-target ratio of 2.02 0.42 while mice with aseptic inflammation induced by zymosan showed a target/non-target ratio of 1,146 0,226. Scintigraphic images revealed a high uptake of the radiopharmaceutical by the kidneys and fast elimination by the bladder. The images also showed higher uptake in the right thigh (infected) in relation to the left (control) for the S. aureus infected mice. In the control groups (C. albicans e zymosan) was not possible to visualize any difference. The results indicate the radiolabeled aptamers as a promising alternative for the development of radiopharmaceuticals for infection.

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.