<p dir="ltr">Tuberculosis remains a global health challenge, ranking as the second leading cause of mortality worldwide in 2022. The resilience of <i>Mycobacterium tuberculosis</i>, the causative agent of tuberculosis, is enhanced by the high expression of efflux pumps that confer antibiotic tolerance and the formation of biofilms that confer resistance to antibiotics. Carbon nanomaterials (CNMs) exhibit a broad-spectrum of antibacterial efficacy, making them promising candidates for combating drug-resistant bacterial strains. The effects of the novel carbon allotropes called fullertubes (C₉₀) on any living cell have not been studied. In our study, we employed <i>Mycobacterium smegmatis</i> as a model organism for <i>M. tuberculosis</i> and exposed it to fullertubes and fullerenes. We explored the impact of these CNMs on efflux activity and biofilm formation through biochemical assays like ethidium bromide transport assay and crystal violet assay. We also investigated their impact on lipid biosynthesis associated with log-phase growth and biofilm formation using metabolic radiolabeling studies. We also investigated the effects of the efflux pump inhibitors (EPIs) piperine, berberine, 1-(1-naphthylmethyl)-piperazine and thioridazine on efflux activity, biofilm formation, and lipid biosynthesis associated with log-phase growth and biofilm formation in <i>M. smegmatis.</i> We utilized metabolic radiolabeling methods using ¹⁴C-palmitic acid and ¹⁴C-acetic acid which are precursors of lipid biosynthesis and analyzed the lipids by silica-thin layer chromatography and autoradiography. Our studies revealed that CNMs do not influence efflux activity. However, efflux pump inhibitors effectively block efflux activity in <i>M. smegmatis</i>. Biofilm formation was decreased by CNMs and EPIs. In biofilm cells, fullertubes increased the incorporation of radiolabeled ¹⁴C-palmitic acid into glycopeptidolipids on the cell surface as well as inside the cell. Piperine and berberine affected the incorporation of the radiolabels into lipids such as trehalose monomycolate, phosphatidylethanolamine and cardiolipin in planktonic and biofilm cells. Our study provides insights into the diverse effects of CNMs and efflux pump inhibitors on <i>M. smegmatis</i>.</p>

L'émergence des zoonoses est à relier directement avec les perturbations générées par l'Homme sur son environnement naturel à plus ou moins grande échelle. Sur l'ensemble des zoonoses émergentes chez l'Homme, la majorité provient d'animaux sauvages. L'étude du rôle de la faune sauvage dans la circulation des agents pathogènes est donc cruciale en particulier quand les interfaces faune sauvage/Homme sont fortes. L'objectif de cette thèse a été de comprendre par des approches éco-épidémiologiques à large échelle, la circulation d'agents pathogènes dans les populations d'un oiseau sauvage en contact étroit avec l'Homme, le goéland leucophée (Larus michahellis). Le premier chapitre présente les différentes méthodes utilisées dans la surveillance d'agents pathogènes ainsi que leurs limites lorsqu'elles sont menées en populations naturelles. Ce chapitre met en évidence au travers d'une étude portant sur les virus influenza aviaires, que la quantification des anticorps maternels dans les œufs est un outil efficace. Le second chapitre consiste à élargir l'échelle spatiale de l'étude afin de mettre en évidence plus finement les facteurs éco-épidémiologiques influençant la transmission des virus influenza aviaires dans et entre les populations de goéland leucophée de l'ouest méditerranéen. Enfin, le dernier chapitre repose sur la comparaison des patrons d'expositions obtenus pour des agents pathogènes au mode de transmission vectoriel : les flavivirus. Cette thèse permet de mettre en évidence les patrons d'exposition de certains agents pathogènes (virus influenza aviaire et flavivirus) et d'appréhender les facteurs éco-épidémiologiques potentiellement impliqués dans leurs circulations. Les résultats permettent d'envisager de futurs axes de recherches, nécessaires pour évaluer plus précisément la dispersion de ces virus en Méditerranée. / The emergence of zoonotic diseases is directly linked to the noise generated by humans on the natural environment to a greater or lesser extent. Of all the emerging zoonoses in humans, the majority comes from wild animals. The study of the role of wildlife in the circulation of pathogens is crucial, especially when wildlife/human interfaces are prominent. The aim of this thesis is to understand, using large scale eco-epidemiological approaches, the circulation of pathogens in a close to human population of wild bird, the yellow-legged gull (Larus michahellis). The first chapter presents the different methods used in the monitoring of pathogens and their limitations when they are conducted in natural populations. This chapter further highlights, through a study of avian influenza viruses, that the quantification of maternal antibodies in eggs is an effective tool. The second chapter expands the spatial scale of the study to highlight the finer eco-epidemiological factors influencing the transmission of avian influenza viruses within and between populations of Western Mediterranean yellow-legged gull. Finally, the last chapter is based on the comparison of exposition patterns obtained for vectorial transmission mode pathogens: flaviviruses. This thesis highlights patterns of exposure for certain pathogens (avian influenza virus and flaviviruses) and enables the understanding of the eco-epidemiological factors potentially involved in their circulations. The results allow to consider future areas of research needed to more accurately assess the dispersion of these viruses over the Mediterranean.

Case reports describe significant norovirus gastroenteritis morbidity in immunocompromised patients. We evaluated norovirus pathogenesis in prospectively enrolled solid organ (SOT) and hematopoietic stem cell transplant (HSCT) patients with diarrhea who presented to Texas Children's Hospital and submitted stool for enteric testing. Noroviruses were detected by real-time reverse transcription polymerase chain reaction. Clinical outcomes of norovirus diarrhea and non-norovirus diarrhea patients, matched by transplanted organ type, were compared. Norovirus infection was identified in 25 (22%) of 116 patients, more frequently than other enteropathogens. Fifty percent of norovirus patients experienced diarrhea lasting ≥14 days, with median duration of 12.5 days (range 1–324 days); 29% developed diarrhea recurrence. Fifty-five percent of norovirus patients were hospitalized for diarrhea, with 27% requiring intensive care unit (ICU) admission. One HSCT recipient developed pneumatosis intestinalis. Three HSCT patients expired ≤6 months of norovirus diarrhea onset. Compared to non-norovirus diarrhea patients, norovirus patients experienced significantly more frequent ICU admission (27% vs. 0%, p = 0.02), greater serum creatinine rise (median 0.3 vs. 0.2 mg/dL, p = 0.01), and more weight loss (median 1.6 vs. 0.6 kg, p < 0.01). Noroviruses are an important cause of diarrhea in pediatric transplant patients and are associated with significant clinical complications.

Baculoviruses are lytic insect viruses. Upon internalisation, the viral genome orchestrates a sequential expression process ultimately leading to lysis of the infected cell. Release of progeny capable of infecting other cells during the process completes the infection cycle. Studies of the infection cycle in cell culture are typically conducted by synchronous infection, i.e. near simultaneous infection of all cells, by means of high virus concentrations. The behaviour of the synchronously infected culture, such as the timing of onset of progeny release, is considered representative for the infection progression within individual cells. In reality, however, the synchronously infected culture only reflects the average behaviour of all infected cells. The infection progresses in individual cells display large variability; this is most obvious in the observation that within the same culture some cells undergo cell lysis at two days post infection while others remain viable up to four days post infection. Such variabilities or asynchronies observed in synchronously infected culture is the topic of this thesis. Using a simple phenomenological model, it is demonstrated that cell death and associated intracellular product release is adequately described assuming that the waiting time from infection to cell death follows a Gaussian distribution with a mean of 59 hours post infection (hpi) and a standard deviation of 15hpi. Unlike other deterministic models developed over the last decade (Licari and Bailey 1992; Nielsen 2000), this stochastic model does not make the biologically inconsistent assumption that cells continue to be metabolically active following loss of membrane integrity. While elegant in its simplicity, the model provides no explanation for the underlying stochasticity. Investigations into the cause of this dispersion of cell death highlighted further asynchronies in the specific recombinant protein yield, in viral DNA content, in virus budding rate, and in cell volume increase instead of clarifying the issue. A modelling framework developed by Licari & Bailey (1992) and later Hu & Bentley (2000) incorporates the number of infectious particles each individual cell receives as a possible source of the dispersions in the host cell responses. However, this was found NOT to be the cause of the observed asynchronies under non-substrate limiting conditions. The timing of cell death, cell volume increase, recombinant product yield, viral DNA content, and virus budding rate is identical in Sf9 cell cultures infected at multiplicities of infection of ~5, ~15, and ~45 infectious particles per cell. Cell cycle variation has previously been suggested as a possible cause for observed asynchronies in baculovirus infections (Brown and Faulkner, 1975). The cell cycle phase is indirectly linked to the cell volume, because a G_2-phase cell prior to division is inherently twice the cell volume of a G_1- phase cell after cell division. By the same logic, it is also apparent that a G_2-phase cell possesses twice the number of ribosomes of a G_1-phase cell and thus a doubled protein production capacity. The effect of the cell cycle or cell volume on the baculovirus infection was determined by splitting an exponentially growing Sf9 cell culture into 5 cell size dependent fractions by centrifugal elutriation. The subsequent infection of these fractions showed (1) no dependency of the timing of cell lysis and cell volume increase and (2) approximately twofold increase of a) recombinant protein yield, b) viral DNA concentration, and c) budded virus yield. The recombinant protein yield showed a strong proportionality to the initial cell volume and the total RNA concentration during the late phase of the infection. As argued in chapter 6, these proportionalities suggest that the observed differences in the responses of the cell fractions to the baculovirus infection are more likely caused by the difference in the protein production capacity than by cell cycle specific molecules. This investigation gave also reason to speculate that infected cells cannot progress beyond the G_2/M phase, and cell cycle progression continues undisturbed until ~8hpi when all cellular DNA replication appears to cease. Resuspended, infected Sf9 cells synchronised by centrifugal elutriation showed an identical cell cycle distribution as the non-infected control cultures for the first ~8hpi; G_1 and G_2/M phase cell proportions remained unchanged, whereas S phase cells progress to G_2/M phase. Subsequently, the non-infected control cells resumed normal cycling whereas all infected cells remained at the same cell cycle phase from 8 to 11hpi. The initial cell cycle arrests in G_2/M phase in both infected and non-infected cultures were caused through medium exchange.

Submitted by Simone Souza (simonecgsouza@hotmail.com) on 2017-10-18T14:04:08Z No. of bitstreams: 1 DISS_2014_Selma Samiko Miyazaki Onuma.pdf: 1498739 bytes, checksum: 80569040824ee0dd609c6d63ad7c4fa7 (MD5) / Approved for entry into archive by Jordan (jordanbiblio@gmail.com) on 2017-11-08T12:33:06Z (GMT) No. of bitstreams: 1 DISS_2014_Selma Samiko Miyazaki Onuma.pdf: 1498739 bytes, checksum: 80569040824ee0dd609c6d63ad7c4fa7 (MD5) / Made available in DSpace on 2017-11-08T12:33:06Z (GMT). No. of bitstreams: 1 DISS_2014_Selma Samiko Miyazaki Onuma.pdf: 1498739 bytes, checksum: 80569040824ee0dd609c6d63ad7c4fa7 (MD5) Previous issue date: 2014-02-27 / Amostras de sangue de 11 onças-pintadas de vida livre (Panthera onca) foram coletadas em duas unidades de conservação federal no Pantanal de Mato Grosso com objetivo de avaliar o perfil sanitário dessas populações. A presença de anticorpos séricos para Ehrlichia spp., Rickettsia spp., Toxoplasma gondii, Neospora caninum e Sarcocystis neurona foi investigada pela Reação de Imunofluorescência Indireta (RIFI); para Leptospira spp. pela Microtécnica de Soroaglutinação Microscópica (SAM) e para Brucella abortus pelo teste do Antígeno Acidificado Tamponado (Rosa Bengala). O DNA genômico em cada amostra de sangue total coletada foi analisada pela Reação em Cadeia pela Polimerase (PCR) com intuito de identificar a presença de DNA de Ehrlichia spp., Rickettsia spp., Hepatozoon spp. e Cytauxzoon spp. Duas fêmeas de carrapato Amblyomma cajennense coletados em um animal foram também testadas pela PCR para detecção de DNA de Ehrlichia spp. e Rickettsia spp. Duas onças foram soropositivas para Ehrlichia spp., com títulos de anticorpos de 80 e 320, respectivamente. Nove animais reagiram positivamente a pelo menos quatro espécies de Rickettsia spp. Dez, 7 e 8 animais amostrados foram soropositivos para T. gondii, N. caninum e S. neurona, respectivamente. Duas onças apresentaram soros reagentes para Leptospira spp. , e o provável sorotipo infectante em ambos os animais foi um isolado brasileiro do sorovar Canicola (L01). Todas as amostras de soro foram negativas para B. abortus. Nenhum DNA da família Anaplasmataceae e do gênero Ehrlichia foi detectado pela PCR ou pela Heminested- PCR nas amostras de sangue, entretanto uma foi positiva na PCR para Rickettsia spp. segundo o gene citrate sintase (gltA), porém negativa para o gene ompA, comum entre riquétsias pertencentes ao grupo da Febre Maculosa. Um A. cajennense apresentou DNA riquetsial, cujo sequenciamento de nucleotídeos demonstrou 99% de similaridade a R. amblyommii. Sete amostras apresentaram DNA de Hepatozoon spp., com 99% de similaridade para H. felis e 10 das 11 amostras foram positivas para DNA de Cytauxzoon spp., com 99% de similaridade para C. felis. O presente estudo mostrou que as onças de vida livre na porção norte do Pantanal foram expostas a inúmeros agentes patogênicos, e que uma abordagem integrada para a conservação da vida selvagem e a integridade da saúde pública é uma questão eminente a fim de determinar intervenções de gestão em áreas protegidas. Este é o primeiro relato da exposição de onças-pintadas de vida livre a N. caninum e S. neurona. / Blood samples from 11 free-living jaguars (Panthera onca) were collected in two federal conservation units in the Pantanal of Mato Grosso state to determine the health profile of these populations . The presence of serum antibodies for Ehrlichia spp., Rickettsia spp., Toxoplasma gondii, Neospora caninum and Sarcocystis neurona was investigated by Immunofluorescence Assay (IFA); for Leptospira spp by the Microscopic Agglutination Test (MAT). and for Brucella abortus by rose Bengal test . Blood genomic DNA in each sample was tested by Polymerase Chain Reaction( PCR ) to identify Ehrlichia spp . , Rickettsia spp . , Hepatozoon spp . and Cytauxzoon spp. Two females of Amblyomma cajennense ticks collected from an animal were tested by PCR for DNA detection of Ehrlichia spp . and Rickettsia spp. Two jaguars were seropositive for Ehrlichia spp ., with antibody titres of 80 and 320, respectively. Nine animals were positive for at least four species of Rickettsia spp. Ten, 7 and 8 of the sampled animals were seropositive for T. gondii, N. caninum and S. neurona, respectively. Two jaguars were seroreactive for Leptospira spp. antigen and the most likely infecting serotype in both animals was a Brazilian isolate of serovar Canicola ( L01 ) . All serum samples were negative for B. abortus. No Anaplasmataceae and Ehrlichia DNA was detected by PCR or by Heminested-PCR. One sample was positive by PCR for Rickettsia spp. according to the citrate synthase gene (gltA), but it was negative for the ompA rickettsial gene of the spotted fever group. An A. cajennense collected showed riquetsial DNA whose sequence showed 99 % similarity to R. amblyommii . Seven samples presented DNA fragments of Hepatozoon spp., with 99% similarity to H. felis and 10 of the 11 samples were positive for DNA fragments of Cytauxzoon spp., with 99 % similarity to C. felis. The present study showed that free-living jaguars in northern Pantanal were exposed to numerous pathogens, and an integrated approach to wildlife conservation and integrity of public health is a prominent issue in order to determine management interventions in protected areas. This is the first report of exposure of free-living jaguars to N. caninum and S. neurona.

Made available in DSpace on 2014-07-29T15:29:11Z (GMT). No. of bitstreams: 1 Dissertacao Talita Silva Ramos.pdf: 3387095 bytes, checksum: 04f2c9013d8026bdbc34d13d75a0c272 (MD5) Previous issue date: 2011-04-27 / The narrowing between humans, domestic and wild animals has increased the dissemination of infectious agents for new hosts. The result of this interaction has lead to zoonosis. Among these animals are birds, which are natural reservoirs, carriers and disseminators of several infectious agents, potentially transmissible to humans. This dissemination can occur through direct and indirect contact with migratory birds originated from smuggling of wild animals. The objective was to detect viral, bacterial and fungal agents with zoonotic potential in wild birds in the county of Goiânia, Goiás. It was carried out a systematic review to assess the State of Goiás in relation to the performance of wild birds as carries of infectious agents to humans. Blood samples and cloacal swabs were collected from 88 different species of birds, as most of them were originated from smuggling and sent to CETAS-IBAMA in Goiânia in the first semester of 2010. The collects were analyzed in the Laboratory of Animal Virology at Federal University of Goiás (UFG) and the bacteriological and fungal analyses were done in the Laboratory of Microbiology at National Service of Industrial Education (SENAI) in Anápolis, Goiás. The blood was stored in tubes without anticoagulant in order to obtain serum and it was carried out a research with Enzyme-Linked Immunosorbent to search for the antibody of influenza virus and the cause of Newcastle disease. In order to isolate and identify filamentous fungus, Candida spp, bacteria, Salmonella sp and Escherichia coli, samples were collected from cloacal excretion through swabs. In the systematic review, three articles were selected using two tests of Relevance. From the interpretation of these studies, it was proved the role of migratory birds as carriers and disseminators in short and long distances of several pathogenic virus to humans. Furthermore, there are no records or even systematic studies about this process in the state of Goiás. From 88 analyzed samples, none of them presented antibodies for the two researched virus. The analysis of samples from cloacal swabs indicated a positivity of 9,1% for Candida tropicalis , 9,1% for Salmonella sp, 1,1% for Aspergillus fumigatus, 11,4% for Candida albicans, 12,5% for Candida krusei, 27,3% for Candida spp, and 43,2% for Escherichia coli. The literature review suggests that further researches should be carried out as the distribution of this zoonosis can possibly be wider, even in the State of Goiás. Regarding the negative results for the viral agents, they indicate that part of the wild birds in Goiânia-Goiás did not have contact with influenza and Newcastle viral antigens yet. Therefore they are not carriers of these photogenes. In relation to presumptive identifications of pathogenic bacteria and fungal agents in wild birds, they confirm the possibility of transmission of these agents among animals and to humans. Besides, this study also reinforces that researches about zoonosis transmitted by wild birds in the state of Goiás are still recent and incipient, highlighting the need of continuation of these studies in the region. / O estreitamento entre a população humana e os animais domésticos com animais silvestres aumentou a disseminação de agentes infecciosos para novos hospedeiros, sendo uma importante consequência, as zoonoses. Entre estes animais, estão as aves, que são reservatórios naturais, portadores e disseminadores de diversos agentes infecciosos, potencialmente patogênicos transmissíveis aos humanos. A disseminação pode ocorrer por meio de contato direto ou indireto com aves migratórias e provenientes do tráfico de animais silvestres. O objetivo foi detectar agentes virais, bacterianos e fúngicos, com potencial zoonótico em aves silvestres no município de Goiânia-Goiás. Foi conduzida uma revisão sistemática para avaliar com ênfase, o Estado de Goiás em relação a atuação das aves silvestres como portadoras de agentes infecciosos para humanos. Amostras de sangue e de swabs cloacais foram coletadas em 88 aves de distintas espécies, sendo a maioria, proveniente do tráfico e encaminhadas ao CETAS-IBAMA de Goiânia. As coletas foram realizadas em cinco incursões ao CETAS no primeiro semestre de 2010. As análises virais foram realizadas no Laboratório de Virologia Animal da UFG e as análises bacteriológicas e fúngicas no Laboratório de Microbiologia do SENAI em Anápolis. O sangue foi coletado em tubos sem anticoagulante para obtenção de soro e com o ensaio imunoenzimático indireto pesquisou-se anticorpos para os vírus influenza e causador da doença de Newcastle. As amostras de excreção cloacal foram coletadas por meio de swabs e realizouse o isolamento e identificação de fungos filamentosos, Candida spp, Salmonella sp e Escherichia coli. Na revisão sistemática, três artigos foram selecionados por meio de dois Testes de Relevância e a partir da interpretação destes estudos foi comprovado o papel das aves silvestres como portadoras e disseminadoras, a curta ou longa distância, de vários agentes infecciosos aos humanos e que não existem registros ou estudos sistemáticos sobre este processo no Estado de Goiás. Das 88 amostras analisadas, nenhuma apresentou anticorpos para os dois vírus pesquisados. As análises das amostras procedentes de swabs cloacais houve uma positividade de 9,1% para Candida tropicalis, 9,1% para Salmonella sp, 1,1% para Aspergillus fumigatus, 11,4% para Candida albicans, 12,5% para Candida krusei, 27,3% para Candida spp e 43,2% para Escherichia coli. Na revisão observou-se que novos estudos devem ser realizados, pois a distribuição destas zoonoses, possivelmente deve ser mais ainda mais ampla, inclusive em relação ao Estado de Goiás. Quanto aos resultados negativos para os agentes virais, estes sugerem que uma parcela das aves silvestres circulantes em Goiânia, ainda não entrou em contato com os agentes virais influenza e Newcastle, não sendo, portanto, portadoras destes patógenos. Em relação às identificações presuntivas de agentes bacterianos e fúngicos, em aves silvestres, estas confirmam a possibilidade da transmissão destes agentes entre os animais, incluindo os humanos. Além disso, este estudo reforça que ainda são incipientes as investigações desenvolvidas na área de zoonoses transmitidas por aves silvestres no Estado de Goiás, ressaltando a necessidade da continuidade destes estudos na região.

<p>  </p> <p>Microbiome studies are varied and involve the examination of microorganisms at different levels: individual cells to determine individual functions, populations of specific microorganisms to determine interactions between organisms, and/or communities of microorganisms for a broader investigation of interactions between organism and environment. These studies are typically done within the context of a particular niche or environment. There are two parts to this dissertation, separated by the types of research involved. First, the analysis of bacterial communities using 16S rRNA sequencing and analysis. In this first part the bacterial communities of the reproductive tract of bulls and the gastrointestinal tract of weanling pigs were studied. The reproductive organs of the male, domestic species had not been studied from an ecological perspective prior to the study. As such, the research was mainly focused on characterizing the bacterial communities found within the prepuce of bulls that were considered to be healthy, or that the breeding soundness exam was satisfactory and the bulls had no clinical disease in the urogenital tract. Through this study two distinct types of bacterial communities were found based on the diversity of the observed taxa; the groups were split into a low diversity group identified by the presence of <em>Bradyrhizobium</em> and a high diversity group distinguished by the abundance of mucosal-associated bacteria found in oral, respiratory, and vaginal communities of cattle. Second, the effects of supplementary, soluble fiber on the intestinal bacterial communities of piglets pre- and/or post-weaning were studied. The rationale behind this study was to determine if pre-weaning fiber could alter the microbiome prior to weaning and the change of diet from liquid to solid. Pre-weaning, supplementary, soluble fiber was found to increase short-chain fatty acid concentrations and bacterial taxa potentially involved in their production. Additionally, bacterial taxa implicated in an increased inflammatory response were reduced in groups fed supplementary fiber. Taken together, the two bacterial community studies highlight the gaps in knowledge for reproductive communities in male animals as well as the potential for reducing weaning stress in pigs. Part two of this dissertation focuses on whole genome sequence analysis as a way to study bacterial populations associated with bovine respiratory disease (BRD), a common and potentially fatal disease in cattle. Identification of BRD has low accuracy and the presence of antibiotic resistant bacteria increases the chance of treatment failure. Using machine learning, the prediction of antibiotic resistance in bacterial isolates from animals with BRD was performed to find potential sequences for use in future molecular assays. While using known resistance genes was helpful for some antibiotics, several of the antibiotics used in treating BRD were better predicted using the machine learning models. Model output sequences should be further tested using molecular methods to determine function and importance before using as an assay target. Put together, the contents of this dissertation should serve as an introduction to bacterial ecology as well as how the concepts can be applied to food animal production systems.</p>

<p dir="ltr"><i>Candida glabrata</i> is a member of the gut microbiota that can become an opportunistic pathogen under certain conditions. It is known for its inherent resistance to azole antifungal drugs and its ability to rapidly develop resistance during treatment. However, the regulatory mechanisms that enable this commensal organism to survive in low-oxygen environments, such as the gut, and to develop antifungal resistance when it becomes pathogenic, are not fully understood. In this study, we demonstrate for the first time the roles of two zinc cluster transcription factors in <i>C. glabrata</i>, Hap1A and Hap1B, in contributing to azole drug resistance in both laboratory strains and drug-resistant clinical isolates, adaptation to hypoxia, and resistance to other antifungal drugs like polyenes and echinocandins under specific conditions.</p><p dir="ltr">Azole drugs, which target the Erg11 protein, are widely used to treat <i>Candida</i> infections. The regulation of azole-induced <i>ERG</i> gene expression and activation of drug efflux pumps in <i>C. glabrata</i> has primarily been linked to the zinc cluster transcription factors Upc2A and Pdr1. Here, we investigated the roles of <i>S. cerevisiae</i> Hap1 orthologs, Hap1A and Hap1B, in <i>C. glabrata</i> as direct regulators of <i>ERG</i> genes upon azole exposure.</p><p dir="ltr">Our research shows that deleting <i>HAP1</i> in the yeast model <i>S. cerevisiae</i> increases sensitivity to fluconazole due to the failure to induce <i>ERG11 </i>expression in the <i>hap1Δ</i> mutant compared to the wild-type strain. Although <i>C. glabrata</i> is closely related to <i>S. cerevisiae</i>, a whole genome duplication (WGD) event allowed <i>C. glabrata</i> to retain two HAP1 ohnologs, while <i>S. cerevisiae</i> lost one copy. Through phylogenetic and syntenic analyses, we identified Hap1A and Hap1B in <i>C. glabrata</i> as ohnologs of Hap1 in <i>S. cerevisiae</i>, which is known to regulate <i>ERG</i> gene expression under both aerobic and hypoxic conditions. Interestingly, deleting <i>HAP1B</i> in <i>C. glabrata</i> increased sensitivity to both triazole and imidazole drugs, similar to Hap1 in <i>S. cerevisiae</i>, while deleting <i>HAP1A </i>did not affect azole sensitivity.</p><p dir="ltr">Gene expression analysis revealed that the increased azole sensitivity in the <i>hap1BΔ </i>strain was due to reduced azole-induced <i>ERG</i> gene expression, leading to lower total endogenous ergosterol levels. Additionally, the loss of <i>HAP1B</i> in <i>C. glabrata</i> clinical isolates like SM1 and BG2, as well as in drug-resistant strains like SM3, also led to increased azole hypersusceptibility. While it was already known that losing <i>UPC2A</i> in <i>C. glabrata</i> increases azole sensitivity, our study is the first to demonstrate that the combined loss of both <i>HAP1B </i>and <i>UPC2A</i> makes <i>C. glabrata</i> strains even more sensitive to azoles than losing either gene alone. Additionally, we show that the loss of both <i>HAP1B </i>and the H3K4 histone methyltransferase <i>SET1</i> increases azole hypersensitivity more than the loss of either gene alone.</p><p dir="ltr">Interestingly, the Hap1A protein is barely detectable under aerobic conditions but is specifically induced under hypoxia, where it plays a crucial role in repressing <i>ERG</i> genes. In the absence of Hap1A, Hap1B compensates by acting as a transcriptional repressor. Our RNA sequencing analysis further showed that losing both <i>HAP1A</i> and <i>HAP1B</i> not only affects genes in the ergosterol biosynthesis pathway but also upregulates iron transport-related genes <i>FET3 </i>and <i>FTR1</i>. Moreover, we found that the hypoxic growth defect caused by the loss of both <i>HAP1A</i> and <i>HAP1B</i> is exacerbated when treated with the echinocandin caspofungin and the cell wall-damaging agent calcofluor white, indicating that these Hap1 ohnologs contribute to maintaining cell wall integrity under hypoxic conditions. Since <i>HAP1A</i> transcript levels remain stable under aerobic conditions, we suspect that Hap1A expression is regulated post-transcriptionally.</p><p dir="ltr">Furthermore, we discovered that the simultaneous loss of both HAP1A and HAP1B leads to increased hypersensitivity to the polyene antifungal drug amphotericin B, though the exact mechanism behind this phenotype remains unclear. Altogether, our study is the first to show that Hap1A and Hap1B have evolved distinct roles, enabling <i>C. glabrata</i> to adapt to specific host and environmental conditions.</p>

<p>Over three million infections were reported in the United States of America in 2019. These infections were caused by either antibiotic-resistant pathogens or <i>Clostridioides difficile</i> and resulted in more than 50,000 deaths. Unfortunately, antibacterial agents are rapidly losing their ability to treat infections and the process of discovering new antibiotics is too slow to cope up with bacterial evolution. Repurposing FDA-approved drugs of well-studied safety, pharmacology and pharmacokinetics represents a faster alternative method of antibacterial drug discovery. Repurposing is more successful and less depleting method of drug discovery than classical de novo method in regard to both cost and time. In the following studies, two major pathogens are targeted, vancomycin-resistant <i>Enterococcus</i> (VRE) and <i>C. difficile</i>. Both bacteria are more prevalent in healthcare settings were more vulnerable population of elderly and immunocompromised individuals reside. In addition, healthcare settings are usually associated with higher frequency of receiving antibiotics which in turn, compromises the integrity of normal microbiota responsible for protection against invading pathogens. Furthermore, hospital stays are associated with exposure to bacterial shedding from other patients. Our aim was to identify FDA-approved drugs with novel ability to eradicate these two bacterial pathogens in the gastrointestinal tract (GIT). Notably, the GIT is considered the actual site of infection in case of <i>C. difficile while it is only a transition site for VRE where the bacteria colonize before causing true infections in other tissues. Studies against both bacteria started with an <i>in vitro</i> screening of FDA-approved drugs and clinical molecules to identify potential candidates for further investigation.</i></p><p><i>For VRE, two drugs where identified with potent inhibitory activity and favorable pharmacokinetic profiles, auranofin and ebselen. Auranofin was approved in the 1960s for the treatment of rheumatoid arthritis due to its anti-inflammatory activity. Auranofin was found to exert potent bacteriostatic activity against both vancomycin-sensitive and vancomycin-resistant <i>Enterococcus</i> strains (minimum inhibitory concentration against 90% of the strains, MIC90 = 1 µg/mL). In addition, bacteria could not develop resistant mutants against auranofin upon prolonged exposure. On the other hand, ebselen is an organoselenium compounds currently in clinical trials for several indications. Similarly, ebselen was found to be a potent inhibitor of VRE growth (MIC90 = 2 µg/mL). In addition, ebselen successfully inhibited bacterial biofilm formation and eradicated mature biofilms. In a mouse model of VRE colonization, both drugs inhibited bacterial shedding and reduced bacterial counts in the GIT of the colonized animals.</i></p><p><i>For <i>C. difficile</i>, auranofin was also found to exert potent inhibitory activity against bacterial growth (MIC90 = 2 µg/mL), toxin production and spore formation. Additionally, it was beneficial in protecting colon cells against <i>C. difficile</i> toxin-induced inflammation. Further, auranofin was found to not promote growth of VRE as seen with the current anticlostridial agents. In addition to auranofin, two more antiprotozoal drugs were found to potently inhibit <i>C. difficile</i> growth, ronidazole and secnidazole. Both drugs are 5-nitroimidazoles approved for human (secnidazole) or veterinary (ronidazole) applications. Secnidazole and ronidazole halted <i>C. difficile</i> growth at very low concentrations (MIC90 = 0.5 and 0.125 µg/mL, respectively). Furthermore, both drugs were superior to metronidazole in bacterial killing and had favorable activities against protective gut microbiota. In addition, they demonstrated efficient protection to mice in a <i>C. difficile</i> infection model. </i></p><p><i>Overall, several drugs were presented to possess favorable activities against <i>C. difficile</i> or VRE. These drugs merit more evaluation as potential candidates for the treatment of infection caused by either bacteria. </i></p><div><i><br></i></div>

<p dir="ltr">The gastrointestinal tract harbors a diverse range of microorganisms, collectively constituting the gut microbiome. <a href="" target="_blank">The maintenance of a symbiotic relationship between the host and these microorganisms is essential to gastrointestinal health. Disruption of the ecological balance within the gut microbiome can result in discomfort or pathological conditions.</a> <a href="" target="_blank">This dissertation explores these alterations within the gastrointestinal tract as potential indicators for specific gastrointestinal diseases.</a> <a href="" target="_blank">In pursuit of this, I collaborated with others to develop a smart ingestible capsule that offers a non-invasive method for enhancing the effectiveness of differential diagnosis and treatment strategies for Inflammatory Bowel Disease (IBD). </a>My contributions encompassed conducting <i>in vitro</i> protein sampling and extraction experiments, as well as enteric coating dissolution tests. Following thorough characterization of the capsule, I advanced to <i>ex vivo</i> sampling experiments. As a proof of concept, the capsule's sampling capabilities have been rigorously validated both <i>in vitro</i> and <i>ex vivo</i> using calprotectin, a key biomarker for monitoring and managing IBD. Future research may explore integrating this technology with other sensors for diverse chemical and gas sensing capabilities, aiming to refine the differential diagnostics of Irritable Bowel Syndrome (IBS) and IBD.</p><p dir="ltr">Simultaneously, the potential transmission of pathogenic microorganisms from the gastrointestinal tract to the environment through fecal matter can lead to substantial public health implications if adequate surveillance is not in place. These pathogens can contaminate water and food sources from various origins, exacerbating the problem. Furthermore, conventional laboratory-based assays, while effective, have extensive turnaround times and require skilled scientists to operate them. In response to this challenge, I have undertaken the development of point-of-care assays, aiming to streamline the detection of fecal contamination. This innovation is designed to mitigate the limitations associated with traditional methods by offering a more rapid and user-friendly approach. The primary objective is to enhance the accessibility of these assays, enabling on-site personnel with varying levels of expertise to utilize them effectively. Through the widespread adoption of these point-of-care assays, the overarching goal is to ensure the consistent provision of safe and reliable water and food supplies to the public.</p>