Candida albicans is an opportunistic pathogen that is present in the normal flora in a majority of individuals. One key factor in C. albicans virulence is the ability to change its morphology from yeast to an elongated or hyphal form. The regulation of this morphogenesis relies in part upon quorum sensing (QS) molecules. C. albicans often exists as part of a mixed culture alongside other microbes and is influenced by their presence as well as the presence of QS molecules that they produce. In this study, a library of diploid homozygous transcriptional regulator knockout (TRKO) mutants were screened to identify strains capable of forming hyphae in the presence of Pseudomonas aeruginosa, Staphylococcus aureus, and Escherichia coli. We identified three strains that showed increased hyphae development compared to wild type C. albicans. The strains identified had deletions of the transcriptional regulating genes Orf19.3928, Orf19.2842 (GZF3), and Orf19.3865 (RFX1). These strains were tested for alterations of filamentation in liquid media, and biofilm formation. All three strains showed increased rates of biofilm formation compared to the wild type. Orf19.3928 showed altered response to farnesol, a marked in biofilm formation and no inhibition of filamentation when farnesol was present in liquid media. The GZF3 deletion strain showed enhanced filamentation with all three bacterial species while the RFX1 deletion strain showed increased filamentation only with E. coli and S. aureus. In spent media, GZF3 showed slight increases in filamentation in E. coli and S. aureus while RFX1 had moderate increases in filamentation in E. coli and S. aureus and slight increases with P. aeruginosa.

Sepsis is the most frequent cause of mortality in most intensive care units. Cardiovascular dysfunction is a major complication associated with sepsis, with high mortality rates up to 70%. Currently, there is no effective treatment approach for sepsis. The integrity of the endothelium is fundamental for the homeostasis of the cardiovascular system. Sepsis induces endothelial cell injury which is the key factor for multiple organ failure. The increased expression of adhesion molecules and chemokines in endothelial cell promotes leukocytes infiltration into the tissue. The loss of tight junction proteins and increased permeability of the endothelial cells will provoke tissue hypoxia and subsequent organ failure. Therefore, preservation of endothelial function is a critical approach for improving sepsis-induced outcome. Here, we showed that endothelial specific protein HSPA12B plays a critical role in the preservation of cardiovascular function in polymicrobial sepsis. HSPA12B is the newest member of HSP70 family which predominantly expresses in endothelial cells. We observed that HSPA12B deficiency (HSPA12B-/-) exaggerated polymicrobial sepsis-induced endothelial dysfunction, leading to worse cardiac dysfunction. HSPA12B-/- significantly increases the expression of adhesion molecules, decreases tight junction protein levels and enhances vascular permeability. HSPA12B-/- alsomarkedly promotes the infiltration of inflammatory cells into the myocardium and inflammatory cytokine production. We investigated the cardioprotective mechanisms of HSPA12B in sepsis induced cardiovascular dysfunction. Exosomes play a critical role in intercellular communication. Exosome is a natural vehicle of microRNAs. We found that exosomes isolated from HSPA12B-/- septic mice induced more expression of adhesion molecules in endothelial cells and inflammation in macrophages. Interestingly, the levels of miR-126 in serum exosomes isolated from HSPA12B-/- septic mice were significantly lowers than in WT septic mice. Importantly, delivery of miR-126 carried exosomes significantly improved cardiac function, suppressed the expression of adhesion molecules, reduced immune cell infiltration in the myocardium, and improved vascular permeability in HSPA12B-/- septic mice. The data suggests that HSPA12B is essential for endothelial function in sepsis and that miR-126 containing exosomes plays a critical role in cardiovascular-protective mechanisms of endothelial HSPA12B in polymicrobial sepsis.

Lyme disease, caused by the bite of a deer tick infected with Borrelia burdorferi, has been increasing in distribution and prevalence annually throughout Maine. Worker’s compensation claims for Lyme disease have also been increasing steadily since the initial claim made in 1999. This research reviewed Maine worker’s compensation claims for Lyme disease from 1999-2011 to determine trends in state distribution and occupation type. Descriptive statistics were calculated to analyze different distributions of occupational Lyme disease. Occupations with the highest distribution of Lyme disease claims were those requiring workers to spend the majority of their time outdoors. A clear trend of claim distribution was seen, which mirrored that of the State of Maine Lyme disease case surveillance data. With the apparent increase in worker’s compensation claims due to Lyme disease and an increased geographic distribution annually, additional prevention and education efforts should be focused toward the higher risk occupations.

Hospital-acquired infections (HAI) are not uncommon in healthcare facilities. They are usually prevented by sanitation techniques and by maintaining a high standard of care. Catheter-associated urinary tract infections (CAUTI) make up a large percentage of hospital-acquired infections and are often the most preventable type of HAI. Patterns in infection rate could provide new ideas on prevention techniques, which might further reduce infection rate, saving lives and cutting costs. CAUTI infection rate was measured from January KJIY to September KJIL and was differentiated based on hospital ward as well as month and season. Overall, ICU units tended to have a higher CAUTI infection rate than ward units, particularly in January, February, April, and May. The CAUTI infection rate was highest in the ICU units during spring and May, and lowest during fall and October. In the ward units, the CAUTI infection rate was highest during summer and March, and lowest during winter and February.

The extensive and sometimes incorrect and noncompliant use of various types of antimicrobial agents has accelerated the development of antimicrobial resistance (AMR). In fact, AMR has become one of the greatest global threat to human health in this era. The broad-spectrum antibiotics aminoglycosides (AGs) display excellent potency against most Gram-negative bacteria, mycobacteria, and some Gram-positive bacteria, such as Staphylococcus aureus. The AG antibiotics amikacin, gentamicin, kanamycin, and tobramycin are still commonly prescribed in the U.S.A. for the treatment of serious infections. Unfortunately, bacteria evolve to acquire resistance to AGs via four different mechanisms: i) changing in membrane permeability to resist drugs from entering, ii) upregulating efflux pumps for active removal of intracellular AGs, iii) modifying the antimicrobial target(s) to prevent drugs binding to their targets, and iv) acquiring resistance enzymes to chemically inactivate the compounds. Amongst all, the acquisition of resistance enzymes, AG-modifying enzymes (AMEs), is the most common resistance mechanism identified. Depending on the chemistry each enzyme catalyzes, AMEs can be further divided into AG N-acetyltransferases (AACs), AG O-phosphotransferases (APHs), and AG O-nucleotidyltransferases. To overcome AME-related resistance, we need to better understand these resistance enzymes and further seek ways to either escape or inhibit their actions. In this dissertation, I summarized my efforts to characterize the AAC(6') domain and its mutant enzymes from a bifunctional AME, AAC(6')-Ie/APH(2")-Ia as well as another common AME, APH(3')-IIa. I also explained my attempt to inhibit the action of various AAC enzymes using metal salts. In an effort to explore the current resistance epidemic, I evaluated the resistance against carbapenem and AG antibiotics and the correlation between the resistance profiles and the AME genes in a collection of 122 Pseudomonas aeruginosa clinical isolates obtained from the University of Kentucky Hospital System. Besides tackling the resistance mechanisms in bacteria, I have also attempted to explore a new antifungal option by repurposing an existing antipsychotic drug, bromperidol, and a panel of its derivatives into a combination therapy with the azole antifungals against a variety of pathogenic yeasts and filamentous fungi.

Hydropathic molecular modeling is a computer-aided molecular design technique for obtaining, representing, and understanding the properties and interactions of biomacromolecular complexes in the biological environment. Hydropathic INTeraction (HINT) is a novel empirical force field to calculate the free energy of intermolecular interaction based on experimentally determined partition coefficients (log Po/w). It includes all the expected interactions between molecules such as hydrogen bonding, hydrophobic, electrostatic, acid-base, and Coulombic interactions, entropy, solvation and others. HINT tools were used to determine, evaluate, and analyze protein-ligand interactions in different research projects: 1) We used these tools to discover small molecule inhibitors of PsaA, a potential target for Streptococcus pneumoniae. We screened and scored potential molecules to obtain hits. After the growth conditions for both the wild type and PsaA mutant of S. pneumoniae were optimized, we then tested our hits. A few compounds passed through the three-stage assay protocol and confirmed the inhibition of PsaA with MICs between 125-250 μM. 2) The SAR of C-3 and C-5 pyrrole-based antitubulin agents at the colchicine-binding site with explicitly solvated models was performed. After docking with GOLD at the colchicine site, post-docking scoring and evaluation were performed with HINT. The total HINT score correlates with binding and activity; similarly, the significance of individual functional groups, protein residues and interactions amongst a collection of compounds can be quantitated. The possibility of water-mediated interactions in a way solvent accessible part of the pocket was considered by subjecting molecular models to MD simulations. Several water molecules were identified to be contributing to the binding and were confirmed by HINT scoring. Finally, using hydropathic molecular modeling tools helped us to understand, evaluate, analyze, and improve protein-ligand interactions in different biological systems.

Syphilis, a systemic sexually transmitted disease, is on the rise in the US, with infection rates the highest recorded since 1994 according to the CDC. Useful laboratory testing is an important diagnostic tool for determining individual syphilis infection and preventing community-wide disease spread. The purpose of this study was to determine the usefulness of a specific automated treponemal test method, the CaptiaTM Syphilis IgG EIA, and the syphilis reverse algorithm interpretation for detecting syphilis infection among patients seeking care in a public health clinic. The study employed a retrospective, nonexperimental descriptive correlational design with data collected between 2012-2013 from 4,077 public health clinic patients with 21% of the patients diagnosed with syphilis infection. There was a statistically significant difference between the CaptiaTM Syphilis IgG and the Fujirebio Serodia TP-PA test results; between the CaptiaTM Syphilis IgG Signal to Cutoff (S/CO) and the MacroVue RPR titer continuous variables; and between the reverse and traditional syphilis interpretation algorithms. The reverse algorithm using the CaptiaTM Syphilis IgG test method provided more useful performance measures with a sensitivity of 82%; specificity of 99%; accuracy of 95%; positive likelihood ratio of 63.06 and negative likelihood of 0.18 than the traditional algorithm using the MacroVue RPR test method. Statistical comparison of the area under the curve (AUC) for the continuous variables, CaptiaTM Syphilis IgG S/CO and RPR titer, concluded that the Syphilis IgG AUC (0.9500) was higher than the RPR titer (0.8155) indicating greater accuracy for detecting syphilis infection. This was the first study to determine that the CaptiaTM Syphilis IgG, the S/CO value, and reverse algorithm are useful diagnostic predictors of syphilis infection among public health clinic patients. The data from this study can be utilized by future researchers and scientists who are developing or improving syphilis detection methods.

The invasive pathways, InlA- and InB-mediated uptake and M cell transcytosis, that Listeria monocytogenes uses to invade the intestine have mainly been studied using infection models that do not truly replicate what occurs during a natural infection. Recently, our lab has developed an oral infection model that is more physiolocally relevant to what occurs during food borne listeriosis. We have sought to evaluate the relative roles of the previously defined invasive pathways, in our oral model of infection. We have done this by utilizing an InlAmCG Lm strain that is able to bind murine E-cadherin, knockout Lm strains, ΔinlA Lm, and ΔinlAΔinlB Lm. We also took advantage of a knockout mice strain CD137-/-that has M cells that are deficient in M cell transcytosis. We were able to show that these invasive pathways are relevant in our oral infection model, that M cell transcytosis is a compensatory pathway for InlA-mediated uptake, and that there might be another mechanism that L. monocytogenes uses to invade the intestines. To confirm this, it is necessary though that the M cell transcytosis deficiency be confirmed in the CD137-/- mice.

Orientador: Rinaldo Poncio Mendes / Resumo: A paracoccidioidomicose (PCM) é micose granulomatosa sistêmica causada por fungos termodimórficos do gênero Paracoccidioides. O presente estudo teve como objetivos avaliar a atividade da liriodenina, extraída de Annona macroprophyllata Donn. Sm, sobre isolados clínicos e cepas padrão de fungos do gênero Paracoccidioides e outras espécies que causam micoses sistêmicas e, através de um estudo-piloto realizado em modelo murino, avaliar a absorção da liriodenina e possíveis efeitos indesejáveis. A concentração inibitória mínima (CIM) e concentração fungicida mínima (CFM) da liriodenina foram determinadas pelo método de microdiluição. As alterações celulares causadas pela liriodenina em P. brasiliensis / cepa padrão Pb18 foram avaliadas pela microscopia eletrônica de transmissão (MET) e de varredura (MEV). Em estudo-piloto, quatro camundongos isogênicos albinos, da linhagem BALB/c, foram utilizados para avaliar a absorção e os efeitos indesejáveis da liriodenina. Os animais, sem infecção, foram divididos em dois grupos: grupo 1, com dois camundongos que receberam a dose de 0,75 mg.kg-1 e grupo 2, com dois camundongos que receberam a dose de 1,50 mg.kg-1, em única tomada. Após seis e 12 horas após a administração, os animais foram sacrificados e amostras de sangue foram coletadas para a determinação dos níveis séricos. Os intestinos foram coletados para exame histológico. O teste de sensibilidade in vitro revelou que a liriodenina possui atividade sobre parte dos fungos do gênero P... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Paracoccidioidomycosis (PCM) is systemic granulomatous mycosis caused by thermodymorphic fungi of the genus Paracoccidioides. The present study aimed to evaluate the activity of liriodenine, extracted from Annona macroprophyllata Donn. Sm on clinical isolates and standard strains of fungi of the genus Paracoccidioides and other species that cause systemic mycoses and, through a pilot study conducted in a murine model, to assess the absorption of liriodenine and possible undesirable effects. The minimum inhibitory concentration (MIC) and minimum fungicidal concentration (CFM) of liriodenine were determined by the microdilution method. Cellular alterations caused by liriodenin in P. brasiliensis / Pb18 standard strain were evaluated by transmission electron microscopy (SEM) and scanning electron microscopy (SEM). In a pilot study, four albino isogenic mice of the BALB / c strain were used to assess the absorption and undesirable effects of liriodenine. The animals, without infection, were divided into two groups: group 1, with two mice receiving the dose of 0.75 mg.kg-1 and group 2, with two mice receiving the dose of 1.50 mg.kg- 1 , in single outlet. After six and 12 hours after administration, the animals were sacrificed and blood samples were collected for the determination of serum levels. The intestines were collected for histological examination. The in vitro sensitivity test revealed that liriodenine has activity on part of fungi of the genus Paracoccidioides, with MIC v... (Complete abstract click electronic access below) / Doutor

Made available in DSpace on 2016-12-23T13:55:58Z (GMT). No. of bitstreams: 1 Marisa Simon Brezinscki - Parte 1.pdf: 8765013 bytes, checksum: 34dcc19e4bb7677d326886ae43d4ad27 (MD5) Previous issue date: 2013-08-12 / Introduction: Skin infectious diseases of fungal, bacterial and viral etiology are frequent in HIV-infected patients. HIV infection may change the epidemiology of infectious diseases, leading to atypical manifestations Objectives: 1 Determine the etiologic agents and the more frequent clinical forms of mycoses in HIV infected patients. 2 - Correlate the occurrence of mycoses with CD4 s levels and viral load. 3-Evaluate the frequency of bacterial and viral skin diseases. Methods: A descriptive cross sectional study, which evaluated 86 patients with HIV infection. Sociodemographic and laboratory data collection, dermatological examination were performed and when necessary, scraping the lesion for direct microscopy and fungal culture, if necessary. Results: The mean age was 44.02 (± 12.17) years, 66.3% were men, and the gender ratio 1.97 men: 1 woman. The average time of diagnosis of HIV infection o was 5.59 years. Injuries caused by fungi, bacteria or viruses in keratinized tissues were observed in 58,14% of the patients (39.53% fungal, 18.6% bacterial and 10.46% viral). Onychomycosis was the most common mycoses, and Candida spp. the most frequent agent in these cases. Trichophyton rubrum was the most frequently isolated agent in the whole sample. Patients with CD4 levels less than 50 cells / uL and viral load greater than 50,000 copies / mL had more mycoses. Viral wart was the most frequent viral skin disease and folliculitis was the most frequent bacterial disease. Conclusions: The etiology of superficial mycoses in HIV patients did not differ from those found in the population immunocompetent. However, in some cases, fungal infections manifested in subtle form, with little inflammatory reaction or with more severe and extensive clinical aspects than those usually observed. The higher frequency of cutaneous mycosis was observed in patients with more advanced degrees of immunosuppression / Introdução: Dermatoses infecciosas de etiologia fúngica, viral e bacteriana são frequentes nos pacientes infectados pelo HIV. A infecção pelo HIV pode alterar a epidemiologia de doenças infecciosas, levando a manifestações atípicas. Objetivos: 1- Determinar os agentes etiológicos e formas clínicas mais frequentes das micoses cutâneas nos pacientes infectados pelo HIV. 2- Correlacionar a ocorrência destas micose com os níveis de linfócitos T CD4 e da carga viral. 3- Avaliar a frequência das dermatoses bacterianas e virais. Métodos: Estudo descritivo tipo corte transversal, que avaliou 86 pacientes com infecção pelo HIV. Foram realizados: coleta de dados sociodemográficos e laboratoriais, exame dermatológico e quando necessário, raspado da lesão para EMD (exame micológico direto) e cultura para fungos. Resultados: A média da idade foi 44,02 (± 12,17) anos; 66,3% eram homens, e a razão de sexo 1,97 homem : 1 mulher. Tempo médio de diagnóstico de infecção pelo HIV foi de 5,59 anos. Lesões causadas por fungos, bactérias ou vírus em tecidos queratinizados foram observadas em 58,14% dos pacientes (39,53% fúngicas, 18,6% virais e 10,46% bacterianas). Micose mais frequente foi a onicomicose, sendo a Candida spp. o agente mais frequente nestes casos. Trichophyton rubrum foi o agente mais isolado em toda a amostra. Pacientes com níveis de linfócitos T CD4 inferiores a 50 células/μL e carga viral maior do que 50.000 cópias/mL apresentavam mais micose cutânea. Dermatose viral mais frequente foi a verruga viral; e bacteriana, a foliculite. Conclusões: A etiologia das micoses superficiais em pacientes com HIV não diferiu daquelas encontradas na população imunocompetente. Contudo, em alguns casos, as micoses se manifestaram de forma sutil, com pouca reação inflamatória ou com quadros clínicos mais graves e extensos do que os habitualmente observados. A maior frequência de micoses cutâneas foi observada nos pacientes com graus mais avançados de imunossupressão