Some pathogenic bacteria including Haemophilus influenzae and Neisseria meningitides produce a protease called IgA1 protease to impair a major antibody, immunoglobulin A1 (IgA1), on human mucosal surfaces. The iga mRNA is initially translated into a precursor containing four distinct domains: a 31-amino acids signal peptide which leads the precursor to the periplasmic space, an 105-kDa protease domain which cleaves host IgA1 molecule, a £]-domain responsible for autotransportation of the protease domain, and a short linker between the protease and the £]-domains. The autotransporter £]-domain can be further divided into three subdomains in Neisseria protease: an extracellular linking region £\-protein and a membrane-embedded £]-core, between which there is a distinguished sequence called surface region. The hydrolytic function of the protease and the transporter role of £]-core had been studied extensively, but the £\-protein and the surface regions were less defined, or had their role characterized. Thus this study is designed to reveal the possible pathogenic functions of the £\-protein and the surface region in bacterial adherence to human cell surfaces. To complete this project, recombinant £\-protein and the surface region were expressed in IgA1 protease-negative E. coli strain (UT5600) respectively and purified to homogeneity. These recombinant proteins were used in cellular assays for bacterial adhesion on human lung cancer cell (A549). Four different invasive strains of pathogenic bacteria (IgA1 protease-positive or negative), were recruited in adherence assays to determine the effect of the purified £\-protein and the surface region on bacterial adherence to A549 cells. Results showed that the both £\-protein and the surface region played a role in bacterial adherence in a species-dependent manner.

Escherichia coli isoladas de infecções do trato urinário (ITU) são conhecidas como E. coli uropatogênicas (UPEC). Dentre as E. coli diarreiogênicas, o patótipo denominado E. coli enteroagregativa (EAEC) é definido pela produção do padrão de adesão agregativa em células epiteliais cultivadas. Estudos recentes mostraram que algumas cepas de UPEC albergam propriedades de virulência de EAEC, indicando que cepas de EAEC podem causar ITU. Assim sendo, o objetivo deste estudo foi analisar as relações filogenéticas entre cepas de EAEC que apresentam marcadores genéticos de E. coli extraintestinais (ExPEC) e cepas de UPEC com e sem marcadores genéticos de EAEC. Para tal, foram selecionadas 92 EAEC, 8 UPEC com e 10 sem marcadores de EAEC. As 92 EAEC foram analisadas quanto à presença dos genes considerados como marcadores de cepas de ExPEC (papA/papC, sfa/foc, afa/dra, iutA, kpsMT II), detectando 30 (32,6%) cepas com esse perfil. Estas 30 cepas foram selecionadas para análises de filogrupos e multilocus sequence type (MLST) junto às cepas de UPEC. Foi observado que 17 (54,4%) cepas de EAEC e 3 (16,6%) de UPEC pertenceram ao filogrupo A, 2 (6,45%) EAEC e 1 (5,5%) UPEC ao filogrupo B1, 3 (9,68%) EAEC e 8 (44,4%) UPEC ao filogrupo B2, 6 (19,35%) EAEC e 2 (11,1%) UPEC ao filogrupo D, 1 (3,2%) EAEC e 4 (22,2%) UPEC ao filogrupo E, 1 (3,2%) EAEC ao filogrupo F e 1 EAEC (3,2%) não pôde ser classificada de acordo com esta metodologia. Comparando os dois grupos de UPEC notou-se que dentre as cepas com marcadores de EAEC 3 (37,5%) pertenceram ao filogrupo E, 2 (25%) aos filogrupos A e D e 1 (12,5%) ao filogrupo B1. Dentre as cepas sem marcadores de EAEC 1 (10%) pertenceu ao filogrupo A, 1 (10%) ao filogrupo E e 8 (80%) ao filogrupo B2. As análises de MLST através do sequenciamento dos genes recA, fumC, icd, mdh, purA, adk e gyrB permitiram determinar 42 sequence types (ST) distintos, dos quais 22 foram descritos neste estudo. Os mais comuns foram o ST 10 (5 cepas) e ST 95 e ST 746 (ambos com 2 cepas cada). A árvore filogenética gerada confirmou esses dados, mostrando o grupamento das cepas de EAEC com marcadores de ExPEC com as cepas de UPEC com marcadores de EAEC. Em resumo, o presente estudo mostrou que um subgrupo de cepas de EAEC está inserido nos mesmos grupos filogenéticos de cepas de UPEC com marcadores de EAEC apresentando, portanto, correlação filogenética. Houve diferenças de distribuição filogenética entre cepas de UPEC com e sem marcador de EAEC. Concui-se que cepas de EAEC podem apresentar potencial uropatogênico, tanto no curso de uma infecção diarreica, quanto em carreadores assintomáticos. / Escherichia coli isolated from urinary tract infections (UTI) are known as uropathogenic E. coli (UPEC). Among the diarrheagenic E. coli, the enteroaggregative E. coli (EAEC) pathotype is defined by the production of the aggregative adherence on cultured epithelial cells. Recent studies have shown that some UPEC strains harbor virulence properties of EAEC, indicating that EAEC strains can cause UTI. Therefore, the aim of this study was to analyze the phylogenetic relationships among EAEC strains that have genetic markers of extraintestinal E. coli (ExPEC) and UPEC strains, with and without genetic markers of EAEC. For that reason, we selected 92 EAEC, 8 UPEC with and 10 without EAEC markers. The 92 EAEC were analyzed for the presence of genes considered as markers for ExPEC strains (papA/papC, sfa/foc, afa/dra, iutA, kpsMT II), detecting 30 (32.6%) strains with that profile. These 30 strains were selected for phylogroup and multilocus sequence type (MLST) analysis with the UPEC strains. It was observed that 17 (54.4%) EAEC and 3 (16.6%) UPEC belonged to the phylogroup A, 2 (6.45%) EAEC and 1 (5.5%) UPEC to the phylogroup B1, 3 (9.68%) EAEC and 8 (44.4%) UPEC to the phylogroup B2, 6 (19.35%) EAEC and 2 (11.1%) UPEC to the phylogroup D, 1 (3.2%) EAEC and 4 (22.2%) UPEC to the phylogroup E, 1 (3.2%) EAEC to the phylogroup F and 1 (3.2%) EAEC could not be classified according to this methodology. Comparing the two groups of UPEC it was observed that among the UPEC strains with EAEC markers, 3 (37.5%) belonged to the phylogroup E, 2 (25%) to the phylogroups A and D and 1 (12.5%) to the phylogroup B1. Among the UPEC strains without EAEC markers, 1 (10%) belonged to the phylogroup A, 1 (10%) to the phylogroup E and 8 (80%) to the phylogroup B2. The MLST analysis by sequencing of recA, fumC, icd, mdh, purA, adk and gyrB genes allowed to determine 42 distinct sequence types (ST), of whom, 22 were described in this study. The most common were ST 10 (5 strains), and ST 95 and ST 746 (both with two strains each). The phylogenetic tree generated confirmed that data, showing the clustering of EAEC strains (harboring ExPEC markers) with the UPEC strains (harboring EAEC markers). In summary, the current study showed that a subgroup of EAEC strains are clustered in the same phylogenetic groups of UPEC strains with EAEC markers and, thus, present phylogenetic correlation. Also, there were differences in phylogenetic distribution among UPEC strains with and without EAEC markers. In conclusion, EAEC strains may have uropathogenic potential, either in the course of a diarrheal infection or in asymptomatic carriers.

Three major topics will be discussed in this dissertation. The first is an optical biosensor for specific diagnosis of bacterial skin and wound infection, followed by a paper microfluidic assay and accompanying monitoring device for monitoring blood coagulation and determining patient-specific heparin and protamine dosing. The final work to be discussed is ongoing work involving the detection of circulating tumor cells (CTCs) using a paper microfluidic detection platform. All of these works involve the development of biosensors for the simultaneous advancement and simplification of diagnosis and analysis of blood and bacterial infection. The aims of each of these projects included significantly decreasing the time to diagnosis and decreasing the reagents, laboratory space, personnel, and other resources needed for detection and diagnosis. The first works are focused on the design, development, and testing of an optical biosensor for the immediate detection of bacterial skin and wound infection, including diagnosing the specific species of bacteria responsible for the infection. The optical biosensor developed allows for diagnosis of a bacterial infection on skin or in a wound in as little as three seconds, in a contact-free, reagent-free manner. The second work focused on the design, development, and testing of a paper microfluidic assay and accompanying Raspberry Pi-based monitoring device for use before, during, and after surgeries requiring the use of cardiopulmonary bypass. The assay monitors the extent of blood coagulation of a whole blood sample and determines patient-specific dose response curves of an anticoagulant and its reversal agent. The final work discussed focuses on developing a paper microfluidic assay for the detection of CTCs from whole blood samples. The goal of this work is to detect multiple morphologies of CTCs from whole blood samples to provide insight on patient prognosis in a rapid, low resource manner.

Target of Myb 1 (TOM1) is an endosomal adaptor protein that plays a role in cargo membrane trafficking for degradation by serving as an alternative endosomal sorting complex required for transport component. TOM1 has also been shown to serve as a novel phosphatidylinositol 5-phosphate (PtdIns5P) effector at signaling endosomes through its VHS domain, delaying cargo degradation in a bacterial infection model. The aim of this thesis is to clarify the structural and functional basis of the autoregulation mechanism of TOM1 to switch from endosomal protein trafficking to the bacterial survival signaling pathway. Our thermal denaturation and spectroscopic studies demonstrate that PtdIns5P reduced thermostability, interhelical contacts, and conformational compaction of TOM1 VHS. The thermodynamic studies indicate that TOM1 VHS endothermically binds to PtdIns5P through two potential noncooperative binding sites, with its acyl chains playing a relevant role in the interaction. These findings suggest that, under Shigella flexneri infection, TOM1 may interact with downstream effectors in a different VHS domain conformational state, thus involving the protein in bacterial survival signaling pathways. In order to obtain molecular details for the interaction of the TOM1 VHS domain for PtdIns5P and Ubiquitin (Ub), the backbone assignment information was obtained by performing NMR experiments, which assigned backbone 1H, 13C, and 15N resonances of the TOM1 VHS domain. With this structural information, our heteronuclear single quantum coherence and molecular dynamics simulations data revealed that TOM1 VHS interacts with PtdIns5P following a fast-exchange regime, with the PtdIns5P binding site predicted to be at a region spanning α-helices 6 to 8. Further mutagenesis and lipid-protein overlay assay studies indicated that K147 plays a critical role in the binding of TOM1 VHS domain to PtdIns5P. TOM1, unexpectedly, did not bind PtdIns5P. Using truncated forms of TOM1 protein, we discovered that neither TOM1 GAT domain nor the C-terminal domain modulated TOM1 VHS's PtdIns5P binding; however, surprisingly, a linker sequence between the TOM1 VHS and GAT domains exhibited an autoinhibition role for TOM1 binding to PtdIns5P. This linker region was observed to induce local conformational changes on the structure of TOM1 VHS domain, especially around α-helices 6 and 8, which are proposed to build up the binding pocket for PtdIns5P. In order to investigate whether the linker region between TOM1 VHS and GAT domain can also regulate the Ub association of TOM1 VHS domain, the binding properties of TOM1 and its domains to Ub were explored. Unexpectedly, the binding affinity of TOM1 VHS-linker for Ub was increased about 10-fold when compared with that for the TOM1 VHS domain, suggesting that the linker enhances the avidity of TOM1 for ubiquitinated cargo. Structural analysis indicated that the linker region may cap the conventional Ub-binding site of TOM1 VHS, thus forming a more compact structure. In summary, this study uncovered a novel intramolecular modulatory mechanism in TOM1 that regulates ligand recognition by its VHS domain. By providing the molecular basis of the TOM1 interactions, we may provide cargo sorting mechanistic insights, create functionally specific mutations, and precisely manipulate TOM1 function under bacterial infection conditions, and other yet-to-be-discovered PtdIns5P-dependent signaling pathways. / Doctor of Philosophy / Membrane trafficking is a delivery network established in a cell to transport proteins (cargoes) from one intracellular place to another one to control their activity. TOM1 is a protein involved in this process, which plays a role in transporting cargoes for degradation. Defects in this trafficking pathway lead to human diseases, such as immunodeficiency and neurodegeneration diseases. TOM1 has also been shown to be beneficial for bacterial survival in human cells. However, how TOM1 switches its role form protein trafficking to bacterial pathogenesis is still unclear. In our study, we discovered an internal region of TOM1 may serve as a switch to shift the role of TOM1 in human cells. In an "on" status, TOM1 favors to transport cargoes, while in an "off" status, TOM1 is used for bacteria survival. This study provides insights in the function of TOM1 which is beneficial for the design of novel therapeutic strategies against TOM1, which will prevent the progress of bacterial infections.

Despite recent advances, radiopharmaceuticals to detect and characterize bacterial infections have a number of limitations. Many of the clinically approved radiopharmaceutical agents are not specific to bacterial infections and accumulate at lesions of inflammation. Hence, new approaches are necessary to detect bacteria with high specificity and selectivity. A library of desferrioxamine B (DFO) derivatives were prepared to create radiolabeled siderophores to create a bacteria-specific imaging probe by exploiting the mechanism bacteria use to scavenge iron, which plays a key role in bacterial growth and biofilm formation Compounds were synthesized using two convenient carbamate forming strategies in 30% to 92% yield. The cold and radioactive gallium (67Ga) complexes were prepared and characterized and their uptake by S. aureus bacteria were assessed in vitro and in vivo. In vivo studies revealed that 67GaDFOethoxycarbamate had uptake comparable to GaDFO that was blockable, showing the compound was actively taken up via the siderophore pathway. In vivo studies in a mouse model resulted in a good infected to non-infected thigh ratio (11:1) and non-specific uptake by the GI tract. Bioorthogonal chemistry was also explored as an approach for imaging infection using trans-cyclooctene (TCO) functionalized vancomycin and a tetrazine functionalized 67GaDFO (67GaDFO-Tz) complex.2,3 In vitro results revealed that allowing vancomycin-TCO to bind S. aureus prior to the addition of 67GaDFO-Tz (pretargeting) showed higher (63%) uptake than with a conjugate formed prior to incubation with the bacteria (direct targeting, 28%). For the bioorthogonal approach, the distribution of the 67GaDFO-Tz was assessed in a S. aureus infection murine model, which showed significant uptake of 67GaDFO-Tz in the GI tract 1 h post intravenous injection. However, uptake in the infected joint was evident at 71 h post infection. The data suggests targeting bacteria using TCO-labeled antibiotics and radiolabeled tetrazines is a feasible strategy, but that further optimization of the vancomycin injection dose and injection time are necessary. / Thesis / Master of Science (MSc)

Over 1.1 billion people smoke worldwide despite the association of smoking with numerous diseases including chronic obstructive pulmonary disease (COPD). The decline in lung function observed in COPD patients is thought to be related to smoke-induced inflammation. COPD patients are also at increased risk of acquiring lung bacterial infections that are associated with exacerbations, characterized by worsened disease symptoms and inflammation. The focus of this thesis is on how cigarette smoke impacts bacterial-host interactions and bacterial community interactions to promote infection and disease. In chapter 3.1, we sought to understand how cigarette smoke primed the lungs towards an amplified inflammatory response to bacterial infection reflective of COPD exacerbations that accelerate disease progression. We present a novel finding that exacerbated neutrophilia elicited by nontypeable Haemophilus influenzae (NTHi) lung challenge in smoke-exposed mice occurred dependent on IL-1α. Smokers and patients with COPD are additionally at increased risk of acquiring bacterial infection that may be related to impaired containment of nasally colonizing pathogens. In chapter 3.2, we found that cigarette smoke predisposed mice to invasive pneumococcal disease (IPD) following nasal pneumococcal colonization associated with attenuated nasal inflammatory responses. To our knowledge, this is the first study to describe the progression from asymptomatic nasal pneumococcal colonization to the development of IPD in the context of cigarette smoking. It has been suggested that smokers have higher rates of pathogen colonization as a consequence of cigarette smoke-induced nasal microbiome dysbiosis. The last study in chapter 3.3 advanced knowledge in the field by testing this hypothesis. We observed that cigarette smoke alone did not alter the mouse nasal microbiome and concluded that microbiome dysbiosis observed in smokers likely occur as a consequence of nasal pathogen colonization. Overall, work presented in this thesis advanced our understanding of how cigarette smoking alters bacterial-host interactions to promote infection and disease. / Thesis / Doctor of Philosophy (PhD) / Over 1.1 billion people smoke worldwide and can develop chronic obstructive pulmonary disease (COPD), a serious inflammatory disease compromising lung function. Additionally, smokers and COPD patients have higher rates of bacterial infection. The goal of this thesis is to understand how smoking impacts our ability to combat infection. Lung infection in COPD patients causes exacerbation, with worsened disease symptoms. Using mouse models, we learned how smoking causes increased lung inflammation following bacterial infection, contributing to damage reflective of COPD exacerbations, and identified a potential intervention. We elucidated smokers may have increased infections due to impaired immune responses in the nose, a major pathogen entry point. It is thought smoking reduced beneficial bacteria that counter pathogen acquisition in the nose. We confirmed smoking did not impact these bacteria, directing research focus towards other ways smokers acquire pathogens. Overall, this thesis advanced knowledge and will help efforts to control disease in smokers.

No description available.

Clostridium difficile infection (CDI) is the most common cause of health-care-associated infectious diarrhea. Recurrence rates are as high as 20–30% after standard treatment with metronidazole or vancomycin, and appear to be reduced for patients treated with fidaxomicin. According to the literature, the risk of CDI recurrence increases after the second relapse to 30–65%. Accurate data for Germany are not yet available. Methods: Based on the research database of arvato health analytics (Munich, Germany), a secondary data analysis for the incidence, treatment characteristics and course of CDI was performed. The database included high granular accounting information of about 1.46 million medically insured patients covering the period 2006–2013, being representative for Germany. The analysis was based on new-onset CDI in 2012 in patients which either received outpatient antibiotic therapy for CDI or were hospitalized. Results: The ICD-10 coded incidence of CDI in 2012 was 83 cases per 100,000 population.

O colapso traqueal é uma obstrução parcial ou total da traqueia caracterizado pelo achatamento dorsoventral dos anéis cartilaginosos e pela frouxidão da membrana traqueal dorsal. Acomete principalmente cães de raças pequenas, de meia idade a idosos, embora também possa ocorrer em cães jovens. O diagnóstico é feito com base nos sinais clínicos e exames complementares. A traqueobroncoscopia permite avaliar o diâmetro da traqueia e dos segmentos brônquicos, principalmente quando as radiografias e fluoroscopia não forem conclusivas e ainda permite a coleta de amostras para citologia, histopatologia e culturas. O objetivo deste estudo foi correlacionar a infecção traqueal com a inflamação da traqueia em cães com colapso de traqueia. A pesquisa foi realizada no HOVET da FMVZ-USP e no Hospital Veterinário Clinivet em Curitiba. A amostra foi constituída por 28 cães, sendo 12 com colapso de traqueia e 16 hígidos para o grupo controle, que propiciou parâmetros de normalidade em relação ao grupo colapso traqueal. Para a coleta de dados utilizou-se a traqueobroncoscopia, com a qual visualizamos a traqueia e graduamos o colapso, colhemos material para cultura bacteriana e citologia. Após a análise dos resultados foi observado diferença estatística significativa nos cães com inflamação e colapso de traqueia. Não foi observado correlação entre a infecção bacteriana e a inflamação na traqueia. Com um teste de dissimilaridade verificou-se que a população bacteriana da orofaringe foi semelhante a da traqueia nos cães do mesmo grupo. Portanto, concluímos que cães com colapso de traqueia tendem a ter a traqueia inflamada, porém não apresentam infecção bacteriana. A composição das bactérias na traqueia pode ser devido à aspiração do conteúdo da orofaringe. / The Tracheal collapse is a partial or total obstruction of the trachea, featured by dorsoventral flattening of the cartilaginous rings and by the laxity of the dorsal tracheal membrane. It mainly affects small breeds, middle-aged and older dogs, although it can also occur in young dogs. The diagnosis is made based on clinical signs and additional exams. The trachealbronchoscopy allows evaluating the trachea diameter and bronchial segments, especially when radiographic and fluoroscopy is not conclusive and still allows the collection of samples for cytology, histopathology and cultures. The objective of this study was correlating the tracheal infection with the tracheal inflammation in dogs with tracheal collapse. The research was conducted in the HOVET FMVZ-USP and Clinivet Veterinary Hospital in Curitiba. The sample consisted of 28 dogs, including 12 with collapsing trachea and 16 healthy subjects in the control group, which allowed normal parameters in relation to the group tracheal collapse. For data collection was used the trachealbronchoscopy, in which was visualized the trachea and the grade of the tracheal collapse was recorded. We also collected samples for cytology and bacterial culture. After analyzing the results we found statistically significant difference in dogs with tracheal collapse and inflammation of the trachea. There was no correlation between bacterial infection and inflammation in the trachea. With dissimilarity test was observed that the bacterial population of the pharynx was similar to the trachea in dogs of the same group. n this study, therefore, concluded that dogs with collapsing trachea tend to have the inflamed trachea, but it does not have bacterial infection. The composition of the bacteria in the trachea may be due to aspiration of pharynx\'s contents.

Inflammation in the lungs can occur for many reasons, from bacterial infections to stretch by mechanical ventilation. In this work we compare and contrast various mathematical models for lung injuries in the categories of acute infection, latent versus active infection, and particulate inhalation. We focus on systems of ordinary differential equations (ODEs), agent-based models (ABMs), and Boolean networks. Each type of model provides different insight into the immune response to damage in the lungs. This knowledge includes a better understanding of the complex dynamics of immune cells, proteins, and cytokines, recommendations for treatment with antibiotics, and a foundation for more well-informed experiments and clinical trials. In each chapter, we provide an in-depth analysis of one model and summaries of several others. In this way we gain a better understanding of the important aspects of modeling the immune response to lung injury and identify possible points for future research.