Hybridization refers to mating between species or between genetically differentiated populations of the same species. Although hybrid offspring may exhibit sterility and/or inviability, hybridization can generate novel genotypic and phenotypic diversities, leading to the origin of new traits and new species, the expansion into ecological niches outside of the parental range (e.g., host range), and altered virulence properties in pathogens. However, the relationship between parental genetic divergence and hybrid performance remains largely unknown. The human pathogenic Cryptococcus (HPC) is an ideal model to study the impacts of parental divergence on the genetic and phenotypic consequences of hybridization. HPC consists of a group of divergent lineages with various degrees of interfertility. These yeasts are the etiologic agents of cryptococcosis, a potentially lethal disease in humans and animals. In this thesis, I examined the effects of parental divergence on cryptococcal hybrids from multiple aspects. I conducted genetic crosses between different lineages to evaluate the mating success and the germination of sexual spores (i.e., basidiospores) under various environmental conditions. Then, I investigated the genotypic and phenotypic diversities among the hybrids under different environmental conditions. Furthermore, I examined the genome stability of diploid inter-lineage hybrids through laboratory experimental evolution and the effect of antifungal drug stress on the loss of heterozygosity (LOH) in these hybrids. We found that parental genetic divergence plays an important role in genotypic and phenotypic diversities among hybrid progeny in HPC. However, our results indicate that parental genetic di-vergence alone can’t explain most of the observed variations. Instead, genetic divergence along with specific parental strains, environmental factors, and their interactions all contributed to hybridization success and to hybrid genotypic and phenotypic variations. My findings will broaden the current understanding of the phenotypic and genotypic consequences of hybridization and explore the connection between genetic architecture and hybrid speciation in the human pathogenic Cryptococcus. / Thesis / Doctor of Science (PhD) / The role of hybridization in evolution can vary widely, giving rise to hybrid vigor and hybrid weakness. Hybridization plays an important role in plants and animals, especially crops, with advantages of increased yield and quality of products. However, the emergence of hybrid vigor in pathogens with increased virulence is an increasing threat to plant, animal, and human healths. My PhD thesis aimed at understanding the effects of parental divergence on hybridization and hybrids in the human pathogenic Cryptococcus. Here, I investigated basidiospore germination rate and hybrid progeny genotypes and phenotypes from diverse genetic crosses in this group of pathogens. My findings contribute towards understanding cryptococcal hybrids and establishing treatment plans against infections by these hybrids.

Developing diagnostic modalities that utilize nanomaterials and miniaturized detectors can have an impact in point-of-care diagnostics. Diagnostic systems that (i) are sensitive, robust, and portable, (ii) allow detection in clinical samples, (iii) require minimal sample preparation yielding results quickly, and (iv) can simultaneously quantify multiple targets, would have a great potential in biomedical research and public healthcare. Bacterial infections still cause pathogenesis throughout the world (Chapter I). The emergence of multi-drug resistant strains, the potential appearance of bacterial pandemics, the increased occurrence of bacterial nosocomial infections, the wide-scale food poisoning incidents and the use of bacteria in biowarfare highlight the need for designing novel bacterial-sensing modalities. Among the most prominent disease-causing bacteria are strains of Escherichia coli, like the E. coli O157:H7 that produces the Shiga-like toxin (Stx). Apart from diarrheagenic E. coli strains, others cause disease varying from hemolytic uremic syndrome and urinary tract infections to septicemia and meningitis. Therefore, the detection of E. coli needs to be performed fast and reliably in diverse environmental and clinical samples. Similarly, Mycobacterium avium spp. paratuberculosis (MAP), a fastidious microorganism that causes Johne's disease in cattle and has been implicated in Crohn's disease (CD) etiology, is found in products from infected animals and clinical samples from CD patients, making MAP an excellent proof-of-principle model. Recently, magnetic relaxation nanosensors (MRnS) provided the first applications of improved diagnostics with high sensitivity and specificity. Nucleic acids, proteins, viruses and enzymatic activity were probed, yet neither large targets (for instance iv bacterial and mammalian cells) nor multiple bacterial disease parameters have been simultaneously monitored, in order to provide thorough information for clinical decision making. Therefore, the goal of this study was to utilize MRnS for the sensitive identification of multiple targets associated with bacterial pathogenesis, while monitoring virulence factors at the microorganism, nucleic acid and virulence factor levels, to facilitate improved diagnosis and optimal treatment regimes. To demonstrate the versatility of MRnS, we used MAP as our model system, as well as several other pathogens and eukaryotic cell lines. In initial studies, we developed MRnS suitable for biomedical applications (Chapter II). The resulting MRnS were composed of an iron oxide core, which was caged within a biodegradable polymeric coating that could be further functionalized for the attachment of molecular probes. We demonstrated that depending on the polymer used the physical and chemical properties of the MRnS can be tailored. Furthermore, we investigated the role of polymer in the enzyme-mimicking activity of MRnS, which may lead to the development of optimized colorimetric in vitro diagnostic systems such as immunoassays and small-molecule-based screening platforms. Additionally, via facile conjugation chemistries, we prepared bacterium-specific MRnS for the detection of nucleic acid signatures (Chapter III). Considering that MAP DNA can be detected in clinical samples and isolates from CD patients via laborious isolation and amplification procedures requiring several days, MRnS detected MAP's IS900 nucleic acid marker up to a single MAP genome copy detection within 30 minutes. Furthermore, these MRnS achieved equally fast IS900 detection even in crude DNA extracts, outperforming the gold standard diagnostic method of nested Polymerase Chain v Reaction (nPCR). Likewise, the MRnS detected IS900 with unprecedented sensitivity and specificity in clinical isolates obtained from blood and biopsies of CD patients, indicating the clinical utility of these nanosensors. Subsequently, we designed MRnS for the detection of MAP via surface-marker recognition in complex matrices (Chapter III). Milk and blood samples containing various concentrations of MAP were screened and quantified without any processing via MRnS, obtaining dynamic concentration-dependent curves within an hour. The MAP MRnS were able not only to identify their target in the presence of interferences from other Gram positive and Gram negative bacteria, but could differentiate MAP among other mycobacteria including Mycobacterium tuberculosis. In addition, detection of MAP was performed in clinical isolates from CD patients and homogenized tissues from Johne's disease cattle, demonstrating for the first time the rapid identification of bacteria in produce, as well as clinical and environmental samples. However, comparing the unique MAP quantification patterns with literatureavailable trends of other targets, we were prompted to elucidate the underlying mechanism of this novel behavior (Chapter IV). We hypothesized that the nanoparticle valency – the amount of probe on the surface of the MRnS – may have modulated the changes in the relaxation times (ΔΤ2) upon MRnS – target association. To address this, we prepared MAP MRnS with high and low anti-MAP antibody levels using the same nanoparticle formulation. Results corroborated our hypothesis, but to further bolster it we investigated if this behavior is target-size-independent. Hence utilizing small-moleculeand antibody-carrying MRnS, we detected cancer cells in blood, observing similar detection patterns that resembled those of the bacterial studies. Notably, a single cancer vi cell was identified via high-valency small-molecule MRnS, having grave importance in cancer diagnostics because a single cancer cell progenitor in circulation can effectively initiate the metastatic process. Apart from cells, we also detected the Cholera Toxin B subunit with valencly-engineered MRnS, observing similar to the cellular targets' diagnostic profiling behavior. Finally, as bacterial drug resistance is of grave healthcare importance, we utilized MRnS for the assessment of bacterial metabolism and drug susceptibility (Chapter V). Contrary to spectophotometric and visual nanosensors, their magnetic counterparts were able to quickly assess bacterial carbohydrate uptake and sensitivity to antibiotics even in blood. Two MRnS-based assay formats were devised relying on either the Concanavalin A (Con A)-induced clustering of polysaccharide-coated nanoparticles or the association between free carbohydrates and Con A-carrying MRnS. Overall, taking together these results, as well as those on pathogen detection and the recent instrumentation advancements, the use of MRnS in the clinic, the lab and the field should be anticipated.

Vibrio vulnificus, an emergent human pathogen, causes septicemia with a mortality rate over 50%. Additionally, symptom onset occurs rapidly, with the incubation time for ingestion cases being around 26 hours. This, combined with the severity of symptoms has led to V. vulnificus being considered the deadliest seafood-associated pathogen, claiming responsibility for 95% of seafood-related deaths. Currently, the molecular mechanisms through which some strains of this bacteria emerge to become pathogens are unknown. The main focus of this study is to expand upon the base of knowledge surrounding this question through comparing virulence phenotypes in environmentally collected strains of V. vulnificus. Specifically, this study will evaluate the pathogenic potential of environmental isolates collected from water and oyster sources in Lee County, Florida, in lieu of the outbreak that occurred in October 2022. To test this, a variety of assays were performed. First, a phylogenetic tree was built to establish the relationships between strains. Next, to study in vitro responses, serum resistance assays and sialic acid growth curves were performed. Then, to further classify the pathogenic potential of these environmental strains, they were tested against THP-1 monocytes differentiated into macrophages for their ability to resist phagocytosis and induce apoptosis. This study found differential responses amongst the environmental isolates, with some exhibiting significant pathogenic potential and others being sensitive to all tested assays. Understanding which strains emerge as pathogens will help determine the prevalence of key virulence factors within natural populations of bacteria and provide critical data on the phenotypic outcomes of differing genotypes.

No description available.

No description available.

<p>The <em>Streptococcus</em> Milleri Group (SMG) asymptomatically colonize the gastrointestinal, female urogenital, and upper respiratory tract in the healthy population, and are therefore traditionally considered commensals. The SMG, however, are also pathogens that cause pyogenic and pulmonary infections. The factors that differentiate pathogenic from non-pathogenic isolates have proven difficult to identify, and consequently the determinants of SMG pathogenicity remain unknown. Characterization of the immune response to the SMG is important towards advancing the understanding of SMG pathogenicity, however there are limited studies that have done so.</p> <p>Herein, we sought to investigate the cytokine profiles produced by human peripheral blood mononuclear cells in response to 35 clinical isolates of the SMG. Cytokine profiles varied across isolates resulting in a spectrum of responses that separated into three subgroups including a high, intermediate, and low response group. The responses were consistent across three individuals with the exception of several differences, which are discussed and warrant further studies on host susceptibility to SMG infections. The high and intermediate response groups were enriched with clinical isolates from invasive infections, which were found to induce significantly higher cytokine production than airway isolates. Cytokine induction was independent of TLR2 activation, suggesting that other pattern recognition receptors are involved in the recognition of and response to the SMG. Phenotypic characteristics, which are used in the clinical identification of the SMG, did not correlate with cytokine induction; therefore phenotypic tests are not sufficient to identify immunostimulatory isolates. The host response to the SMG characterized in this study provides foundational knowledge for future studies to investigate the mechanism of recognition as well as the function of downstream effector responses in the control of colonization and infection.</p> / Master of Science (MSc)

Pathogenic E. coli infections cause approximately one quarter of disease losses in commercial turkey flocks. A small subgroup of E. coli causes most infections. Epidemiologic studies of this disease have been hindered by a lack of reliable markers to discriminate between pathogenic and fecal E. coli and by the diversity of poultry strains. Reliance on antimicrobials to control E. coli infections has caused widespread antimicrobial resistance. One hundred five clinical E. coli were obtained, and 1104 isolates were collected from fecal specimens of 20 flocks in an integrated turkey operation. Biochemical fingerprinting and antimicrobial susceptibility tests were performed on all isolates, and somatic antigen serologic testing and PCR for potential virulence genes were conducted on 299 strains including all clinical isolates and fecal isolates that had similar traits to clinical isolates. Most avian E. coli infections were caused by a few clonal strains that were uncommon in normal fecal flora. The potential virulence genes iss, K1 and tsh were detected more frequently among clinical than fecal isolates; however, the pattern of occurrence did not suggest that these genes were useful markers for identifying pathogenic strains. Syndromes consistent with colibacillosis were the most commonly reported illness and principal rationale for antimicrobial therapy in sampled flocks. Most clinical E. coli isolates were resistant to gentamicin, sulfamethoxazole and tetracycline. Although resistance to fluoroquinolones and β-lactam antibiotics occurred less frequently, the potential for resistance to emerge to these antimicrobials was evident. A Bayesian model to estimate sample size confirmed the diversity of avian fecal E. coli strains. Studies are needed to define risk factors for infection with and identify markers for avian pathogenic E. coli strains. These research priorities are complementary and may lead to the identification of new interventions to prevent this important infectious disease of poultry. / Ph. D.

Microbial infections of medical implants occur in 10% of the more than 20 million surgical procedures carried out annually in the United States. The additional treatments required to address these infections generate more than $11 billion in additional patient costs, increase recovery time, and decrease overall patient quality of life. As the population ages, the number of necessary and voluntary surgical procedures increases; The rate of infection increases proportionately. While treatments are available, the biofilm mode of growth confers resistance to antimicrobial therapies up to 500 times greater than that of planktonic microbes. Currently, the only guaranteed method of removing an established microbial implant infection is through surgical excision of the implant and surrounding tissues. While removing the original infection, additional colonization and pathogenesis may take place. This research explores the a priori assumption that a medical implant infection cannot occur unless a microbial cell is capable of adhering to the implant surface. From that assumption, the following sections will focus primarily on identifying the necessary and sufficient factors influencing microbial adhesion, discretizing those factors into measurable quantities, and developing methods by which those factors may be mitigated or eliminated. Following is a brief summary of each major topic treated within this research period. Development of a Benchmark System: We have characterized the interactions between Pseudomonas aeruginosa ATCC 10145 and Candida parapsilosis ATCC 90018 using a novel method of cellular immobilization, which emphasizes minimal chemical modification of the cell surface. This research describes the very different force-separation interactions seen between C. parapsilosis and both a common medical implant material (viz., silicone rubber) and a nascent P. aeruginosa biofilm grown on the same material. This study was the first step in developing an ab initio technique which may be used to determine the relative affinity of a microbial cell for an implant material surface. The Role of the Substrate: Microbial adhesion to a medical implant device involves two major components, being the microbe itself, and the substrate to which it adheres. Each of the two has specific and unique surface chemical and textural characteristics which, when combined, allow for microbial colonization and subsequent infection. The goal of this study was to identify correlations between the adhesive strength of Staphylococcus epidermidis to a variety of chemically and texturally distinct substrates, and common surface characterization parameters (e.g., surface roughness and water contact angle). Relationships to adhesive strength did not demonstrate statistically significant or consistent trends. To extend upon the correlation parameters, we have employed a Discrete Bonding Model, which characterizes the surface texture according to Mandelbrot fractal theory. Correlations between the adhesive strength and the observational scale show stronger relationships, indicating a significant contribution of the surface texture to a microbe's ability to colonize a surface. Finding a Surface That Cannot Be Touched: Historically, AFM force-separation curves demonstrating only repulsive behavior on extension of the piezoactuator have been largely ignored, in terms of quantitative modeling of the interactions. In bacterial systems, such behavior describes the majority of the force profiles recorded by the instrument. As a result of the former lack of study, the latter data sets have remained unanalyzed and unanalyzable. Building on existing mathematical models, we have developed an analytical method by which the point of zero separation between a surface (viz., the microbial cell wall) coated with a polymer brush and an AFM probe may be quantitatively identified.

Kyoto University (京都大学) / 0048 / 新制・課程博士 / 博士(工学) / 甲第16388号 / 工博第3469号 / 新制||工||1524(附属図書館) / 29019 / 京都大学大学院工学研究科都市環境工学専攻 / (主査)教授 田中 宏明, 教授 清水 芳久, 教授 藤井 滋穂 / 学位規則第4条第1項該当

Les Virus de l'Immunodéficience Humaine (VIH) sont le résultat de plusieurs transmissions inter-espèces de SIV (Virus de l'Immunodéficience Simienne) de Primates Non Humains (PNH) à l'Homme. Les SIV les plus proches du VIH-1 sont le SIVcpz et le SIVgor qui infectent naturellement les chimpanzés et les gorilles. Les SIVsmm retrouvés chez les mangabés enfumés d'Afrique de l'Ouest sont les plus proches du VIH-2. Actuellement, au moins 13 transmissions du singe à l'Homme ont été documentées, 4 à l'origine des 4 groupes du VIH-1 (groupe M, N, O et P) et 9 pour le 9 VIH-2 (A-I). La question du réservoir à l'origine du VIH-1 chez l'Homme est partiellement résolue. Les chimpanzés, Pan troglodytes troglodytes, du sud-est et centre sud du Cameroun sont respectivement les réservoirs du VIH-1 M pandémique chez l'Homme ainsi que du VIH-1 groupe N. En ce qui concerne les groupes O et P, il n'y a actuellement pas de réponse définitive. Les SIVgor sont bien les virus les plus proches phylogénétiquement des VIH-1 O et P. Cependant, de plus amples recherches sont nécessaires pour identifier les ancêtres directs des variants O et P. Ces recherches supplémentaires aideront aussi à élucider l'origine du SIVgor chez les gorilles, et à savoir si ce sont les gorilles qui ont transmis les virus O et/ou P à l'Homme, ou s'il existe toujours un réservoir des ancêtres O et P chez les chimpanzés. Des études supplémentaires sont aussi nécessaires afin de mieux comprendre les mécanismes d'adaptation à un nouvel hôte et l'impact des infections SIV chez les grands singes. Dans ce but, l'étude du récepteur accessoire pour le VIH, l'intégrine α4β7, pourrait aussi jouer un rôle pour l'infection du SIV/VIH. Cette intégrine facilite également la migration du virus vers l'intestin. Une étude récente a montré des substitutions d'acides aminés chez les Primates du Nouveau Monde (PNM) qui empêche l'adhérence du liant. Ainsi, les polymorphismes de cette intégrine et son rôle dans l'infection SIV chez les Primates de l'Ancien Monde (PdAM) sont encore inconnus. L'objectif majeur de cette thèse était de mieux documenter et mieux comprendre l'infection SIV chez les gorilles sauvages en Afrique Centrale. Sur plus de 6.000 échantillons testés, nous avons constaté que seuls les gorilles (Gorilla gorilla gorilla) du sud Cameroun sont infectés par le SIVgor. Parmi eux, nous avons identifié les ancêtres du VIH-1 P chez des populations du sud-ouest Cameroun. Nous avons aussi mis en évidence que les gorilles sont à l'origine du VIH-1 groupe O. Les analyses fonctionnelles du facteur de restriction APOBEC3G ont montré que celui-ci protège les gorilles des infections SIVcpz, expliquant en partie la faible prévalence de SIVgor. Nous avons évalué une nouvelle technologie sérologique, le Luminex®, en utilisant des antigènes spécifiques de la lignée SIVgor. Ces résultats ont été comparés avec ceux que nous pouvons obtenir avec l'INNO-LIATM qui est une technique de référence basée sur des réactions croisées entre anticorps SIV et antigènes VIH-1. Nous avons aussi évalué la faisabilité de la technologie de séquençage de deuxième génération Illumina® pour étudier les viromes de deux gorilles. Nous n'avons pas pu obtenir la séquence du SIVgor dans l'échantillon de l'individu infecté. Cependant, en comparant les résultats obtenus entre les deux gorilles étudiés, nous avons pu constater un probable déséquilibre de la réplication des virus entériques seulement pour le gorille infecté par le SIVgor. Enfin, nous avons décrit la diversité de la sous-unité α4 de l'intégrine α4β7 chez les PdAM. En conclusion, ces travaux de thèse ont apporté de nouvelles connaissances majeures sur l'infection SIV chez les gorilles et ont contribués à élucider l'origine des quatre groupes VIH-1. / Human Immunodeficiency Viruses (HIV) are the result of numerous interspecies transmissions of different SIV (Simian Immunodeficiency Virus) from Non-Human Primates (NHP) to humans. SIVcpz and SIVgor from chimpanzees and gorillas are most closely related to HIV-1, and SIVsmm from sooty mangabeys in West Africa to HIV-2. At least 13 cross-species transmissions from NHP to humans have been reported, 4 leading to the 4 HIV-1 group (M, N, O and P) and 9 for the 9 HIV-2 groups (A-I). Today the origin of HIV-1 group M and N is elucidated and their simian ancestors, have been identified in chimpanzee (Pan troglodytes troglodytes) populations in southeast and south-central Cameroon, respectively. HIV-1 group O and P are most closely related to SIVgor from gorillas but their direct ancestors have not been identified yet. More studies are thus needed to clarify the origin of HIV-1 group O and P in humans as well as on the origin of SIVgor in gorillas. These studies will also elucidate whether HIV-1 group O and P have been transmitted by chimpanzees or gorillas and whether simian ancestors of these HIV groups and the ancestor of SIVgor still circulates in today's chimpanzee populations. More studies are also needed to understand viral and host factors related adaptation in the new host and the impact of SIV infection in general in apes. As such, α4β7 integrin has been recently described as a new HIV-1 receptor that facilitates virus migration to the Gut-Associated Lymphoid Tissue (GALT). In a recent study, amino acid substitutions were observed in the α4 binding site in New World Primates (NWP), that can reduce the activity of this receptor. The impact of the genetic diversity of this integrin in Old World Primates (OWP) and its role in SIV infection is still unknown. Therefore, characterizing the polymorphisms profiles in OWP could bring new insights into progression of the pathogenic and non pathogenic SIV infections. The main objective of this thesis was to better characterize and understand SIV infection in wild gorillas in Central Africa. On more than 6,000 fecal samples from gorillas collected across Central Africa, we showed that only gorillas from southern Cameroon are infected with SIVgor and we identified the ancestors of HIV1 group P in gorilla populations from southwest Cameroon. We also provided evidence that gorillas are at the origin of HIV-1 group O in humans. Functional analysis of the restriction factor APOBEC3G showed that its protects gorillas from SIVcpz infections and can explain the low prevalence in gorillas. We evaluated a new antibody detection approach in faecal samples, based on Luminex® technology that use SIVgor specific antigens, comparing with the actual serological test INNO-LIATM HIV confirmatory assay, based on cross-reactive SIV antibodies with HIV antigens. We also evaluated the feasibility of virus sequencing in faecal samples with the Illumina® technology to study viromes of gorillas. We studied two samples, one of a SIVgor infected individual and one from an uninfected gorilla. Although the SIVgor sequence was not retrieved from the infected individual, we observed a tendency to enteric virus replication disorder in the infected animal that has not been seen in the uninfected one. Finally, we also documented here the genetic diversity of the α4 subunit from OWP. In this thesis we documented more in detail different aspects of SIV infection in gorillas and contributed to elucidate the origin of all HIV-1 groups.