Exserohilum turcicum is the hemibiotrophic causal agent of Northern leaf blight of maize and sorghum. Despite the global importance of this yield-limiting pathogen, knowledge regarding genes contributing to disease development and race-specificity is limited. Therefore, this study aimed to identify genes involved in host colonization during biotrophic and necrotrophic phases of infection, as well as race-specific differences in gene expression. RNAseq of maize seedlings inoculated with a race 13N or 23N E. turcicum isolate was conducted to identify genes contributing to fungal pathogenicity, and expression was validated for four effector candidates. A population genetic study was undertaken of isolates from maize and sorghum to select isolates for sequencing of three putative effectors. Fungal biomass positively correlated with the percentages of E. turcicum reads mapped and indicated a lifestyle switch from biotrophy to necrotrophy between 7 and 13 dpi. Transcriptome sequencing enabled identification of cell wall degrading enzymes, peptidase-encoding genes, secondary metabolite biosynthesis genes and candidate effectors likely contributing to the pathogenicity of E. turcicum. Profiling of Ecp6 and candidate effector SIX13-like revealed increased expression at 5 and 7 dpi compared to 2 and 13 dpi. Evidence of host specificity was obtained from microsatellite haplotypes and sequencing of SIX13-like. Identification of candidate effector SIX13-like is consistent with the colonization of E. turcicum through the xylem of susceptible hosts and possibly indicates specificity of E. turcicum to either maize or sorghum. This study identified E. turcicum genes putatively involved in pathogenicity and describes a hypothetical model of the E. turcicum – maize interaction. / Thesis (PhD)--University of Pretoria, 2019. / The financial assistance of the National Research Foundation (NRF South Africa, grant unique numbers 85847, 88785, 92762 and 93671) toward this research is hereby acknowledged. Opinions expressed and conclusions arrived at, are those of the authors and are not necessarily to be attributed to the NRF. / Plant Science / PhD / Unrestricted

Background Hepatitis B is a world health problem. The virus is a blood pathogen that transfers through blood and body fluids. Hepatitis B is common in the west pacific area and in Vietnam nurses are at higher risk to get infected by hepatitis B. Aim The aim with the study is to describe Vietnamese nursing student´s perception of caring for patients with hepatitis B. Method It is a qualitative study with semi-structured interviews. Result There is a lack of knowledge about hepatitis B in the community and self-education is needed in the care. Fear of getting infected among nurses and a mistrust for the vaccine were found. The awareness of getting infected by needles and sharp instrument were also found. Conclusion Lack of knowledge is a problem in the society, in the health care and also experienced by the nursing students. For the future the nursing students want to educate patients in how to live with the virus, but they see many challenges. Lack of knowledge leads to stigma that infected patients are exposed to by health care and the society, those actions of stigma affects the nursing students in how they see and treat the virus.

Pathogen-associated molecular pattern (PAMP) recognition occurs by plasma membrane located receptors that induce among other processes nuclear gene expression. The plant FLS2-BAK1 receptor complex binds the bacterial PAMP, flg22 and induces a series of defense responses. The resulting signal transduction events occur through the activation of two MAPK signaling cascades, which trigger a rapid and strong activation of MPK3, MPK4 and MPK6. Cellular responses to pathogens are regulated by the activated MAPKs, which lead to the eventual phosphorylation of cytoplasmic and nuclear substrates. These MAPK substrates in turn respond to phosphorylation by reprogramming the expression of defense genes. A large scale phosphoproteomics screen of nuclear proteins in wild type and mpk mutant plants in response to flg22 revealed several novel putative targets of MAP kinases. This thesis is aimed at identifying the role of two of these chromatin associated proteins in plant immunity and their signaling mechanisms. The chromatin associated proteins we chose to study here are LITTLE NUCLEI/CROWDED NUCLEI (LINC/CRWN), LINC1 and the AT-HOOK MOTIF CONTANING NUCLEAR LOCALIZED 13 (AHL13) proteins. We demonstrate that these two chromatin associated proteins play a positive regulatory role in jasmonic acid signaling and immunity. Knock out mutants for both genes exhibit impairment in early and late innate immune reposes to both PAMP and hemibiotrophic pathogen strains. We also demonstrate that these mutants are compromised in regulating the expression of genes involved in jasmonic acid (JA) signaling and responses and genes involved in the biosynthesis both the indole and aliphatic glucosinolate (GS) pathways. Moreover, Pst DC3000 hrcC triggers JA and JAIle accumulation in these mutants, whereas salicylic acid (SA) levels are unchanged. We were also able to identify and validate two novel MAPK targeted phosphosites in AHL13 that affect the protein stability of AHL13 and we establish its role as a MPK6 substrate that affects jasmonic acid biosynthesis and PTI responses. Together this work identifies two novel signaling components involved in the regulation of jasmonic acid homeostasis and immunity.

La détection rapide et sensible des agents pathogènes est d’une très grande importance pour la biosécurité. Les biopuces sont bien adaptées à cet effet, car elles permettent la détection multiplexe des cibles. Une limitation cruciale des biopuces est leur manque de fiabilité et de sensibilité. L’objectif de cette thèse est de développer une architecture reproductible de biopuces à base de couche mince de silicium amorphe carboné (a-SiC:H) déposée sur un réflecteur en aluminium pour une détection fiable et sensible des pathogènes. Nous avons choisi comme système modèle l’interaction de la toxine alimentaire ochratoxine A (OTA) avec son aptamère AntiOTA de longueur 36mer. Les aptamères (simples brins d’ADN) sont de plus en plus utilisés comme sondes en raison de leur grande spécificité et affinité vis-à-vis d’une large gamme de cibles (i.e. protéines, bactéries…). La stratégie de fabrication consiste en un greffage de monocouches organiques d’acides carboxyliques via des liaisons Si-C robustes, suivi de l’accrochage covalent des aptamères par un couplage peptidique. Les processus de greffage ont été mis au point sur silicium cristallin permettant la quantification des couches greffées par spectroscopie infrarouge en mode ATR (Attenuated total reflexion). La quantification IR des interactions OTA – AntiOTA a été montrée pour la première fois sur des surfaces par IR-ATR. La spécificité de l’aptamère a été démontrée en utilisant une molécule chimiquement similaire (warfarin), pour laquelle l’AntiOTA ne montre aucune affinité. Ces protocoles bien contrôlés ont été transférés sur l’architecture de la biopuce a-SiC:H. Les aptamères immobilisés sont hybridés avec des brins complémentaires marqués avec des fluorophores. En présence de l’OTA une déshybridation des brins complémentaires est attendue, conduisant à une diminution du signal fluorescent. Différentes longueurs de brins complémentaires ont été comparées, montrant jusqu’à 13% de diminution due à l’interaction de l’OTA. / Rapid and sensitive detection of pathogenic targets play a crucial role in biosecurity. Biochips are ideal for this, as they allow easy and multiplex detection of targets. A crucial limitation in biochips is that they often suffer from low reliability and sensitivity. The goal of this thesis is to develop a stable and reproducible architecture for biochips based on an amorphous silicon carbon alloy (a-SiC:H) deposited on an aluminium back-reflector for reliable and sensitive detection of pathogens. On these biochips we introduced the interaction of the food and feed toxin ochratoxin A (OTA) with its 36mer aptamer AntiOTA as a model system. Aptamers (single strands of DNA) are ideal as probes for biochips as they display high specificity and affinity towards a wide range of targets (i.e. proteins, bacteria…). The well-controlled multi-step fabrication process consists of the reliable photochemical grafting of acid-terminated organic monolayers on silicon surfaces by robust Si C bonds, which in turn were functionalized with aptamers by stable peptide coupling. Carrying out this process on crystalline silicon allowed monitoring and quantification of every step by infrared spectroscopy (IR-ATR). The interaction OTA – AntiOTA was shown for the first time on surfaces by IR, and an IR in situ calibration allowed the quantification of OTA which was bound by the aptamers on the surface. The specificity of AntiOTA towards OTA was demonstrated by using a chemically similar molecule (warfarin), for which AntiOTA shows no affinity. The well-controlled protocols were transferred to the a-SiC:H biochip. The immobilized aptamers were hybridized with complementary and fluorescent-labeled DNA-strands. In presence of OTA, dehybridization of the complementary strands is expected, resulting in a decrease of fluorescent signal. Different lengths of complementary strands were compared, exhibiting up to 13% signal decrease due to OTA.

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第19774号 / 農博第2170号 / 新制||農||1040(附属図書館) / 学位論文||H28||N4990(農学部図書室) / 32810 / 京都大学大学院農学研究科応用生命科学専攻 / (主査)教授 植田 充美, 教授 栗原 達夫, 教授 矢﨑 一史 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

No description available.

Bacterial pathogens are a major cause of diseases in human, agricultural plants and farm animals. Even after decades of research they remain a challenge to health care as they are known to rapidly evolve and develop resistance to the existing drugs. Systems biology is an emerging area of research where all of the components of the system, their interactions, and the dynamics can be studied in a comprehensive, quantitative, and integrative fashion to generate predictive models. When applied to bacterial pathogenesis, systems biology approaches will help identify potential novel molecular targets for drug discovery. A pre-requisite for conducting systems analysis is the identification of the building blocks of the system i.e. individual components of the system (structural annotation), identification of their functions (functional annotation) and identification of the interactions among the individual components (interaction prediction). In the context of bacterial pathogenesis, it is necessary to identify the host-pathogen interactions. This dissertation work describes computational resources that enable comprehensive systems level study of host pathogen system to enhance our understanding of bacterial pathogenesis. It specifically focuses on improving the structural and functional annotation of pathogen genomes as well as identifying host-pathogen interactions at a genome scale. The novel contributions of this dissertation towards systems biology of bacterial pathogens include three computational tools/resources. “TAAPP” (Tiling array analysis pipeline for prokaryotes) is a web based tool for the analysis of whole genome tiling array data for bacterial pathogens. TAAPP helps improve the structural annotation of bacterial genomes. “ISO-IEA” (Inferred from sequence orthology - Inferred from electronic annotation) is a tool that can be used for the functional annotation of any sequenced genome. “HPIDB” (Host pathogen interaction database) is developed with data a mining capability that includes host-pathogen interaction prediction. The new knowledge gained due to the implementation of these tools is the description of the non coding RNA as well as a computationally predicted host-pathogen interaction network for the human respiratory pathogen Streptococcus pneumoniae. In summary, the computation tools and resources developed in this dissertation study will enable building systems biology models of bacterial pathogens.

Bacteria inhabit diverse environmental niches and consequently, must modulate their metabolism to adapt to stress. The nucleotide second messengers guanosine tetraphosphate (ppGpp) and guanosine pentaphosphate (pppGpp) (collectively referred to as (p)ppGpp) are essential for survival during nutrient starvation. (p)ppGpp is synthesized by the RelA-SpoT homologue (RSH) protein family and coordinates the control of cellular metabolism through its combined effect on over 50 proteins. While the role of (p)ppGpp has largely been associated with nutrient limitation, recent studies have shown that (p)ppGpp and related nucleotides have a previously underappreciated effect on different aspects of bacterial physiology, such as regulating bacterial interactions with its host. This thesis focuses on the coordination of virulence gene expression and evasion of host immunity by (p)ppGpp in Salmonella enterica serovar Typhimurium. In the first data chapter, I describe the role of (p)ppGpp in mediating bacterial resistance to killing by the human complement system. I identified that (p)ppGpp activates ppnN, a nucleotide metabolism associated enzyme, and the biosynthesis of lipopolysaccharide O-antigen to protect Salmonella from cell lysis by complement. The second data chapter compares and contrasts the stringent response of an invasive clinical isolate of Salmonella Typhimurium to a strain of Salmonella Typhimurium that causes acute gastroenteritis using RNA-sequencing. Critical analysis of our transcriptomics dataset showed that flagellar-based motility is differentially regulated by (p)ppGpp in the two strains of Salmonella. Together, these findings demonstrate that (p)ppGpp has significant functional roles beyond mediating adaptation to nutrient limitation. / Thesis / Doctor of Philosophy (PhD)

No description available.

A needlestick injury is defined by the Center for Disease Control (CDC) as a percutaneous injury due to accidental handling of a sharp. The CDC estimates that approximately 400,000 needlestick incidences occur each year in United States healthcare facilities, and reports from other developed countries, such as the United Kingdom and Spain, share similar frequencies. Further, the World Health Organization (WHO) estimates two million international healthcare workers are exposed annually to infectious disease as a consequence of a needlestick event, resulting in 37.6% and 39% of hepatitis B and hepatitis C cases, respectively. In the United States, federal and state legislation have greatly reduced incidence rates since the late 1980s, providing education, better protocols and effective post-exposure management. Additionally, the introduction of national surveillance databases led to stronger epidemiological support for the causation of needlestick injury and consequently, a stronger national awareness. In an effort to better protect healthcare workers, corporations such as DuPont and BD have further reduced needlestick incidences in the United States by designing products ranging from safety-engineered syringes to adhesive strips surrounded in strong synthetic materials such as Kevlar® and Lycra®. These devices are instrumental in minimizing the needlestick problem in both the clinic and in the operating room. As part of the current United States legislation, healthcare organizations are mandated to implement and utilize these safety-engineered syringes and needles. Despite the rise in protective equipment, national database surveillance and federal/state legislature, the incidence rate remains high as hundreds of thousands of injuries persist each year. We sought to find other solutions for better protecting healthcare workers through the implementation of golden orb weaver spider silk in personal protective equipment. This silk, gathered from the Nephila clavipes, is one of the strongest and toughest biomaterials in known existence. Its characteristically high energy absorption makes it an ideal material for reinforcing gloves and other protective equipment for healthcare workers. We believe that products made from this silk would serve as strong barriers against needlestick injury and bloodborne pathogen exposure. We are in the process of designing and fabricating such a glove and completed preliminary strength testing to ensure the superiority of our material. Tensile testing conducted at Tufts' Department of Biomedical Engineering suggests that our silk possesses the same mechanical profile as N. clavipes silk found in published literature. We plan on utilizing Fourier-transform infrared (DSC-FTIR) microspectroscopy to study the protein structure and possibly conducting enzyme degradation assays to assess the property changes under unique conditions. This information combined with our patented extraction and reinforcing methodology will provide the groundwork for partnering with industry leaders to make this product a reality and help eliminate the incidence of needlestick injury.