Soilborne plant pathogens are responsible for many of the major crop diseases worldwide. However, plant root tips are generally resistant to pathogen infections. The goal of this dissertation research is to understand the mechanism of this natural resistance by testing the hypothesis that root caps and root border cells control the rhizosphere community through the biological products which they deliver to the soil. Specific objectives of this dissertation project are 1) identifying, isolating, and characterizing the genes important for border cell development and for root exudates delivery, and 2) analyzing the function of extracellular macromolecules in root exudates in root tip-fungal pathogen interaction. The expression of a primary cell wall synthesis gene, PsFut1, encoding Pisum sativum fucosyltransferase, was characterized during border cell production, and the impact of silencing this gene on border cell development was examined. Another gene, BRDgal1, encoding β-galactosidase, was identified and characterized in Pisum sativum during this study. It was shown that this β-galactosidase is specifically produced in and secreted from root border cells. The microarray transcriptional profiling in M. truncatula and mRNA differential display analysis in pea plants were carried out following the induction of border cell production to gain a broader understanding of the genes which potentially influence border cell development. In order to study the commonality of border cell production across different plant species, the expression of rcpme1, the marker gene for border cell production, was compared between the garden pea and a gymnosperm species, the Norway spruce (Picea abies). To accomplish the second objective, the focus of this study was shifted from border cell development to mucilaginous root exudates excreted by border cells and root cap cells. This resulted in a breakthrough in the understanding of the mechanisms of root tip resistance. The presence of extracellular DNA in the root mucilage was discovered and its requirement for root tip resistance to fungal infection was demonstrated. Extracellular proteins in the root mucilage were identified and they were shown to be also required for the root tip resistance to fungal infection. This work provided new insights into understanding plant defense mechanisms.

Traumatic brain injury (TBI) is a relatively unknown disease considering the tens of millions of people affected around the world each year. Many TBI patients die from their injuries and survivors often suffer from life-long disabilities. The primary injury initiates a variety of cellular and molecular processes that are both beneficial and detrimental for the brain, but that are not fully understood. The focus of this thesis has been to study the role of astrocytes in clearance of dead cells after TBI and to identify injury specific proteins that may function as biomarkers, by using cell cultures, animal models and in cerebrospinal fluid (CSF) from TBI patients. The result demonstrates a new function in that astrocytes, the most numerous cell type in the brain, engulf dead cells after injury both in cell cultures and in adult mice and thereby save neurons from contact-induced apoptosis. Astrocytes are effective phagocytes, but degrade the ingested dead cells very slowly. Moreover, astrocytes express the lysosome-alkalizing proteins Rab27a and Nox2 as well as major histocompatibility complex class II, the receptors on which antigens are being presented. By lowering the pH of the lysosomes with acidic nanoparticles, the degradation increases, but the astrocytes still remained less effective than macrophages. Taken together, the data indicates that the low acidification in astrocytes can preserve antigens and that astrocytes may be able to activate T cells. The expression and secretion of injury-specific proteins was studied in a cell culture model of TBI by separate mass spectrometry analysis of cells and medium. Interestingly, close to 30 % of the injury-specific proteins in medium are linked to actin, for example ezrin of the ezrin/radixin/moesin (ERM) protein family. Ezrin, but none of the other ERM proteins or actin, is actively secreted after injury. Extracellular ezrin also increases in CSF in response to experimental TBI in rats and is present in CSF from TBI patients, indicating that ezrin is a potential biomarker for TBI.

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No. of bitstreams: 2 Dissertação - Andrea Santana de Oliveira - 2016.pdf: 1927842 bytes, checksum: 5b65d88f78b5077ed835f4eb96131b65 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2016-09-30 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Staphylococcus saprophyticus is a Gram-positive bacterium responsible for genitourinary infections, mainly affecting young sexually active women. Along with Escherichia coli is responsible for 90% of infections in fertile women, however, can cause infections in men and women of all ages. The repertoire of proteins secreted by pathogenic microorganisms is used to ensure success in the establishment of infection and persistence in the host. In this sense, this study aimed to comparative characterization of extracellular proteome of 3 clinical strain of S. saprophyticus, in order to detect possible differences in the secretion of proteins related to virulence and adaptation of the microorganism. The strains used in the study are called ATCC 15305, 7108 and 9325. Ultra-Performance Liquid Chromatography coupled to Mass Spectrometry in tandem (UPLC-MSE) have identified a total of 159 proteins. Among them, 44 were found in exoproteome of 3 strain, while 20 only in strains ATCC 15305 and 7108, 11 in ATCC 15305 and 9325, and 12 in 7108 and 9325. Fifteen peptides were expressed exclusively by S. saprophyticus 9325, 21 by ATCC 15305 strain and 36 by 7108. The three strain secreted molecules with biological function related to the glycolytic pathway, such as the triosephosphate isomerase (TPI), enolase, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and fructose-bisphosphate aldolase, molecules clearly involved in extracellular processes from other pathogenic organisms, proteins known as "moonlighting". Proteins involved in defense against stress, such as catalase, alkyl hydroperoxide reductase subunit C (AhpC), superoxide dismutase (SOD), were also detected in the analysis, among others related to metabolic processes such as lactic fermentation, cell wall synthesis, protein processing, and iron metabolism. Staphylococcal secretory antigen A (SsaA), a immunogenic molecule previously detected in ATCC 15305 strain, was not detected in strain 7108, as confirmed by Western-blotting assay. PCR reactions with specific primers and genomic DNA of the 3 strains and subsequent sequencing ssaA gene, showed that the strain has the gene under conditions identical to those found in S. saprophyticus ATCC 15305 and 9325, but is not able to secrete the antigen into the extracellular milieu, under environmental conditions in study. In exoproteome S. saprophyticus 7108 was found to take a greater amount of unique proteins, including four of the class of peptidases, enzymes often identified as potential virulence factors of bacterial. Thus, the differences found in repertoire of extracellular proteins of three clinical strain of S. saprophyticus, demonstrate a metabolic flexibility used by this uropathogen to promote pathogenesis in human genitourinary tract. / Staphylococcus saprophyticus é uma bactéria Gram-positiva responsável por infecções do trato geniturinário, acometendo principalmente mulheres jovens sexualmente ativas. Junto com Escherichia coli, é responsável por 90% das infecções em mulheres férteis, entretanto, pode causar infecções em homens e mulheres de todas as idades. O repertório de proteínas secretadas por microrganismos patogênicos é utilizado para garantir sucesso no estabelecimento da infecção e na persistência dentro do hospedeiro. Nesse sentido, este trabalho teve como objetivo principal a caracterização comparativa do proteoma extracelular de 3 cepas clínicas de S. saprophyticus, com a finalidade de detectar possíveis diferenças na secreção de proteínas relacionadas à virulência e adaptação do microrganismo. As cepas utilizadas no estudo são denominadas de ATCC 15305, 7108 e 9325. Por meio da Cromatografia Líquida de Ultra Desempenho acoplada à Espectrometria de Massas in tandem (UPLC-MSE), foram identificadas um total de 159 proteínas. Dentre elas, 44 foram encontradas no exoproteoma dos 3 cepas, enquanto que 20 apenas nas cepas ATCC 15305 e 7108, 11 em ATCC 15305 e 9325, e 12 nas cepas 7108 e 9325. Quinze peptídeos foram expressos exclusivamente por S. saprophyticus 9325, 21 pelo ATCC 15305 e 36 pela cepa 7108. Os três cepas secretaram moléculas com função biológica relacionada à via glicolítica, como a triose-fosfato isomerase (TPI), enolase, gliceraldeído-3-fosfato desidrogenase (GAPDH) e frutose-bifosfato aldolase, moléculas claramente envolvidas em processos extracelulares de outros organismos patogênicos, conhecidas como proteínas “moonlighting”. Proteínas envolvidas na defesa contra o estresse, como a catalase, a alquil hidroperóxido redutase subunidade C (AhpC), a superóxido dismutase (SOD), também foram detectadas na análise, dentre outras relacionadas à processos metabólicos como fermentação lática, síntese de parede celular, processamento de proteínas e metabolismo do ferro. O antígeno estafilocócico secretado A (SsaA), uma molécula imunogênica detectada previamente na cepa ATCC 15305, não foi detectada na cepa 7108, dado confirmado por ensaio de Western-blotting. Reações de PCR com primers específicos e DNA genômico das 3 cepas e posterior sequenciamento do gene ssaA, mostrou que a cepa possui o gene em condições idênticas ao encontrado em S. saprophyticus ATCC 15305 e 9325, porém não é capaz de secretar o antígeno para o meio extracelular, nas condições ambientais em estudo. No exoproteoma de S. saprophyticus 7108 foi encontrada uma maior quantidade de proteínas exclusivas, incluindo quatro da classe das peptidases, enzimas apontadas muitas vezes como potenciais fatores de virulência bacteriana. Desta forma, as diferenças encontradas no repertório de proteínas extracelulares de 3 cepas clínicas de S. saprophyticus, demonstram uma flexibilidade metabólica utilizada por este uropatógeno em promover a patogênese no trato geniturinário humano.

Proteins modularity enhances the multi-functionality and versatility of proteins by providing such properties as multiple and various ligand-binding sites, increased ligand affinity through the avidity effect, and the juxtaposition of ligand-binding modules near catalytic domains. An NMR-based "dissect-and-build" approach to studying modular protein structure and function has proven very successful, whereby modules are initially characterized individually and then correlated with the overall function of a protein. We have used the dissect-and-build approach and NMR to study two modular protein systems. Chapter 2 details the NMR solution structure of the weak-lysine-binding kringle IV type 8 (KIV8) module from the apolipoprotein(a) (apo(a)) component of lipoprotein(a) was determined and its ligand-binding properties assessed. In vitro studies have demonstrated the importance of the apo(a) KIV7 and KIV8 modules in mediating specific lysine-dependent interactions with the apolipoproteinB-100 (apoB-100) component of LDL in the initial non-covalent step of lipoprotein assembly. Notable differences identified in the lysine binding site (LBS) of the KIV8 were deemed responsible for the differential modes of apoB-100 recognition by KIV7 and KIV8. In addition, the KIV8 structure has brought to light the importance of an RGD sequence at the N-terminus of the apo(a) KIV8 module, which may mediate important apo(a)-integrin interactions. In Chapters 3-6, structure-function studies of the CpGH84C X82 and the CpGH84A dockerin-containing modular pair were conducted to understand how the varying modularity unique to the C-terminal regions of the secreted multi-modular family 84 glycoside hydrolases influences the spreading of Clostridium perfringens. Identification of a CpGH84C cohesin module (X82), and the structural characterization of a dockerin-containing modular pair provides the first evidence for multi-enzyme complex formation mediated by non-cellulosomal cohesin-dockerin interactions. The formation of large hydrolytic enzyme complexes introduces a novel mechanism by which C. perfringens may enhance its role in pathogenesis. / Thesis (Ph.D, Biochemistry) -- Queen's University, 2007-09-27 11:46:38.753