M.Sc. / Lipopolysaccharides (LPS) are ubiquitous, indispensable components of the cell surface of Gram-negative bacteria that have diverse roles in bacterial pathogenesis of plants. LPS as pathogen-associated molecular pattern (PAMP) molecules can be recognized by plants to directly trigger some defense—related responses. LPS can also alter the response of plants to subsequent bacterial inoculation; these delayed effects include alterations in the expression patterns of genes coding for some pathogenesis related (PR) proteins, promotion of the synthesis of the antimicrobial conjugates, and prevention of the hypersensitive reaction caused by avirulent bacteria. Prevention of the response may allow expression of resistance in the absence of catastrophic tissue damage. LPS from Burkholderia cepacia (LPSB. cep.) have been found to trigger a strong response in plants resulting in the activation of genes coding for some pathogenesis related proteins, receptor-like kinases and resistance (R) proteins. LPS are tripartite amphipathic molecules, consisting of a Lipid A moiety that is embedded in the outer leaflet of the phospholipids/protein bilayer, a core oligosaccharide, and a polysaccharide consisting of repeating units (0-Antigen/O-side chain). Typically the Lipid A consists of a bisphosphorylated glucosamine disaccharide which is substituted by amide- and ester-bound fatty acids and / or acyloxyacyl groups. The core region, a non-repetitive oligosaccharide, is usually connected to the Lipid A part via one 3-deoxy-D-manno-oct-2-ulosonic (Kdo) residue. The core is attached in turn to the 0- Antigen that consists in most cases of a repetitive polysaccharide and that represents the major part of LPS. The bond between the Lipid A section and the Kdo residue of the core is labile under mild acid hydroysis conditions; and this allows for the fractionation of the LPS molecule into a Lipid A part and an 0-Antigen part, attached to the core. Thus far the eliciting (active) parts of LPSB. cep. have not yet been identified. In general, it is known that the Lipid A is more conserved from one organism to another as compared to the 0-Antigen. In animals, Lipid A is believed to be the active part as it was found to elicit some defense-related responses. In plants, Lipid A was also found to trigger defense responses. Several structures of the 0-Antigens from different bacteria have been characterised, but their biological activities have not yet been investigated in detail.

South Africa is at present encountering one of the worst Tuberculosis (TB) epidemics in the world, accentuating the need for intervention to eradicate TB. Various studies have established that certain population groups are at risk for increased susceptibility to infection with Mycobacterium tuberculosis (M. tuberculosis). This predominantly occurs in populations, like the native African population groups, who were not exposed to TB until the disease arrived in their country with European settlers, colonialists and missionaries. These population groups consequently lack the natural resistance to infection, which other populations developed through years of exposure to the pathogen. Several susceptibility-associated genetic polymorphisms have been proposed to explain differential susceptibility to TB. HLA class II molecules play a pivotal role in the activation of the host immune response against M. tuberculosis. Consequently numerous HLA class II genes have been found to be associated with TB. Among the most commonly observed associations is that of HLA-DR2 with TB, which has been observed in various population groups. Although this association has been observed to transcend ethnic barriers, inter-population variation has also been established regarding HLA-TB associations. In this study, the possible association of HLA class II polymorphisms, specifically of the HLA-DRB1, DQB1, DRB3, DRB4 and DRB5 loci, with TB susceptibility was investigated in the Venda population of South Africa. This was achieved by conducting both a case-control and family-based association study. The results obtained in this study established a unique association between HLA-DRB1*1302, DQ7 and TB susceptibility. A marginally significant association was also observed with DRB1*1301 and DQ6d and possible TB resistance. The above-mentioned results, which were observed in the case-control group, could not be replicated in the family-based study. It was therefore concluded from the results obtained in this study that employing both a case-control and family-based analysis when undertaking an association study is the most beneficial option. / Prof. Liza Bornman

The work presented in this thesis forms part of a continuing programme concerned with the structural determination of capsular polysaccharides of some Enterobacteriaceae. Since bacteria of this family are pathogenic to Man, work in this laboratory has focused on the structural elucidation of Klebsiella and, more recently, Escherichia coli ( E. coli) capsular polysaccharides ( K-antigens ). To date, some 74 K-antigens have been distinguished serologically within the genus E. coli and the structures of approximately 70% are known. In general, the K-antigens of the E. coli are characterized by a wide variety of constituent monosaccharides arranged in repeating units. In this thesis the structural elucidation of the capsular polysaccharides of E. coli serotypes 08: KI02: H- and 08: K47: H2 is presented. A variety of chemical techniques has been employed in the structural analysis, and are discussed. The thesis also includes extensive two-dimensional n.m.r. studies on the E. coli KI02 and K47 polysaccharides, as well as on a modified KI02 polymer produced after a lithium-ethylenediamine degradation of the native polysaccharide.

Previous studies with conjugates containing the NH2-terminal and COOH-terminal antigenic determinants of oxidized ferredoxin from C. pasteurianum indicated a need for at least two determinants to stimulate DNA synthesis in sensitized lymphocytes. This suggested a mechanism involving cell cooperation, a possibility which has been investigated here by selectively inactivating cells binding one or the other of the determinants. Cells from immunized guinea pigs were tested in vitro for their capacity to bind antigen or to be stimulated by it before and after "antigen suicide" with radioiodinated conjugates containing the NH2-terminal or COOH-terminal determinants of oxidized ferredoxin. A microculture system for assessing antigen induced stimulation of 3H-thymidine uptake by lymphocytes was developed for this work. The data show that: 1) Lymphocytes from unimmunized guinea pigs bind both NH2-terminal and COOH-terminal determinants at a frequency of about 10-4. In immune animals the proportion of antigen binding cells increased about 4-6 fold. The frequency of cells binding the determinants depends markedly on the specific activity of antigens employed. 2) Both T and B lymphocytes bind the antigenic determinants from oxidized ferredoxin. 3) Specific inactivation of cells binding either determinant was achieved by antigen suicide with ¹²⁵I-NH₂-terminal or ¹²⁵I COOH-terminal s-BSA conjugates. Synergy occurs between the NH2-terminal binding cells and COOH-terminal binding cells in the proliferative response of sensitized lymph node cells challenged with oxidized ferredoxin in vitro. Evidence from B cell depletion studies indicates that this is a T cell-T cell interaction. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

Evidências epidemiológica e imunológica sugerem que o desenvolvimento da imunidade natural contra doença meningocócica pode está associado com a reação cruzada de antígenos em comuns com Neisseria meningitidis e outras bactérias comensais, como Neisseria lactamica. O Objetivo deste trabalho foi de investigar a imunogenicidade de antígenos de vesículas de membrana externa (OMV) de N. lactamica, com ou sem a presença de Bordetella pertussis (BP), utilizada como adjuvante. Grupos de camundongos neonatos da linhagem BALB/c foram imunizados com antígenos de N. lactamica. Os resultados de nossos estudos mostraram o predomínio de altos títulos de anticorpos dos isótipos IgG e IgM com alta e intermediária avidez, depois das imunizações pela via (i.n) com N. lactamica. A análise do soro por immunoblot mostrou proteínas com reatividade cruzada entre as espécies do gênero Neisseria e os anticorpos monoclonais utilizados neste trabalho. Estes resultados sugerem que antígenos de N. lactamica e N. meningititdis em comum, possam ser importantes na imunidade natural contra doença meningocócica, e no desenvolvimento de vacina. / Immunological and epidemiological evidences suggest that the development of natural immunity to meningogoccal disease may be associated with crossreactive antigens together with Neisseria meningitidis and other commensal bacteria, like Neisseria lactamica. The present study aimed to investigate the immunogenicity of antigens of outer membrane vesicles (OMV) of N. lactamica with or without the presence of Bordetella pertussis (BP) used as an adjuvant. Groups of neonate BALB/c mice were immunized intranasally antigens of N. lactamica. The results of our studies showed the predominance of high titers of antibodies of IgG and IgM isotipes with high and intermediate avidity after intranasal immunization with N. lactamica. Immunoblot analysis of serum showed cross-reactivity proteins between the species of the gender Neisseria and the monoclonal antibodies used in this study. These results suggest that antigens of N. lactamica and N. meningitidis in common may be important in natural immunity against meningogoccal disease and in the development of vaccine.

The transformation from normal to malignant phenotype in human cancers is associated with aberrant cell-surface glycosylation. Mucin 1 (MUC1), the heavily glycosylated cell-surface mucin, is altered in both, expression and glycosylation pattern in many cancers. The presence of truncated glycan structures, often capped by sialic acid, commonly known as tumor-associated carbohydrate antigens (TACAs), play key roles in tumor initiations, progression, and metastasis. Accumulating evidence suggests that expression of TACAs is associated with escape of immune defenses. Human macrophage galactose-type lectin (hMGL, HML, CD301 or CLEC10A), a C-type lectin expressed by antigen presenting cells (APC), is a receptor of mucin-type TACAs, -GalNAc (Thomsen nouvelle antigen; Tn; CD175) and its 2,6-sialylated derivative (sTn; CD175s). To date, the relative contributions of these glycans, as well as underlying peptide backbone, and different degrees of valency, on binding thermodynamics and kinetics with hMGL remains elusive. In order to discern the subtle utility of these distinct features, chemical syntheses of the MUC1, HGVTSAPDTRPAPGSTAPPA tandem repeat sequence, and its site-specific serine (Ser) and threonine (Thr) glycosylated analogs were carried out. Circular dichroism (CD) spectroscopy experiments detected increasing structural order of the Thr glycopeptides compared to its nonglycosylated analogs. Isothermal titration calorimetry (ITC) data analysis of lectin binding to the Thr glycopeptides invariably showed enthalpy-driven processes. Affinity enhancement of the Thr glycopeptides for hMGL occurred relative to free GalNAc, revealing an increasing trend in affinity by one order of magnitude, for mono- (KD = 6-8 μM) to triglycosylated (KD = 600 nM) MUC1 peptides. To delineate the relevance of the solvent structure in the protein carbohydrate recognition process, experiments in D2O were performed, exposing enthalpy-entropy compensation differences. KinITC analysis highlighted prolonged complex lifetimes. Furthermore, atomic force microscopy (AFM) based dynamic single-molecule force spectroscopy (SMFS) provided molecular level insight into the energy landscapes governing recognition of the MUC1(Tn)-hMGL complexes. In summary, our results suggest that contact with hMGL critically depends on the type of TACA, nature of the vicinity surrounding the glycan, and its density. This highlights the importance and current efforts in design of prophylactic and therapeutic cancer vaccines with special emphasis on the synthetic glycopeptide vaccines. / Includes bibliography. / Dissertation (Ph.D.)--Florida Atlantic University, 2021. / FAU Electronic Theses and Dissertations Collection

Cancer is the second leading cause of death in the United States. The risk of cancer development and subsequent death from the disease increases sharply in the population over 55 years of age(1). Coagulation problems also increase with age and have been implicated in increasing the metastatic spread of cancer. Although cancer treatment has improved constantly it still remains quite toxic and improvement are needed Mouse mammary tumor is a common form of cancer used for experimental animal study. This investigation was designed to study the nature of leukocytic response to mouse mammary tumor implants in C3H/HeJ mice with and without anticoagulants. The main thrust of the research was in three areas: first, to develop a background in light microscope morphology of spontaneous mouse mammary tumor and its change with first and second passage into normal mice; second, to analyze change in leukocytic response in sham operated, tumor- and liver-implanted mice; and third, to analyze changes in leukocytic response to tumor implants with and without anticoagulation.

Human platelets respond to various macromolecules in the plasma. Uptake of specific ligands, and antibodies to various epitopes on the platelet plasma membrane, has been observed. The platelet canalicular system has been shown to be involved with this uptake. Recently, investigators have speculated on the role of endocytosis in platelets to account for the presence of plasma proteins such as fibrinogen and immunoglobulin within platelet organelles. Antibodies binding to cell-surface antigens on platelets can lead to a redistribution of these antigens. When antibodies, specific for platelet cell-surface receptors, bind to platelets they may either undergo endocytosis into intracellular vacuoles, or may merely become sequestered within the canalicular system of platelets. The present study investigated whether endocytosis occurs in platelets. Such a process would lead to the endocytic uptake of a fluid-phase marker and would involve internalization and recycling of cell surface membrane. A fluid-phase marker (FITC-dextran) was used to measure any constitutive endocytic activity. In addition, a suitable membrane marker was used to determine whether membrane internalization occurred. This involved a technique whereby radioactive galactose was covalently attached to cell-surface glycoconjugates. A monoclonal antibody to the platelet receptor, GPIIbIIIa, was used in conjunction with the membrane marker in order to determine if membrane internalization was involved during the subsequent redistribution of the receptor-antibody complex. Immunocytochemical techniques using electron-dense probes were employed to localise the sites to which this receptor-antibody complex became redistributed. In comparison with reported rates of endocytic uptake of fluid-phase marker in other cell types, no significant endocytic activity could be detected with platelets, after taking their relatively small volume into account. Similarly, membrane internalization was not detected with resting platelets. Following challenge of the platelets with anti-GPIIbIIIa antibody, no membrane internalization could be measured during redistribution of the receptor-antibody complex. The compartment to which the receptor-antibody complex was redistributed could be identified morphologically as the canalicular system. The present data provide evidence for a process of sequestration of receptor-antibody in the canalicular system of resting platelets. It remains possible that other mechanisms exist within the platelet system for uptake of extracellular material as this study dealt exclusively with the platelet response to a specific antibody. These results may have implications with respect to the interaction of platelets with anti-platelet antibodies in the normal state, as well as with clinical disorders involving elevated levels of platelet-associated IgG. As far as can be deduced from the available literature, these data represent the first use of a covalent membrane marker in conjunction with uptake of macromolecules to study endocytic events in human platelets.

The purpose of these investigations was to isolate and purify the largest of several antigens demonstrable in rabies-infected suckling mouse brains. The biochemical and biophysical properties of the antigen were studied with a view to elucidating its contribution to the intracellular synthesis and the structure of the virus particles. Extracts of normal and infected suckling mouse brains were purified by precipitation at pH 4.5 and freed of the smaller antigens by centrifugation prior to digestion with RNAase, DNAase and trypsin. The large antigen was purified by enzyme treatment, preparative ultracentrifugation, exclusion chroma tography and gradient centrifugation and appeared as rings varying in diameter between 8 and 12 mμ when examined by electron microscopy. Methods for the chemical estimation of pentose, deoxypentose and nitrogen were modified to meet the requirements of this investigation, and these techniques were used to determine the composition of the purified antigen. The antigen is a ribonucleoprotein, and found to be resistant to RNAase, DNAase, trypsin and chymotrypsin. A purified solution of the antigen contained 7.3μg RNA/ml., 11.4μg protein/ml and probably a trace of DNA. The success of this programme has resulted in the accumulation of certain original information which has been used in clari£ying the nature and structure of the largest soluble antigen.

No description available.