Mastitis is the most prevalent and major cause of economic losses in dairy farms. Bovine mastitis caused by strains of <i>S. aureus</i> is a major economically important disease affecting the dairy industry worldwide. <i>S. aureus</i> is one of the most common udder pathogens that cause either clinical or sub-clinical mammary gland infections. Different treatment regimes have failed to cure <i>S. aureus</i> intramammary infections. Most mastitis vaccination strategies have focused on the enhancement of systemic humoral immunity rather than strengthening local intramammary immunity. Vaccines aimed at enhancing intramammary immunity of dairy cows against <i>S. aureus</i> mastitis have had limited success. Commercially available vaccines show various degrees of success and work in research laboratories with experimental vaccines suggest that in part, the failure of these vaccines lies in the limited antigenic repertoire contained in the vaccine formulations. Moreover, not only does variation in the antigenic composition but also presence of capsular polysaccharide in most pathogenic strains and decreased activity of immune effectors in milk affect the success of vaccines. In addition to these, the ability of <i>S. aureus</i> to attach and internalize into mammary epithelial cells, enables bacteria to escape from the effect of immunity and antibiotics by being hidden in the intracellular niche and thereby causing chronic recurrent intramammary infection. <i>S. aureus</i> also has the ability to become electron-transport-defective and to form slow-growing small colonies that are non haemolytic and less virulent. These small colony variants might hide from the immune surveillance in the intracellular area and revert to the parental strain causing chronic recurrent infections. If immunization targets antigenic molecules that are conserved throughout all pathogenic strains, even the small colony variants can be controlled since the immune system will clear the parental strain which causes lethal infection. Thus, immunization trials should focus on conserved immunogenic antigen molecules among pathogenic strains formulated with an adjuvant and delivered by a route of immunization to induce maximum stimulation of the immune system. Moreover, immunization should focus on inducing Th1 responses, which is protective against <i>S. aureus</i> mastitis. It has been reported that proteins with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity might be used as such antigens to induce protection against parasitic and microbial infections. Previous study in our laboratory on mastitis-causing streptococci indicates that GapC proteins of <i>S. uberis</i> and <i>S. dysgalactiae</i> have potential as vaccine antigens to protect dairy cows against mastitis caused by environmental streptococci. Two conserved cell wall associated proteins with iii glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity, GapB and GapC have been identified from <i>S. aureus</i> isolates from bovine intramammary infections. The overall goal of this study was to improve our understanding on intramammary immunity using the GapC and GapB proteins of <i>S. aureus</i> as model antigens for mastitis and to determine the regulation of expression of <i>gapB</i> and <i>gapC</i> genes and their roles in the pathogenesis of bovine <i>S. aureus</i> mastitis. We hypothesized that strengthening local intramammary immunity using GapB and GapC proteins of <i>S. aureus</i> as antigens will protect against bovine <i>S. aureus</i> mastitis. To test this hypothesis we took the approach of using the <i>gapB</i> and <i>gapC</i> genes and constructed plasmids encoding GapB, GapC and GapB::GapC (GapC/B) chimeric proteins. We set six objectives to test our hypothesis using these proteins to enhance the intramammary immunity. In aim 1 we constructed plasmids encoding the GapB, GapC proteins and also constructed a chimeric gene encoding the GapC and GapB proteins as a single entity (GapC/B chimera) as the basis for a multivalent vaccine. In this objective the humoral and cellular immune responses to GapC/B were compared to the responses to the individual proteins alone or in combination in C57 BL/6 mice. Our results showed that the GapC/B protein elicited strong humoral and cellular immune responses as judged by the levels of total IgG, IgG1, IgG2a, IL-4 and IFN-ã secretion and lymphocyte proliferation. These results strongly suggest the potential of this chimeric protein as a target for vaccine production to control mastitis caused by <i>S. aureus</i>. In aim 2 we continued our studies on GapC/B by testing the effects of DNA vaccination with plasmids encoding the individual gapB and gapC genes as well as the gapC/B protein gene with or without a boost with the recombinant proteins. The results showed that DNA vaccination alone was unable to elicit a significant humoral response and barely able to elicit a detectable cell-mediated response to the recombinant antigens but subsequent immunization with the proteins elicited an excellent response. In addition, we found that DNA vaccination using a plasmid encoding the GapC/B chimera followed by a boost with the same protein, although successful, is less effective than priming with plasmids encoding GapB or GapC followed by a boost with the individual antigens. In aim 3 we optimized immune responses in cows by comparing route of vaccination (subcutaneous versus intradermal), site of vaccination (locally at the area drained by the supramammary lymph node versus distantly at area drained by parotid lymph node. Our results showed that both subcutaneous and intradermal immunizations with the GapC/B protein at the area drained by the supramammary and parotid lymph nodes resulted in significantly increased serum and milk titers of total IgG, IgG1, IgG2, iv and IgA in all vaccinated groups as compared to placebo. The anti-GapC/B IgG1 serum and milk titers were significantly higher in all vaccinated group as compared to the placebo group. These results indicated that vaccination at the area drained by the supramammary lymph node resulted in better immune responses. In aim 4 we tested different formulations of the GapC/B antigen with adjuvants such as PCPP, CpG, PCPP + CpG and VSA-3. We found that the VSA-3 formulation induced the best immune responses in cows. In this objective we also monitored immune responses longitudinally over one lactation cycle to determine the duration of immune responses by measuring IgG, IgG1, IgG2, and IgA on monthly blood and milk samples. We found that the duration of immune responses was about four months. In aim 5 we tested the role of GapC in the virulence of <i>S. aureus</i> mastitis using the <i>S. aureus</i> wild type strain RN6390 and its isogenic GapC mutant strain H330. Our results from both in vitro adhesion and invasion assays on MAC- T cells and in vivo infection of ovine mammary glands showed that GapC is an important virulence factor in <i>S. aureus</i> mastitis. In aim 6 we examined the role of sar and agr loci on the expression of <i>gapC</i> and <i>gapB</i> genes by qRT- PCR using <i>S. aureus</i> RN6390 and its isogenic mutants defective in agrA, sarA and sar/agr (double mutant) at exponential and stationary phases of growth. Our results showed that both <i>gapB</i> and <i>gapC</i> expression were down regulated in the mutant strains, indicating that the expression of the <i>gapB</i> and <i>gapC</i> genes is controlled by the universal virulence gene regulators, agr and sar. We also checked the role of environmental factors such as pH, growth media, and oxygen tension on the expression of <i>gapB</i> and <i>gapC</i> using q-RT-PCR. Our results showed that the expression of <i>gapB</i> and <i>gapC</i> genes in different strains of <i>S. aureus</i> was not consistent under the above-mentioned environmental conditions.

The transport of phosphate (Pi) between subcellular compartments is central to metabolic regulation. Although some of the transporters involved in controlling the intracellular distribution of Pi have been identified in plants, others are predicted from genetic and biochemical studies. The Arabidopsis thaliana genome encodes a family of six proteins that share similarity with SLC17/type I Pi transporters, a diverse group of animal proteins involved in the transport of Pi, organic anions and chloride. Heterologous expression in yeast, and gene expression and localization studies in plants were used to characterize all six members of this Arabidopsis family, which we have named PHT4. All of the PHT4 proteins mediate Pi transport in yeast with high specificity. Bioinformatic analysis and localization of PHT4-GFP fusion proteins indicate that five of the proteins are targeted to the plastid inner envelope membrane, and the sixth resides in the Golgi apparatus. PHT4 genes are expressed in both roots and leaves although two of the genes are expressed predominantly in leaves and one mostly in roots. These expression patterns, together with Pi transport activities and subcellular locations, suggest roles for PHT4 proteins in the transport of Pi between the cytosol and chloroplasts, heterotrophic plastids and the Golgi apparatus.

Contaminants in aqueous environments exist in phases that are sorbed to suspended or colloidal material and that are dissolved in solution. Polymer flocculants can be used to remove suspended or colloidal material along with sorbed contaminants, but they remove little of the dissolved contaminants. In the study presented here, development of polymers to sorb contaminants from aqueous solution during the flocculation process was investigated. Atrazine and phosphate (H₂PO₄ ̄) were chosen as test contaminants. For a given test contaminant, multiple copies of a functional group that interacted with that contaminant were inserted into the polymer backbone of a polyacrylamide flocculant. The functional groups inserted into the polymer structure acted as a trap for the dissolved contaminant. The traps were a cyclic secondary amine that interacted with atrazine, and a thiourea that interacted with phosphate. Modified flocculants with different configurations and densities of trapping groups were made and evaluated for removal of the test contaminants from aqueous suspensions. The suspensions consisted of bentonite or kaolinite in water with a known concentration of a test contaminant. The atrazine source was labeled with ¹⁴C and concentrations were measured using a scintillation counter. The source of phosphate used was NaH₂PO₄ and ion chromatography was used to measure the aqueous concentrations of phosphate. In general, the modified polymer flocculants containing trapping groups removed significantly more atrazine and phosphate from suspension compared to the control polymer flocculants ([alpha] =0.05). While the amount of modified polymers needed to achieve significant removal of the test contaminant were higher than the Environmental Protection Agency limit for concentration of polyacrylamide flocculants in water, it was possible to enhance the polymers sorbtion and removal of contaminants from solution during the flocculation process.

Zugl.: Giessen, Universiẗat, Diss., 2009.

Reproduction de : Thèse de doctorat : cMicrobiologie et biocatalyse industrielles : Toulouse, INPT : 2007. / Titre provenant de l'écran-titre. Bibliogr. 236 réf.

Thèse de doctorat : Enzymologie Moléculaire et Biologie Structurale : Nancy 1 : 2008. / Titre provenant de l'écran-titre.

Phosphorus (P) has long been acknowledged as a vital nutrient for living organisms and is a key factor responsible for the fresh water eutrophication. Our understanding of the phosphorus cycle has been limited by: (1) the common assumption that all P in the environment occurs primarily as phosphates and (2) by the limited analytical methods available to identify P speciation. In an attempt to understand the distribution and chemistry of phosphorus within a freshwater system we must be able to identify individual P species. To this end, we used a coupled High Performance Liquid Chromatograph (HPLC) - Inductively Coupled Plasma Mass Spectrometer (ICPMS) to determine concentrations of orthophosphate (+5), phosphite (+3) and hypophosphite (+1) in aqueous samples using methods modified from IC techniques developed by Ivey & Foster (2005) and Pech, et al. (2009) and Atlas et al. (in prep). The identification of different P species provides insight pertaining to contamination, bioavailability and sustainability within a freshwater system. Thirty-two individual water samples were collected from six different bodies of freshwater in the Tampa Bay area between the months of November 2012 to March 2013. The freshwater samples collected were from river and pond/swamp water locations. Two sampling sites were chosen at each location. At each site, one sample was collected from the water's surface and a second sample was collected from the sediment pore water. When depth was sufficient a third sample was obtained from the midpoint between the surface and sediment. Analytical results show that redox reactions of P occur in all freshwater samples collected as identified by HPLC-ICP-MS analysis. Our data show that the distribution and concentration of reduced P is controlled primarily by pH, and secondarily by water circulation, ORP and sediment type. Our results also imply biologic influence as a potential primary control of reduced P flux. Additional samples must be collected in order to quantify and differentiate the processes controlling P speciation. The ability to identify P speciation raises many questions concerning the validity of current methods used to measure P; other forms of reduced P may be present. Additional sample analysis will be necessary to determine how and if reduced forms of P affect the P cycle.

published_or_final_version / Dentistry / Master / Master of Dental Surgery

published_or_final_version / abstract / toc / Dentistry / Doctoral / Doctor of Philosophy

published_or_final_version / Biochemistry / Master / Master of Philosophy