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.

In this study we attempt to use phytoremediation techniques to treat the contaminated soil of cadmium. The experiment is divided into two stages. In the first stage, we selected three different species of plants which could tolerate heavy-metals: vetiver (Vetiveria zizanioides), Pteris ensiformis cv. 'Victoriae' according to the past records, and Alternanthera philoxeroides (Mart.) Griseb, which were sampled from the metal contaminated site in Hunei, Kaohsiung county. These three species were planted in three pots with 10, 20 and 30 mg Cd kg-1 in soil respectively. After 9 weeks of the growth, the vetiver was found accumulating the highest Cd and grew better than the other two species. Therefore, we selected the species of vetiver in the second stage of experiment. First, the species of vetiver was planted in the pots with concentrations of 30 and 50 mg Cd kg-1 in soil respectively. Then the pots were put in the greenhouse for incubation. After the test was run for 210 days, we found that the species of vetiver was helpful in the increasing the number of species and amounts of each species of microbe ( total bacteria, fungi and actinomycete ), as well as dehydrogenase activity. Meanwhile, it was effective to decrease the bioavailability of cadmium. In addition, the infection rate of mycorrhizal fungi was increased , which showed that the species of vetiver could resist the cadmium stress in soils and stimulate the soil fertility. Finally, we use molecular biotechniques of PCR-DGGE to observe the microbial diversity in the contaminated soil. We found that the pots with 30 mg Cd kg-1 in soil had more number of bands than the pots with different Cd concentrations in soil, while the pots without vegetation was found more fruitful than vegetated pots. These experimental results indicated that the pots planted with the species of vetiver under this situation would help some special microorganisms to grow, and thus that the microbial diversity was reduced. The results also showed that the pots planted with vetiver with initial cadmium concentrations of 30 and 50 mg Cd kg-1 respectively, in soil exhibited the degradation rate of about 30 percent for both. It was not satisfied to this result in this study. However, the phytoextraction rates of cadmium were measured equal to 7.8 and 8.9 percent, respectively. According to these results, we suggested that the plant, which could hyperaccumulate heavy metals, might be used to increase the removable ability of cadmium in the future.

Thesis (Ph. D.)--University of Virginia, 2000. / Spine title: Initiation of DNA replication. Department of Biochemistry and Molecular Genetics. Includes bibliographical references (142-171). Also available online through Digital Dissertations.

published_or_final_version / Medicine / Master / Master of Philosophy

Despite recognition of aldehyde dehydrogenase (Aldh) as a surrogate marker in isolating primitive hematopoietic stem and progenitor cells (HSPC) [1], its role in HSPC regulation, particularly during embryonic development, remains unclear. In this study, we examined the role of Aldh during embryonic hematopoiesis in zebrafish, which has emerged as a model for hematopoietic studies. [2] Wild--?type and transgenic [Tg(gata1:gfp),Tg(fli1:gfp)] zebrafish embryos were microinjected with anti--?sense morpholinos (MO) at 1--?cell to 4--?cell stage and evaluated by morphology, flow cytometry, in situ hybridization (ISH) and Q-RT-PCR. In addition, human CD34+ cells, which were enriched with hematopoietic stem cells (HSC), were isolated from umbilical cord blood samples for analysis of ALDH16A1 expression. It was subsequently compared with CD34- cells which were devoid of HSC activity. When aldh16a1 was knocked down by anti-sense morpholino (the embryos were referred herewith aldh16a1MO embryos), gene expression associated with erythropoiesis was significantly reduced at 18hpf .(gata1:0.70±0.03fold; p=0.002) (α-embryonic hemoglobin: 0.48±0.04fold; p=0.003) (β-embryonic hemoglobin: 0.56±0.03fold; p=0.001). Angiogenesis was also perturbed at 48 and 72hpf. Furthermore, human ALDH16A1 was significantly upregulated (4.79±1.00fold; p=0.00006) in CD34+ (enriched with HSC) as compared to CD34- (devoid of HSC) populations in umbilical cord blood. Aldh16a1 is important for the maintenance of primitive hematopoiesis at early (18hpf) and angiogenesis at later (48,72 hpf) embryonic stages. As angiogenesis plays an important role in pathophysiology of malignancies, novel therapy against ALDH16A1 might be exploited in therapeutic intervention in cancer treatment. Moreover, a specific role of zebrafish aldh16a1 in primitive erythropoiesis and a higher level of ALDH16A1 expression in human HSC-enriched cells suggested a conserved mechanism whereby ALDH regulates hematopoiesis. / published_or_final_version / Medicine / Master / Master of Research in Medicine

The abundance of tumour initiating cells (TIC) has been suggested to be an important prognostic indicator in cancers. Both SOX2 and ALDH have been individually reported to be putative TIC markers but their combined status is unclear and their usefulness in the prognostication of non-small cell lung cancer (NSCLC)has not been reported. This study investigated the patterns of ALDH and SOX2 protein expression in NSCLC using immunohistochemistry. Expression was graded using semi-automated signal capturing and image analysis software. ALDH and SOX2 were expressed in 41% and 43% of all NSCLC, respectively. ALDH was expressed in 36% of adenocarcinomas (AD)and 65% of squamous cell carcinomas (SCC), while SOX2 was expressed in 36% of AD and 80% of SCC., respectively. Taking all cases into consideration, the expression of ALDH and SOX2 significantly correlated with each other (p=0.003). No prognostic value of the abundance of ALDH and SOX2-expressing cancer cells was found with regard to all NSCLC or in AD. In contrast, for SCC, a significantly better prognosis with longer cancer-specific survival (CSS) and disease-free survival was found in tumours with higher ALDH expression, while a longer CSS was found in those with higher SOX2 expression. Contrary to the hypothesis that a high TIC content indicated by high combined ALDH and SOX2 expression would predict poor patient outcome, amongst all NSCLC, the combined phenotype of SOX2+/ALDH-was associated with the worst prognosis compared with the SOX2+/ALDH+(p=0.026) and SOX-/ALDH-(p=0.048),while no significant difference was observed with the SOX-/ALDH+ phenotypes. In view of the tight correlation between ALDH and SOX2 protein levels, in vitro studies were performed to investigate whether ALDH could be an upstream regulator of SOX2 expression. Pharmacological inhibition of ALDH enzyme function led to down-regulation of SOX2 mRNA and nuclear protein expression in lung cancer cell lines, indicating a regulatory role of ALDH on the SOX2 stemness pathway in lung cancer. In summary, the findings implicate complex factors are likely to be involved in determining the expression levels of ALDH and SOX2 in clinical lung cancers and their mechanisms affecting patient survival remain to be clarified. Further investigations on the specificity of ALDH/SOX2 as TIC marker, TIC interaction with the tumour micro-environment, and potential complex antagonistic functions of ALDH in TIC maintenance are required. / published_or_final_version / Pathology / Master / Master of Medical Sciences

published_or_final_version / Biochemistry / Master / Master of Philosophy

published_or_final_version / Biochemistry / Master / Master of Philosophy

published_or_final_version / Zoology / Doctoral / Doctor of Philosophy

Thermodynamic and kinetic experiments have been performed at ionic strength 0.30 to elucidate the relationship between the structure of pig heart H₄-LDH (lactate dehydrogenase) and its catalytic function. Calorimetry and fluorescence were used to determine all the thermodynamic parameters for binary and ternary complex formation. TFL (trifluorolactate) and oxamate were employed as nonreactive analogs of the substrates lactate and pyruvate, respectively, to examine ternary complex formation in the absence of the ensuing redox step. At pH 6 where there is no apparent change in the protonation state of LDH upon binary complex formation, LDH binds NADH more tightly than NAD due to an entropy effect, i.e., only 1.1 out of the 3.1 kcal/mole difference in free energy changes is enthalpic in origin. The heat capacities of LDH·NAD (-150 ± 30 cal/K-mole) and LDH·NADH (-220 ± 40 cal/K-mole) formation at pH 6 and 25°C are relatively small and similar. These results suggest the importance of charge interactions in coenzyme binding. Structural information indicates that Arg-106, a positively charged residue of a loop of polypeptide in LDH which at equilibrium alternates between two conformations, open (extended into solvent) and closed (part of the active site), interacts unfavorably with the positively charged nicotinamide ring of NAD when the loop is in the closed conformation. Thermodynamic experiments demonstrate the suitability of TFL as an analog of lactate. TFL displays the correct specificity by binding to LDH·NAD more tightly (Kₐ = 400 M⁻¹) than to LDH·NADH (Kₐ = 34 M⁻¹) at pH 8 and 25°C. This binding requires that an enzymic residue with a pK = 6.7 not be protonated in accordance with the role of His-193 in analog binding in crystalline ternary complexes. Since the free energy change of the redox step is small, the difference in the free energy changes of formation of LDH·NAD·TFL and LDH·NADH·oxamate from LDH+NAD+TFL and LDH+NADH+oxamate, respectively, should approximate the free energy change of the actual enzymic reaction. The free energy and enthalpy changes of this model reaction are within 10% of the values of the actual reaction. Steady-state kinetic experiments further support the use of TFL as an analog of lactate. At pH 8 and 25°C TFL acts mainly as competitive inhibitor of lactate during lactate oxidation. The difference between the TFL dissociation constant (2.5 mM) and its inhibition constant (8.0 mM) means that TFL is not a simple dead-end inhibitor, i.e., LDH·NAD·TFL must be connected to the productive pathway of the reaction at more than one point. This is consistent with the existence of two conformational states of LDH·NAD. Additional support for the existence of two conformational states of LDH comes from analysis of the heat capacity changes of ternary complex formation. The large negative heat capacity changes at 25°C of TFL binding to LDH·NAD at pH 8 (-150 cal/K-mole) and of oxamate binding to LDH·NADH at pH 8 (-330 cal/K-mole) and pH 6 (-420 cal/K-mole) are partly attributed to a reaction heat effect arising from a shift in the conformational equilibrium of LDH to one in which the loop is in the closed position. As shown by calorimetry and fluorescence, phosphate binds to a single class of sites of LDH. The thermodynamic parameters of this process at pH 6 and 25°C are ΔG = -30 kcal/mole, ΔH = -5.1 kcal/mole, and ΔS = -7.0 cal/K-mole. Binding is not at the active site.