This thesis discusses the structure-function studies on triosephosphate isomerase (TIM) from Plasmodium falciparum (Pf), directed towards understanding the roles of highly conserved residues by site derected mutagenesis. Chapter 1 provides an introductory overview to the relevant literature on triosephosphate isomerase. In addition, this Chapter provides an analysis of conserved residues in TIM, and amino acid diversity at specific positions in the structure using a dataset of 503 TIM sequences. Chapter 2 reports the work on the completely conserved residue, C126 in TIM, which is proximal to the active site. Five mutants, C126S, C126A, C126V, C126M and C126T have been characterized. Crystal structures of 3-phosphoglycolate (PGA) bound C126S mutant and the unliganded forms of the C126S and C126A mutants have been determined at a resolution of 1.7 Å to 2.1 Å. Kinetic studies reveal a ~5 fold drop in kcat for the C126S and C126A mutants, while a ~ 10 fold drop is observed for the other three mutants. All the mutants show reduced stability at lower concentration and higher temperature. Chapter 3 presents the kinetic and structural characterization for the E97Q and E97D mutants of Pf TIM. A 4000 fold reduction in kcat is observed for E97Q, 100 fold reduction for the E97D mutant, while a ~ 9000 fold drop in activity for the control mutant, E165A. A large conformational change for the critical K12 side chain is observed in the crystal structure of the E97Q mutant, while it remains unchanged in the E97D structure. The results are interpreted to invoke a direct role for E97 in the catalytic proton transfer cycle, eliminating the need to invoke the formation of the energetically unfavorable imidazolate anion at H95. Chapter 4 reports investigations with position 96 by the biochemical and structural characterization of single mutants, F96Y, F96A and the double mutants, F96S/S73A and F96S/L167V. F96Y showed ~100 fold drop in activity, F96A revealed ~10 fold drop in activity, while F96S/S73A showed 100 fold lower activity than that of the wild type enzyme. Interestingly, the double mutant F96S/L167V proved to be a partial pseudorevertant, showing 10 fold higher activity than the single mutant, F96S. Chapter 5 describes the cloning, and preliminary kinetic and biophysical characterization of the enzyme, Dm TIM. A survey of disease causing mutations in TIM and the relationship of these sites of mutation to the active site and the dimer interface of TIM is presented in this Chapter.

Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / The development of polymers with the ability to transport and release drugs and bioactives has been growing rapidly because of their unique properties in protecting and enhancing solubilization of drugs in physiological media. In this work, polyesters derived from β-cyclodextrin were developed in two different systems: The former consisted of cyclodextrin cross-linked with citric acid and subsequently functionalized with glutamic acid. The latter was obtained from β-cyclodextrin esterified with gluthamic acid. The polyesters were characterized by Fourier Transform Infrared Spectroscopy (FTIR), Solid-state Cross-Polarization Magic Angle Spinning Carbon-13 Nuclear Magnetic Resonance (CP/MAS 13C–NMR) and Thermogravimetric Analysis (TGA) for the confirmation of the formation of polymers and the esterification of glutamic acid with β-cyclodextrin molecules. The formation of inclusion complex of the polymers in solution with orange methyl was evaluated to verify the activation of β-cyclodextrin cavities. The polymers were used as reducer and stabilizer in the synthesis of silver nanoparticles (AgNPs). The characterization of the AgNPs was performed by UV-Vis spectroscopy, and bands between 350-500 nm evidenced of the obtainment of silver nanoparticles. Analyses by transmission electron microscopy revealed AgNPs with spherical morphology and diameter around ~ 5 to ~60 nm, corroborating with the results of UV-Vis. / O desenvolvimento de polímero com a capacidade de transportar e liberar fármacos e compostos bioativos vem crescendo rapidamente, devido suas propriedades únicas em proteger e aumentar a solubilização em meios fisiológicos. Neste trabalho, foram desenvolvidos poliésteres derivado de β-ciclodextrina em dois sistemas distintos. O primeiro sistema foi obtido a partir de β-ciclodextrina reticulado com ácido cítrico e posteriormente funcionalizado com ácido glutâmico. O segundo foi a partir de β-ciclodextrina esterificada com ácido glutâmico. Os poliésteres foram caracterizados por espectroscopia na região do infravermelho com transformada de Fourier (FTIR), ressonância magnética nuclear no estado sólido sob polarização cruzada e rotação com ângulo mágico (RMN 13C CP/MAS) e análise termogravimétrica (TG), para a confirmação da formação dos polímeros e a esterificação do ácido glutâmico com as moléculas de β-ciclodextrina. A formação de complexo de inclusão dos polímeros com o alaranjado de metila foi avaliada em solução, comprovando-se a ativação das cavidades de β-ciclodextrina. Os polímeros foram testados quanto a habilidade de promover a formação/estabilização de nanopartículas de prata (AgNPs) a partir de nitrato de prata em solução. A caracterização das AgNPs foi realizada por espectroscopia de UV-Vis, e as bandas observadas entre 350-500 nm são evidências da presença de nanopartículas de prata. As imagens de microscopia eletrônica de transmissão revelaram AgNPs de morfologia esférica com diâmetro em torno de ~5 a ~60 nm, corroborando com os resultados de UV-Vis. / São Cristóvão, SE

The subject of the thesis is study of possible applications of isoform of glutamic acid polymer (-PGA). The theoretical part is focused on the properties of this biopolymer and potential applications in various areas. Producers and mechanisms of biosynthesis are also mentioned. In the experimental part, the polymer was firstly characterised by following methods: FT-IR spectroscopy, TGA, DSC and SEC-MALS. Its isoelectric point, antimicrobial activity and solubility in various solvents were also determined. The biopolymer was also precipitated by divalent cations and its interaction with oppositely charged CTAB surfactant was studied. The main experimental study was researching the effect of -PGA on viability of Saccharomyces cerevisiae and Lactobacillus rhamnosus under stress conditions by flow cytometry. The performed stresses included ethanol exposure, high temperature and freezing stress, in which its effects were compared to conventional cryoprotectants. The cells of the mentioned microorganisms were also stressed osmotically and exposed to model gastrointestinal juices - gastric, pancreatic and bile. The protective effects of -PGA on the cells were recorded in ethanol stress on Lactobacillus rhamnosus. Its excellent cryoprotection properties were confirmed and its protective effect of gastric juice exposure on Saccharomyces cerevisiae cells was also observed. At the end of the experimental part, -PGA/alginate beads suitable for encapsulation of probiotic bacteria and -PGA/chitosan nanoparticles for encapsulation of biologically active substances.

Indiana University-Purdue University Indianapolis (IUPUI) / Acute and chronic activation of both ionotropic and metabotropic glutamate (glut) receptors is implicated in many neurodegenerative disorders including AD, dementia, epilepsy, stroke and neurotrauma. TRH and glut receptors (ionotropic & metabotropic) receptors are differentially coexpressed in granule and pyramidal neurons of the hippocampus. The author shows TRH to be protective when added to cultured pituitary adenoma (GH-3) cells and neuron-like pheochromocytoma (PC12) cells either prior to, during, or after glut-induced toxicity (Endo. Soc. Abs. 01), and also shows that the possible neuroprotective mechanism may involve heterologous downregulation of the metabotropic glut receptors, using superfused hippocampal slices and noting a reduction of Gαq/11 (SFN Abs. 02). He has also demonstrated that TRH protected against glut toxicity in fetal cortical cultures (Endo. Soc. Abs. 04). To extend these studies he used 14-day cultured rat fetal hippocampal neurons (Day E17) to determine if TRH is protective against toxicity induced by specific ionotropic and metabotropic glut agonists. Neuronal viability and integrity were assessed by trypan blue exclusion and LDH release after 18 hrs following 30 min exposure to glut agonists. Ten µM dihydroxyphenylglycine (DHPG, a Group 1 receptor agonist) + 30 µM N-methyl-D-aspartate (NMDA)-induced toxicity (42% vs contr. P<0.05); whereas, concurrent and continued treatment with 10 uM but not 1uM 3Me-HTRH resulted in less neuronal death and damage (86% vs contr P<0.05; 53% vs contr. P>0.05) respectively. DHPG treatment alone (10 µM) for 30 min. was non-toxic by both criteria (90% vs contr. P<0.05). The data suggest that TRH may be a selective modulator of glut-induced toxicity.

Indiana University-Purdue University Indianapolis (IUPUI) / Introduction: During glutamate excitotoxicity, overstimulation of glutamate receptors leads to sustained elevation in cytosolic Ca2+ ([Ca2+]c), or delayed Ca2+ dysregulation (DCD), which is causally linked to cell death. There are two major hypothetical mechanisms for DCD: the continuous activation of N-methyl-D-aspartate-subtype of the ionotropic glutamate receptors (NMDAR) and the reversal of the plasmalemmal Na+/Ca2+ exchanger. However, the contribution of each of these mechanisms in DCD is not completely established. Major results: Neurons exposed to excitotoxic glutamate produced DCD, an increase in cytosolic Na+ ([Na+]c), and plasma membrane depolarization. MK801 and memantine, noncompetitive NMDAR inhibitors, added after glutamate, completely prevented DCD; however AP-5, a competitive NMDAR inhibitor, failed to do so. The NMDAR inhibitors had no effect on lowering elevated [Na+]c or on restoring plasma membrane potential, which are conditions suggesting NCXrev could be involved. In experiments inducing NCXrev, MK801 and memantine completely inhibited Ca2+ dysregulation after glutamate while AP-5 did not. Inhibition of NCXrev, either with KB-R7943 or by preventing the increase in [Na+]c, failed to avert DCD. However, NCXrev inhibition combined with NMDAR blocked by AP-5 completely prevented DCD. Overall, these data suggested that both NMDAR and NCXrev are essential for glutamate-induced DCD, and inhibition of only one mechanism is insufficient to prevent collapse of calcium homeostasis. Based on the data above, we investigated a NMDA receptor antagonist currently in clinical trials for reducing the effects of glutamate excitotoxicity, ifenprodil. Ifenprodil is an activity-dependent, NMDAR inhibitor selective for the NR2B subunit. We found that ifenprodil not only inhibited the NR2B-specific NMDAR, but also inhibited NCXrev. If ifenprodil is combined with PEAQX, a NMDAR inhibitor selective for the NR2A subunit, low concentrations of both inhibitors completely prevent DCD. Conclusion: The inhibition of a single Ca2+ influx mechanism is insufficient in preventing DCD, which requires simultaneous inhibition of both the NMDAR and NCXrev. These findings are critical for the correct interpretation of the experimental results obtained with these inhibitors and for better understanding of their neuroprotective actions.

Poly-gamma-glutamic acid (γPGA), which is a biodegradable, non-immunogenic and unusual anionic amino-acid polymer consist of D- and L-glutamic acid units, was exploited for a wide array of useful applications. Bacillus are well known cellular system important for fermentation to synthesize γPGA, which is used as thickener, drugs carrier, cryoprotectant, humectant, biological adhesive, flocculants, or heavy metal absorbent. This study focused on the isolation of Bacillus spp. that is possible to produce γ-PGA from different soil samples from different places in Vietnam. Study the effect of precursors, temperature, carbon sources, times and pH on γ-PGA production. From 31 soil samples and 4 straws samples, strain 20.2 which produced the highest γ-PGA yields (riches 15.2 mg/ml), was identified as Bacillus sp. 20.2 by molecular biology method. The suitable conditions for growing of Bacillus sp. 20.2 strain to produce γ-PGA are at 37°C, pH 7 after 72 hours. Citric acid instead of glucose in a GSP medium is better for producing γ-PGA by strain Bacillus sp. 20.2. / Poly-gamma-glutamic acid (γ-PGA) là một polymer amino-acid gồm D và L-glutamic acid, có khả năng phân hủy sinh học, không gây miễn dịch, đã được ứng dụng rộng rãi trong công nghiệp, y học. Bacillus subtilis được biết đến là hệ thống tế bào ý nghĩa quan trọng trong quá trình lên men để tổng hợp γ-PGA. γ-PGA hòa tan trong nước, phân hủy sinh học và không độc đối với con người và môi trường. γ-PGA ổn định với nhiều protease vì các protease thường không nhận acid γ- glutamic (Obst et al., 2004). γ-PGA có cấu trúc đồng phân đơn giản, không gây miễn dịch. Do đó, γ-PGA đã được quan tâm ứng dụng trong các lĩnh vực như y học, công nghiệp thực phẩm, mỹ phẩm và đặc biệt là xử lý nước nhiễm kim loại nặng. Trong nghiên cứu này chúng tôi tập trung phân lập, tuyển chọn các chủng Bacillus có khả năng sinh tổng hợp PGA cao. Sau đó định danh và đánh giá khả năng sinh tổng hợp PGA từ chủng đã phân lập được. Kết quả cho thấy từ 34 mẫu rơm và đất, chúng tôi đã phân lập được chủng với mã số 20.2 có khả năng sinh PGA cao nhất đạt 15.2 mg/ml. Chủng này đã được định danh bằng phân tích trình tự gene 16S rRNA và thuộc loài Bacillus sp. Môi trường thích hợp sinh tổng hợp PGA là GSP ở điều kiện 37oC pH7 sau 72 giờ nuôi cấy.

L'étude qu'on a entreprise a pour but d'analyser les facteurs de virulence des espèces "Classiques" et "nouvelles" de Brucella. Dans cette perspective, on a analysé les génomes des espèces récemment découvertes : Brucella inopinata BO1 et Brucella inopinata-like BO2, isolés pour la première fois de patients humains sans réservoir animal connu. On a découvert que ces deux espèces possèdent des profils de restriction uniques. De plus, BO2 possède deux chromosomes de taille identique, un profil jamais décrit pour une autre espèce de Brucella. L'analyse de la réplication intracellulaire de ces deux espèces révèle que BO2 ne se réplique pas dans les macrophages humains et murins alors que BO1 se réplique d'une façon similaire à Brucella suis 1330, ce qui confirme la potentielle implication de BO1 dans la pathogenèse chez l'homme. Sur un autre niveau d'analyse, on a été à la recherche de facteurs de virulence potentiels dans d'autres espèces de Brucella notamment Brucella microti et Brucella suis sur les niveaux génomique et post-transcriptionnel. Sur le niveau génomique, on a découvert que le système GAD (glutamate decarboxylase) confère une résistance à l'acidité à Brucella microti lors de son passage dans l'estomac. Sur le niveau post-transcriptionnel, on a isolé, séquencé et identifié les petits ARNs noncodant associés à la protéine chaperone Hfq, qui joue un rôle important dans la virulence de Brucella. / We have undertaken in this study a multidimensional analysis of the virulence factors of "Classical" and new "Brucella species". In this objective, we have analysed the genomes of newly described species Brucella inopinata BO1 and Brucella inopinata-like BO2 isolated for the first time from human patients with no known animal reservoir. We found that these two species have unique restriction profiles. In addition, BO2 has a unique chromosomal distribution with two chromosomes of the same size, never seen before in Brucella. Analysis of the intracellular replication of these strains reveals that BO2 is unable to replicate in neither human nor mouse macrophages while BO1 successfully entered and replicated as efficiently as Brucella suis 1330 confirming the potential virulence of this species for humans. On an other level of analysis, we looked for potential virulence factors in other Brucella species including Brucella microti and Brucella suis at the genomic and post-transcriptional level. At the genomic level we discovered that the glutamate decarboxylase system confers resistance to acidity to Brucella miroti during its transit in the stomach. On the post-transcriptional level, we isolated, sequenced and identified small noncoding RNAs associated to the chaperone protein Hfq, known to play a role in the virulence of Brucella.

Mycobacterium tuberculosis, the causative agent of pulmonary tuberculosis, infects one-third of the world’s human population. Despite the multiplicity of antimicrobial mechanisms mounted by its host, M. tuberculosis shows a remarkable ability to survive either by evoking survival strategies or by interference with critical macrophage functions that are required to successfully respond to the infection. It has been postulated that the outcome of exposure to M. tuberculosis (in terms of disease symptoms) largely depends upon the selective gene expression of tuberculosis bacilli along with activation of specific signaling pathways in the infected host cells during different phases of infection. In this perspective, determination of the complete genome sequence of Mycobacterium tuberculosis has provided crucial information with respect to the physiology of this bacterium and the pathogenesis of tuberculosis. However, putative functional annotation to all hypothetical proteins coded by M. tuberculosis genome remains complex. One important outcome of the genome-sequencing project was the discovery of two new multigene families designated PE and PPE. About 10% of the M. tuberculosis coding capacity is devoted to the PE and PPE genes, named for the Pro-Glu (PE) and Pro-Pro-Glu (PPE) motifs near the N terminus of their gene products. In addition to these motifs, proteins of PE family share N-terminal domains of approximately 100 amino acids, whereas the PPE proteins possess an N-terminal domain of about 180 amino acids. Many PE and PPE proteins are composed only of these N-terminal homologous domains. However, other members possess an additional C-terminal segment of variable length, often composed of multiple copies of polymorphic GC rich sequences (PGRS). The uniqueness of the PE genes is further illustrated by the fact that these genes are restricted to mycobacteria. However, despite their abundance in mycobacteria, very little is known regarding the expression or the functions of PE family genes. Although the PE and PPE families of mycobacterial proteins are the focus of intense research, no precise function has so far been unraveled for any member of these families. In perspective of above-mentioned observations, we have chosen Rv0754 as a representative PE family gene. Rv0754 was shown to be upregulated in tubercle bacilli upon infection of bone marrow derived macrophages as well as in M. tuberculosis isolated from alveolar macrophages of infected mice. In the current investigation, we demonstrate that Rv0754 is hypoxia responsive gene based on promoter or transcript expression analysis. Further, extensive bioinformatics analysis predicated that Rv0754 posses possible Phosphoglycerate Mutase domain, an enzyme known for its significant role not only in the glycolytic pathway of the carbohydrate metabolism, but also for the crucial cell fate decision during conditions like oxidative stress as well as infection. Experimental data clearly suggests that hypoxic environment dependent expression of Rv0754 imparts resistance to macrophages from oxidative stress. These findings could be attributed to the presence of catalytically active Phosphoglycerate Mutase domain of Rv0754. More often, sophisticated regulation/modulation of key signaling events regulate the critical cell fate decisions during oxidative stress. In this context, TLR2 dependent triggering of PI3K-ERK1/2- NF-κB signaling axis by Rv0754 may be operative in imparting resistance to oxidative stress. Further, Rv0754 triggers COX-2 expression by activating PI3K-ERK1/2-NF-κB cascade in mouse macrophages. These observations are of relevance as Rv0754 is associated with cell wall and is exposed outside the surface of the bacterium suggesting the possible access to intracellular compartments of the infected macrophages. Additionally, Rv0754 elicited humoral antibody reactivities in a panel of human sera or in cerebrospinal fluid samples obtained from different clinical categories of tuberculosis patients. DNA immunizations experiments in mice clearly suggested that Rv0754 is an immunodominant antigen demonstrating significant T cell and humoral reactivity. These observations clearly advocate that Rv0754 protein is expressed in vivo during active infection with M. tuberculosis and that the Rv0754 is immunogenic. Taken together, our findings suggest that Rv0754 is a novel PE_PGRS protein with unique features which could generate conditions that favor survival of the mycobacteria.

One-third of the world’s population is latently infected with Mycobacterium tuberculosis, which causes over 2 million deaths every year. The current live attenuated vaccine, Bacille Calmette-Guerin (BCG), protects against miliary tuberculosis in children, but fails to consistently protect against pulmonary tuberculosis in adults. The global resurgence of tuberculosis, together with the HIV pandemic and emerging multi-drug resistance, has heightened the need for an effective vaccine. Completion of the M. tuberculosis genome sequence paved way for identification of many new candidate antigens for protective vaccine against tuberculosis. This includes the discovery of two multigene families of proteins PE and PPE which constitute 10% of the coding capacity of the M. tuberculosis genome. Members of the PE and PPE protein families are characterized by highly conserved N-terminal domains and the C-terminus, however, exhibit considerable variation in the number of residues as well as in the sequence. Till date, little is known about the functional role of the proteins of PPE or PE family in the biology of M.tuberculosis. Some of the PE_PGRS proteins have been found to be associated with the cell wall and influence interactions with other cells. PE and PPE family of proteins are of potential interest from the point of view of immune response, since they show antigenic variation which may play a role in immune evasion. Very little is known about the immunogenecity of these two classes of proteins and only few proteins have been shown to be potent B or T cell antigens, like Rv3873, Mtb39 and Rv0915c. Two proteins from PE_PGRS subfamily, Rv1759c and Rv3367 are expressed during infection and show antibody response in humans and rabbits, respectively. Rv1196 and Rv0915c from PPE family have been shown to be good T cell antigens. Another study has shown that the PE domain of PE_PGRS protein Rv1818c upon immunization into mice induces good cell mediated immune response in mice, whereas the PGRS domain is responsible for good humoral response. In humans there is increasing evidence to suggest that CD8+ T cells are elicited in response to infection with mycobacteria. CD8+ CTL may play an important role through several mechanisms. They produce potent anti-bacterial cytokines such as IFN-γ and TNF-α in response to antigenic stimulation and IFN-γ is critical for immunity to TB. Thus, identification of antigens and peptides that induce T cell responses could be useful for designing new vaccines to protect against TB. Relatively few epitopes in mycobacterial antigens have so far been identified for human CD8 T cells. In this regard, release of genome sequences of M. tuberculosis has provided an opportunity to identify proteins with vaccine potential that could give immune protection in individuals with different HLA backgrounds. Objectives and scope of the present work 1. Prediction of putative T cell antigens in PE and PPE family of proteins of Mycobacterium tuberculosis through immuno-informatics approach 2. Evaluation of immune response to three of the PE and PPE proteins in mouse model. 3. Evaluation of immune response against chosen PE and PPE proteins of Mycobacterium tuberculosis with Human Peripheral Blood Mononuclear Cells (PBMCs) from PPD positive healthy donors and TB patients. 4. Immune response to multi-epitope DNA vaccine construct for Mycobacterium tuberculosis. Prediction of MHC class I peptides from PE and PPE proteins. In an effort to identify potential T cell antigens from PE and PPE family of proteins, we have carried out a systematic in silico analysis of the 167 different PE and PPE proteins. Employing immuno-informatics approach, a set of HLA class I binding peptides have been identified from these proteins. Further, their binding abilities have been ascertained using independent methods such as molecular modeling and structural analysis methods. The nonameric sequences from PE and PPE families of proteins were predicted to contain high percentage of binding peptides to human class I HLA, whereas PE_PGRS proteins show relatively low level of binding. This difference is seen in spite of PE and PE_PGRS being Sub-families of the same family, PE. Seventy-one high- as well as low-affinity peptides from both PE and PPE proteins have been analyzed for structural compatibility with crystal structures of HLA in terms of intermolecular energies and were found to correlate well with the corresponding affinities predicted by the BIMAS algorithm. Most of the peptides binding to HLA are specific with very few promiscuous binders. Identification of T cell epitopes from three of the PE/PPE proteins using DNA immunization This work describes the evaluation of immune responses to three of the PE and PPE proteins in mouse model. Three of PE and PPE proteins, coded by Rv1818c, Rv3812 and Rv3018c genes were chosen based on immuno-informatics approach. They were cloned, expressed in prokaryotic and mammalian expression vectors and recombinant protein expressing stable cell lines were made. T lymphocytes from DNA immunized mice recognize synthetic peptides from chosen proteins in vitro, indicating that these peptides are being processed and presented by MHC molecules to T cells. By MHC stabilization assay, 5 of the synthetic peptides were found to stabilize the MHC class I molecules on the cell surface for more than 6 hrs, validating the computational prediction. Recognition of T cell epitopes derived from PE/PPE proteins by human PBMCs This work describes the evaluation of immune response against three of PE and PPE proteins of Mycobacterium tuberculosis with Human Peripheral Blood Mononuclear Cells (PBMCs) from PPD positive Healthy donors and TB patients. Proliferation response of PBMCs from ten PPD positive healthy donors as well as from ten TB patients, indicated that the peptides from PE and PPE proteins of Mtb can sensitize naive T cells and induce peptide specific IFN-γ and also the T cell response to the chosen peptides was both HLA class I restricted and CD8 mediated. After the peptide specific expansion, significant percentage of CD8+ T cells were shown to secrete IFN-γ and stained positive for perforin. Antigen specific CD8+ T cells were found to have cytolytic potential in addition to their cytokine function. Immune response to a multiepitope DNA vaccine in mouse model Minigene poly-epitope vaccine constructs coding for nine peptides derived from identified T cell antigens of PE and PPE proteins and three of the experimentally mapped epitopes from M tuberculosis was designed and constructed. The minigene was used to immunize mice and the immune response was tested. The DNA primed splenocytes recognized the full length poly-epitope protein as well as the individual peptides. T cell response to epitopes was enhanced by mere presence in multi-epitope construct compared to full length antigens. Human PBMCs derived from both PPD+ve and TB patients also recognized the peptides in vitro. It is thus obvious that a large cocktail of proteins are required to achieve reasonable population coverage. Besides, this work suggests the feasibility of designing haplotype specific subunit vaccine, which can be given to individuals with known HLA haplotype. The haplotype specific vaccines can be combined to target a population where the distribution of HLA alleles is known. This work also indicates that use of single or limited number of genes in a DNA vaccine may not be suitable to cover a given population.

The fetal lung epithelium secretes fluid into the potential pulmonary air-spaces by actively transporting chloride (Cl¯) into the lung lumen. This Cl¯-driven fluid secretion declines with the progression of lung development. Recent studies demonstrate that the A-type γ-aminobutyric acid receptor (GABAAR), a Cl¯ channel, and glutamic acid decarboxylase (GAD65/67), key GABA-synthesizing enzymes, are expressed in adult pulmonary epithelial cells (ECs), forming an autocrine GABAAR signaling system. My thesis study revealed that GABAAR π- and β2- subunits are expressed in high levels in the fetal rat lung epithelium and decline at birth, consistent with pattern of fluid secretion. Immunohistochemistry showed distinct profiles of expression for GABAAR subunits and GAD65/67. Treatment of alveolar ECs with dexamethasone reduced the GABAAR π-subunit expression. These results suggest that the GABAAR signaling in the fetal pulmonary epithelium is developmentally regulated and the GABAAR expression and GABAAR-mediated Cl¯ secretion in pulmonary ECs may be regulated by glucosteroids.