Submitted by Ana Damasceno (ana.damasceno@unipampa.edu.br) on 2017-05-12T17:36:02Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Caracterização química, atividades antioxidante, antileucêmica e antimicrobiana da própolis âmbar sul brasileira.pdf: 1920627 bytes, checksum: a4863fc2a0ca638e21de691cb6125515 (MD5) / Made available in DSpace on 2017-05-12T17:36:02Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Caracterização química, atividades antioxidante, antileucêmica e antimicrobiana da própolis âmbar sul brasileira.pdf: 1920627 bytes, checksum: a4863fc2a0ca638e21de691cb6125515 (MD5) Previous issue date: 2017-03-27 / A própolis é um composto utilizado pelas abelhas, com a finalidade de vedar a colmeia e evitar contaminações, que se destaca por suas atividades biológicas as quais têm sido muito estudadas para fins terapêuticos. Quando produzida por abelhas da espécie Apis mellifera a substância é composta por cerca de 50 % de resina vegetal misturada a enzimas presentes em sua saliva e cera. Este produto natural pode variar dependendo da origem botânica/geográfica e mais de 300 compostos já foram descritos. No caso do Brasil, existe grande variabilidade da composição química que é facilmente explicada pela sua grande biodiversidade. O objetivo deste trabalho foi analisar o perfil químico e as atividades antioxidante, antileucêmica e antimicrobiana da própolis de São Gabriel/Rio Grande do Sul, a qual denominamos própolis Âmbar, e comparar suas propriedades com as própolis brasileiras Vermelha e Verde. As análises do perfil químico foram realizadas pelas técnicas de GC-MS, HPLC e quantificação dos flavonoides e fenóis totais. A atividade antioxidante foi aferida pelas técnicas DPPH°, ABTS°+ e FRAP. A atividade antileucêmica foi analisada nas linhagens celulares K562, Jurkat e U937 pelos parâmetros de IC50, viabilidade, apoptose e ciclo celular. E a atividade antimicrobiana foi analisada aferindo-se o crescimento das espécies E. coli e S. aureus. Os resultados obtidos da análise por GC-MS das própolis Âmbar, dos dois anos coletados, identificam um total de 99 compostos dentre os quais apenas 16 foram identificados para as própolis Verde e Vermelha. Também foi possível notar que grande parte dos compostos encontrados são descritos para o gênero Eucalyptus que parece ser uma fonte vegetal importante para a produção da própolis Âmbar. Quanto aos fenóis totais, flavonóides totais e atividade antioxidante, as própolis apresentaram resultados diferentes entre si, sendo os valores obtidos para a própolis Âmbar sempre menores que os encontrados para as própolis Verde e Vermelha. Porém quanto à atividade antileucêmica a própolis Âmbar apresentou resultados similares a própolis Vermelha nas análises de IC50 e viabilidade. E na análise do efeito antimicrobiano todas as própolis igualaram seu efeito em concentrações acima de 500 µg/mL, apresentando também atividades semelhantes no tratamento com E.coli na concentração de 100 µg/mL. Tipificação, identificação da origem geográfica/botânica e quantidade de estudos sobre suas atividades biológicas agregam valor à própolis. Esperamos com este trabalho expandir o conhecimento técnico-científico da própolis Âmbar contribuindo com o desenvolvimento regional e ampliando o conhecimento sobre própolis. / Propolis is a compound used by bees to seal the hive and prevent contamination that stands out because of its biological activities which have been studied for therapeutic purposes. When produced by Apis mellifera bees specie the substance is composed by about 50% vegetable resin mixed with enzymes present in its saliva and wax. This natural product may vary depending on the botanical/geographical origin and more than 300 compounds have already been described. In Brazil there is a great variability of chemical composition that is easily explained by the Brazilian biodiversity. The goal of this work was to analyze the chemical profile and antioxidant, antileukemic and antimicrobial activities of São Gabriel/Rio Grande do Sul propolis, which we call Amber propolis, and to compare its properties with the Brazilian propolis Red and Green. The chemical analyzes were performed by total flavonoids and total phenols quantification, GC-MS and HPLC. The antioxidant activity was measured by DPPH°, ABTS°+ and FRAP techniques. The antileukemic activity was analyzed in the K562, Jurkat and U937 cell lines taken into account IC50, viability, apoptosis and cell cycle parameters. The antimicrobial activity was analyzed by E. coli and S. aureus growth. The results obtained from the GC-MS of Amber propolis, collected in two years, identified a total of 99 compounds among, from these only 16 were identified for the Green and Red propolis. It was also possible verify that most of the compounds found are described for the genus Eucalyptus which seems to be an important source of compounds for the Amber propolis production. Values for total phenolics, total flavonoids and antioxidant activity, were different among the propolis, being the values obtained for the Amber propolis always smaller than those found for the Green and Red propolis. However, regarding the antileukemic activity, the propolis Amber presented similar results to the Red propolis for IC50 analyzes and viability. In the analysis of the antimicrobial effect, all the propolis presented similar effects above 500 μg/mL and also presenting some levels of activity at 100 μg / mL in E. coli. Typification, identification of the geographical/botanical origin and quantity of studies on its biological activities add value to propolis. We hope with this work to expand the technical-scientific knowledge of propolis Amber contributing to regional development and expanding knowledge about propolis.

Honey has been used to heal wounds since ancient times. There are many references in ancient literature that cite honey for its medicinal uses. It is used as an alternative agent to cure infections of wounds, burns, ulcers etc. Researchers have shown some of the antimicrobial properties of honey when used as an ointment. When applied to an affected area, it helps to promote the growth of healthy tissue. One of the factors on which the quality of the honey depends, is its geographical origin. Based on the location, honey types can vary as much as 100-fold from each other in color, aroma, viscosity, and antimicrobial properties. The important components in honey that play an essential part in healing wounds and contributing to the antimicrobial properties are enzymes. Their presence allows honey to kill various types of pathogenic bacteria, viruses, fungi etc. A higher antimicrobial effect is seen in monofloral honey (when a single plant species is the source of nectar), which is often more potent than other types of honey in terms of antibacterial activity. Resistance of pathogens to these antimicrobial actions has never been shown, which makes honey a more promising source of antimicrobial research. Presently, infections of burns and wounds are very challenging to treat, especially when they are caused by antibiotic-resistant bacteria. The purpose of this study was to examine the antimicrobial properties of honey from Utah and other locales, and to identify promising antimicrobial activities that could be useful in treating infections caused by resistant bacteria.

<p>This investigation focused on Rhoicissus tomentosa, belonging to the family, Vitaceae in an attempt to assess the phytochemistry of this plant which is widely used by traditional healers in South Africa to ensure the safe delivery during pregnancy and childbirth (Hutchings et al., 1996).</p>

<p>This investigation focused on Rhoicissus tomentosa, belonging to the family, Vitaceae in an attempt to assess the phytochemistry of this plant which is widely used by traditional healers in South Africa to ensure the safe delivery during pregnancy and childbirth (Hutchings et al., 1996).</p>

Thesis (MSc Food Sc)--University of Stellenbosch, 2001. / ENGLISH ABSTRACT:Aspalafhus linearis is an indigenous fynbos plant cultivated in the Clanwilliam area of the Western Cape, South Africa. The rooibos tea that is prepared from this plant, has become popular worldwide mainly due to the alleged health properties. Studies on the anti-microbial properties of green, black and oolong teas have shown that these teas have strong anti-microbial activity against a wide range of microbes. No studies have been done on the anti-microbial activity of rooibos tea and the aim of this study was to determine what impact rooibos tea extracts would have on the growth of different food spoilage and potential pathogenic microbes. Water and ethyl acetate extracts of fermented and unfermented rooibos tea were used to determine the inhibitory effect on the growth of an Escherichia coli strain. The E. coli culture was grown in tea-MRS with either added fermented or unfermented rooibos tea extracts. Both the water and ethyl acetate extracts showed a strong inhibitory effect against the E. coli strain in that there was a decrease in the final bacterial cell density (Nmax)(from 0.59 00 to 0.25 00) and the maximum specific growth rate (~max)(from 1.12 h-1 to 0.20 h-1) and an increase in the doubling time (~) (from 0.59 h to 1.80 h) and lag time (tlag)(from 4.81 h to 6.60 h) as the concentration of the soluble solids of the tea extracts was increased from 0.5 to 5.0 g.r1 . Furthermore, it was found that the fermented rooibos tea had a much stronger inhibitory effect (69% decrease in growth at 5.0 g.r1 soluble solids) compared to the unfermented rooibos tea extracts (35.1% decrease in growth at 5.0 g.r1 soluble solids). The resulting data indicated that rooibos tea had a very strong inhibitory effect on the growth of the E. coli strain. It was also found that the water extracts of rooibos tea showed a stronger inhibitory effect on the growth of the E. coli than the ethyl acetate extracts, indicating that the antimicrobial activity of rooibos tea is not exclusively due to the polyphenolic content - individual compounds. It was also determined that the rooibos tea water extracts showed a bacteriostatic action against the E. coli strain in that as soon as the tea is no longer part of the growth medium, the E. coli resumed a normal growth pattern. The data obtained showed that the inhibitory effect of rooibos tea water extracts (69% decrease in growth) against the growth of E. coli was more pronounced than that found when black tea water extracts (25.7% decrease in growth) at the same concentrations were used.Rooibos tea water extracts (0.5 - 5.0 g.r1) of fermented and unfermented tea were also used to determine the inhibitory effect on other food spoilage microbes and potential pathogens. Strains of Staphylococcus aureus, Bacillus cereus, Listeria monocytogenes, Streptococcus mutans, Saccharomyces cerevisiae and Zygosaccharomyces rouxii were grown in the presence of fermented and unfermented rooibos tea water extracts. The effect that fermented rooibos tea had on the growth of all the microbes tested was in the following order: Staph. aureus (90.8% decrease in growth) > L. monocytogenes (89.2% decrease in growth) > Strep. mutans (84.1% decrease in growth) > B. cereus (80.3% decrease in growth) > Sacch. cerevisiae (77.7% decrease in growth) > E. coli (69.0% decrease in growth). The rooibos tea clearly had an inhibitory effect on the growth of all the microbes, with the exception of the Z. rouxii strain where the presence of the tea water extracts was found to enhance the growth. The inhibitory effect of rooibos tea on the growth of these microbes was shown by changes in the growth parameters with Nmax and IJmaxshowing decreases, while the ld and tlagincreased as the concentration of the tea soluble solids was increased. As with E. coli, the fermented rooibos tea water extracts showed the stronger inhibitory effect on the growth of the various microbes. The data obtained in this study suggests that rooibos tea is not effective as an anti-microbial agent against all yeast species, but will strongly retard the growth of specific Gram-positive and Gram-negative bacteria. As long as rooibos tea is present, strong anti-microbial activity will be observed at a cup of tea concentration of 2.5 g.r1 soluble solids. These results may be of value to support the health claims associated with rooibos tea and may in the future lead to the use of rooibos tea as a "natural" food preservative. / AFRIKAANSE OPSOMMING:Aspalathus linearis is 'n inheemse fynbosplant wat gekultiveer word in die Clanwilliam area van die Wes Kaap, Suid-Afrika. Rooibostee, wat gemaak word van hierdie plante, het baie gewild geword wereldwyd a.g.v. die gesondheidsaspekte van hierdie tee. Studies toon dat groen, swart en oolong tee sterk anti-mikrobiese aktiwiteit het teen 'n wye reeks mikrobes. Aangesien daar voorheen geen studies gedoen is op die anti-mikrobiese aktiwiteit van rooibostee nie, was die doel van hierdie studie om die effek van rooibostee te bepaal op die groei van verskillende voedselbederwers en potensiele patogeniese mikrobes. Water- en etielasetaat-ekstrakte van gefermenteerde en ongefermenteerde rooibos tee is gebruik om die inhiberende effek op die groei van Escherichia coli te bepaal. Escherichia coli is gegroei in tee-MRS met bygevoegde gefermenteerde of ongefermenteerde rooibostee-ekstrakte. Seide die water- en etielasetaatekstrakte van rooibostee het 'n sterk inhiberende effek gewys teen E. coli en dit word gestaaf deur 'n afname in die finale bakteriese seldigtheid (Nmax)(vanaf 0.59 00 tot 0.25 00) en die maksimum spesifieke groeitempo (lJmax) (vanaf 1.12 h-1 tot 0.20 h-1) en 'n toename in die verdubbelingstyd (~) (vanaf 0.59 h tot 1.80 h) en die sloerfase (tlag)(vanaf 4.81 h tot 6.60 h) 5005 wat die konsentrasie van oplosbare vastestowwe van die tee toeneem van 0.5 tot 5.0 g.r1 . Verder is daar gevind dat die gefermenteerde rooibostee 'n baie sterker inhiberende effek het (69% afname in groei by 5.0 g.r1 oplosbare vastestowwe) in vergelyking met die ongefermenteerde rooibostee-ekstrakte (35.1% afname in groei by 5.0 g.r1 oplosbare vastestowwe). Die resultate van die data dui aan dat rooibos tee 'n baie sterk inhiberende effek het op die groei van die E. coli spesie. Die waterekstrakte van rooibostee het 'n sterker inhibisie getoon teen die groei van E. coli as die etielasetaat-ekstrakte, wat aandui dat die anti-mikrobiese aktiwiteit van rooibostee nie eksklusief toegeskryf kan word aan die polifenoliese samestelling nie. Daar is ook gevind dat rooibostee water-ekstrakte 'n bakteriostatiese effek het teen E. coli, want sodra die tee ekstrakte nie meer teenwoordig is in die groeimedium nie, hervat E. coli normale groei. Die data wys ook dat die inhiberende effek van rooibostee water-ekstrakte (69.0% afname in goei) teen E. coli baie sterker is as die van swart tee water-ekstrakte (25.7% afname in groei) by dieselfde konsentrasies.Rooibostee water-ekstrakte (0.5 - 5.0 g.r1) van gefermenteerde en ongefermenteerde rooibostee is ook gebruik om die inhiberende effek te bepaal teen ander voedselbederwers en potensiele patogene. Spesies van Staphylococcus aureus, Bacillus cereus, Listeria monocytogenes, Streptococcus mutans, Saccharomyces cerevisiae en Zygosaccharomyces rouxii is gegroei in die teenwoordigheid van gefermenteerde en ongefermenteerde rooibostee waterekstrakte. Die effek wat gefermenteerde rooibostee het op die groei van die getoetste mikrobes is 5005 volg: Staph. aureus (90.8% afname in groei) > L. monocytogenes (89.2% afname in groei) > Strep. mutans (84.1% afname in groei) > B. cereus (80.3% afname in groei) > Sacch. cerevisiae (77.7% afname in groei) > E. coli (69.0% afname in groei). Rooibostee het 'n duidelike inhiberende effek gehad teen al die organismes, behalwe teen Z. rouxii spes ie, waar die teenwoordigheid van rooibostee die groei van die organisme bevorder het. Die inhiberende effek van rooibostee teen die groei van hierdie mikrobes word ondersteun deur die groei parameters waar die Nmaxen IJmaxafgeneem het terwyl die ~ en tlagtoegeneem het 5005 wat die konsentrasie van die oplosbare vastestowwe toeneem. Die gefermenteerde rooibostee water-ekstrakte het ook 'n sterker inhiberende effek op die groei van die verskillende mikrobes net 5005 met E. coli. Die data wat verkry is van hierdie studie dui aan dat rooibostee nie effektief sal wees as 'n anti-mikrobiese middel teen aile gis spesies nie, maar dit sal die groei van spesifieke Gram-positiewe en Gram-negatiewe bakterie sterk vertraag. So lank as wat rooibostee teenwoordig is, sal sterk anti-mikrobiese aktiwiteit waargeneem word by 'n koppie-tee konsentrasie van 2.5 g.r1 oplosbare vastestowwe. Hierdie resultate kan help om die gesondheidseienskappe geassosieer met rooibostee te ondersteun en help om die gebruik van rooibostee as 'n "natuurlike" preserveermiddel te bevorder. dedicated to my parents

Made available in DSpace on 2014-06-11T19:22:59Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-06-08Bitstream added on 2014-06-13T20:29:34Z : No. of bitstreams: 1 silva_avr_me_rcla.pdf: 1706887 bytes, checksum: eda68ea29b93397b581e427121535611 (MD5) / Neste trabalho estudou-se a estrutura, função e mecanismo de ação do peptídeo antibacteriano Protonectarina-MP (isolado de veneno da vespa social Protonectarina sylveirae) tendo seu resíduo C-terminal nas formas ácida (-OH) e amidada (-NH2). Os peptídeos foram sintetizados, utilizando-se a estratégia Fmoc, purificados por cromatografia líquida de alta performance. O monitoramento do material sintético foi feito por espectrometria de massas ESI-MS e por seqüenciamento através de Química Degradativa de Edman. A estrutura secundária foi investigada pelo uso de espectroscopia de dicroísmo circular e modelagem molecular. Atividade lítica (extravasamento) e interação do resíduo de triptofano em vesículas foram investigadas pelo uso de espectrômetro de fluorescência. Foram realizados ensaios sobre as interações desses peptídeos em meio de vesículas zwitteriônicas e aniônica, formando complexos proteolipossomos que foram submetidos à troca isotópica H/D monitorada por espectrometria de massas ESI-MS e MS/MS. Além disso, foram realizados ensaios biológicos de atividade hemolítica, de desgranulação de mastócito, de liberação da enzima citoplasmática Lactato Desidrogenase e de atividade antimicrobianas. Os dados de CD revelam uma tendência dos peptídeos se estruturarem em hélice-α em ambiente hidrofóbico e em ambiente de membranas. Porém, o mesmo não pode ser observado em meio aquoso. Os modelos obtidos para ambos os peptídeos por modelagem molecular mostram uma estruturação em hélice-α anfipática. Nos ensaios de atividade lítica em vesículas, os peptídeos apresentaram um processo com cooperatividade positiva, com curvas de dose-resposta que mostram uma dependência sigmoidal com a concentração do peptídeo. Os resultados da fluorescência do triptofanos mostram um deslocamento da emissão para a região de onda do azul para o peptídeo... / In the present work was studied the structure, function and mechanism of action of the antibacterial peptide Protonectarina-MP (isolated from venom of social wasp Protonectarina sylveirae) with its carboxyamidation (-NH2) and carboxyl-free (-OH) Cterminal forms. The peptides were manually synthesized on-solid phase by using Fmoc strategy and purified under HPLC. The homogeneity of the synthetic material was analyzed by ESI mass spectrometry and Edman Degradation Chemistry. The secondary structure was investigated through circular dichroism (CD) spectroscopy and molecular modeling. Lytic activity and peptides interaction with the membranes was also investigated through tryptophan emission, by fluorescence spectrometry. The interaction of peptides with zwitterionic and anionic vesicles was investigated through the combination of H/D exchange and ESI-mass spectrometry. Some biological activities, like: mast cell degranulation, release of cytoplasmic enzyme lactate dehydrogenase, hemolysis and antibiosis were investigated for both peptides. The CD spectra revealed that the peptides in hydrophobic environments or in presence of biological membranes have the tendency to form helix conformations; however, organized structures were not observed in aqueous or buffer solutions. The models obtained by molecular modeling show that both peptides form an amphipathic α-helix. The peptides presented a positive cooperative process in the lytic activity of vesicles, with dose-response curves presenting a sigmoidal dependence with the peptide concentration. The results of the fluorescence of tryptophans showed a shift of the emission wavelength to the blue region of the peptide Protonectarina-MP (-NH2), which was not observed for its analogue presenting the C-terminal residue in free acid form. This is indicating a greater interaction of the amidated peptide in membranes, when compared to the peptide... (Complete abstract click electronic access below)

Magister Scientiae - MSc / This investigation focused on Rhoicissus tomentosa, belonging to the family, Vitaceae in an attempt to assess the phytochemistry of this plant which is widely used by traditional healers in South Africa to ensure the safe delivery during pregnancy and childbirth (Hutchings et al., 1996). / South Africa

Sweden faces shortages in antibiotics. Shortages are caused due to a variety of reasons. Due to low profit margins and opportunity costs, antibiotic supply chains may experience a lack of competition. Lack of competition across the various stages of supply chains leads to fragility in the supply chain which ultimately results in shortages. Lack of communication is another such factor leading to shortages. Incorporating an AI/IT system across the supply chain would help prevent the occurrence of shortages by addressing such factors.  PLATINEA, an innovation platform, aims to address the threat of anti-microbial resistance by ensuring a steady supply of antibiotics. Their work package 4 is dedicated to eliminating risk factors or causes of shortages that arise from supply chains of antibiotics. PLATINEA has drafted a mind map to identify the risk factors or causes of shortages in Sweden. This thesis revolves around conducting a cost benefit analysis for implementing an AI/IT tool that addresses the risk factors and causes of shortages identified from the mind map that stem from the Swedish supply chains of antibiotics. A model consisting of a breakdown in costs and benefits was created. The model not only helped us frame the various costs and benefits, but also evolved during the research to help us structure our results better. An AI/IT tool has been devised keeping the risk factors and causes of shortage in mind. This tool has four versions that have varying levels of integration and automation. Semi-structured Interviews were conducted with experts in the field of artificial Intelligence and machine learning. calculation based on historical data were made to determine costs of shortages and to some extent, visualize the extent of costs involved in antibiotic resistance. Based on the information gathered from the interviews and literatures, the costs and benefits identified in the model are addressed, including the significant benefit of reducing cost of shortages.

No description available.

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.