Dyslipidemia can be a comorbidity of both insulin-resistance and atherosclerosis. Hypertriglyceridemia is common in hyperglycemia and is associated with hypoalphalipoproteinemia (low HDL) and with altered nucleotide or purinergic signaling. We therefore hypothesized that extracellular nucleotides may affect hepatic lipoprotein metabolism. Our studies confirm this view and show that nucleotides regulate cellular proteolytic pathways in liver cells and thereby control lipoprotein secretion and their metabolism by hepatic lipase (HL). Treatment of liver cells with the nucleotide, adenosine diphosphate (ADP), stimulates VLDL-apoB100 and apoE secretion, but blocks HDL-apoA-I and HL secretion. ADP functions like a proteasomal inhibitor to block proteasomal degradation and stimulate apoB100 secretion. Blocking the proteosome is known to activate autophagic pathways. The nucleotide consequently stimulates autophagic degradation in liver cells and increases cellular levels of the autophagic proteins, LC3 and p62. Confocal studies show that ADP increases cellular LC3 levels and promotes co-localization of LC3 and apoA-I in an autophagosomal degradation compartment. ADP acts through the G-protein coupled receptor, P2Y13, to stimulate autophagy and block both HDL and HL secretion. Overexpression of P2Y13 increases cellular LC3 levels and blocks the induction of both HDL and HL secretion, while P2Y13 siRNA reduce LC3 protein levels and cause up to a ten-fold stimulation in HDL and HL secretion. P2Y13 gene expression regulates autophagy through the insulin receptor (IR-β). A reduction in P2Y13 expression increases the phosphorylation of IR-β and protein kinase B (Akt) >3-fold, while increasing P2Y13 expression inhibits the activation of IR-β and Akt. Experiments with epitope-labeled apoA-I and HL show that activation of purinergic pathways has no effect on the internalization and degradation of extracellular apoA-I and HL, which confirms the view that nucleotides primarily impact intracellular protein transport and degradation. In conclusion, elevated blood glucose levels may promote dyslipidemia by stimulating purinergic signaling through P2Y13 and IR-β and perturbing the intracellular degradation and secretion of both HDL and VLDL.

Although spinal cord injury (SCI) is a debilitating condition that presents a large socioeconomic problem in the United States, there is currently no treatment that reliably reduces morbidity and mortality. Current research is aimed at identifying mechanisms involved in the pathophysiology of SCI and using this knowledge to develop rational treatments. We have observed plasma membrane compromise in the acute (within 10 minutes), sub-acute (3 days), and chronic phases (5 weeks) in a rat model of contusion SCI and postulate that it negatively affects neurological outcome. Holes/tears in the plasma membrane were assessed with a dye exclusion assay, in which a fluorescent cell-impermeant dye was injected into the cerebrospinal fluid prior to sacrifice; therefore, cellular uptake of the dye is indicative of plasma membrane compromise. As early as 10 minutes after SCI, widespread uptake of permeability markers was evident in neuronal cell bodies as well as axonal projections. The number of permeable cells and the size of the membrane breaches (measured by using permeability markers of various sizes) varied with distance from the injury site, with larger disruptions located closer to the epicenter. Greater cellular uptake was observed when the impact force was increased (200 > 150 > 100 kdyn > sham). At longer time points (3 days and 5 weeks), substantial permeability marker uptake was observed in axons but not in cell bodies. Cells with increased permeability displayed a variety of pathomorphological alterations, including swelling, blebbing, retraction bulb formation, neurofilament loss, and fragmentation, suggesting that increased plasma membrane permeability is detrimental to cell survival and function. We therefore investigated a clinically-relevant treatment strategy designed to restore plasma membrane integrity. Animals were treated with citicoline, a molecule utilized in the endogenous synthesis of phosphatidylcholine (the major membrane component in mammalian cells). Citicoline has been shown to be beneficial in numerous studies of neurological disease, improving overall outcome by increasing phospholipid synthesis and attenuating phospholipid destruction (by reducing phospholipase A2 activity). However, these mechanisms have not been explored in a model of SCI. When compared to injured animals receiving vehicle (saline) injections, citicoline treatment after SCI did not have a statistically significant effect on cytoplasmic PLA2 activity (at 24h post-injury), the density of permeable axons (at 3 days post-injury), or the lesion volume (at 3 days post-injury). Since citicoline may improve neurological outcome after SCI through mechanisms we did not directly assess, we then conducted a longer-term study to evaluate the overall efficacy of citicoline treatment in terms of longer-term functional and histological consequences. Citicoline did not have a biologically significant effect on behavioral recovery (evaluated during open field locomotion, grid walk and hyperalgesia testing weekly for up to 5 weeks post-injury) or lesion volume (at 5 weeks post-injury). The lack of citicoline-mediated effect may be attributed to experimental parameters (e.g., dosing or sensitivity of outcome measures) or biological inefficacy. Although we were not able to demonstrate that citicoline improves outcome after SCI, the finding that plasma membrane damage occurs in a persistent fashion and is associated with pathophysiological cellular alterations may provide fundamental knowledge necessary for developing treatments targeted at membrane repair. Future work examining the complex mechanisms causing prolonged membrane damage after SCI and evaluating strategies for manipulating these pathways (potentially using citicoline in combination with other pharmacological agents) may lead to a clinically effective therapy.

Thesis (Ph.D.) -- University of Texas Southwestern Medical Center at Dallas, 2005. / Vita. Bibliography: References located at the end of each chapter.

Made available in DSpace on 2015-07-22T20:50:43Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-05-27. Added 1 bitstream(s) on 2015-08-11T03:25:25Z : No. of bitstreams: 1 Publico-00212.pdf: 1245886 bytes, checksum: 4848d0e58992c5a9ff4b387a14c2534a (MD5) / Objetivos: Analisar a expressão das proteínas CD10 e NM23 por estudo imunohistoquímico do tecido do carcinoma colorretal e da mucosa adjacente. Avaliar a relação da expressão dessas proteínas com os aspectos anatomopatológicos da neoplasia, estadiamento clínico, ocorrência de metástases hepáticas e prognóstico dos doentes. Método: Cento e trinta doentes operados por carcinoma colorretal foram analisados. Bloco de tissue microarray foi confeccionado com tecido neoplásico e com a mucosa não neoplásica adjacente. Estudo imuno-histoquímico foi realizado com anticorpos monoclonais NM23 e CD10 no tecido neoplásico e no tecido não neoplásico da mucosa adjacente. A leitura foi realizada por aparelho de escaneamento de lâminas. A imunoexpressão foi avaliada pelo percentual de células coradas e foram obtidos escores de intensidade. Foram considerados como positivos para CD 10 os tumores que expressavam o marcador em mais de 10% das células neoplásicas. Para NM 23 considerou-se dois grupos divididos em fortes expressores (mais de 50%) e expressores fracos (menos de 50%) das células coradas. Os resultados foram relacionados com as características morfológicas e histopatológicas do carcinoma colorretal, estadiamento clínico, presença de metástases hepáticas e com o prognóstico. No estudo estatístico foram utilizados os testes de Mann-Whitney, Kruskal- Wallis e exato de Fisher. A sobrevivência foi avaliada utilizando a curva de Kaplan- Meier, e o desfecho de comparação entre as curvas foi calculado pelo teste de Long rank. Resultados: Ambos os marcadores CD10 e NM23 apresentaram expressão maior no tecido do carcinoma do que na mucosa não neoplásica adjacente (p<0,0001 para ambos). A imunoexpressão tecidual das proteínas NM23 e CD10 não apresentou relação com o grau de diferenciação celular (p=0,57 e p=0,48, respectivamente), invasão vascular (p=0,85 e p=0,67, respectivamente), invasão linfática (p=0,41 e 0,73, respectivamente), infiltração perineural (p=0,46 e p=0,24, respectivamente) e com o estadiamento pela classificação TNM (p=0,19 para ambos). A imunoexpressão de CD10 no tecido do carcinoma colorretal foi maior (p=0,05) nas neoplasias exofíticas do que nos tumores não exofíticos. A expressão das proteínas NM23 e CD10 não apresentou relação com a incidência de metástases linfonodais (p=0,08 e 0,30, respectivamente). A expressão tecidual dos marcadores NM23 e CD10 não se relacionou com a ocorrência de metástases hepáticas (p=0,59 e p=0,31, respectivamente). A sobrevivência livre de doença mostrou relação significante (p=0,01) com a maior intensidade de imunoexpressão da proteína NM23 no tecido do carcinoma colorretal, o mesmo não ocorrendo com a imunoexpressão da proteína CD10 (p=0,18). A sobrevivência global não mostrou relação com as expressões das proteínas NM23 e CD10 (p=0,13 e p=0,24, respectivamente). Conclusões: O tecido neoplásico do carcinoma colorretal expressou mais intensamente as proteínas NM23 e CD10 do que a mucosa não neoplásica adjacente. A imunoexpressão de CD10 no tecido do carcinoma colorretal foi maior (p=0,05) nas neoplasias exofíticas do que nos tumores não exofíticos. A expressão das proteínas NM23 e CD10 não se relacionou com os demais aspectos anatomopatológicos da neoplasia, com a presença de metástase hepática e com o estadiamento do carcinoma colorretal. Os doentes com imunoexpressão aumentada da proteína NM23 apresentaram sobrevivência livre de doença significativamente maior. A intensidade da imunoexpressão tecidual da proteína CD10 não influenciou a sobrevivência livre de doença e a sobrevivência global não se relacionou com a imunoexpressão das proteínas NM23 e CD10. / Aims: To analyze the tissue expression of the proteins CD 10 and NM 23 through the immunohistochemichal study of the colorectal carcinoma and evaluate the expression relation of these proteins with the anatomopathological aspects of the neoplasia, clinical staging, occurrence of hepatic metastasis and patients’ prognostic. Method: One hundred and thirty operated patients of colorectal carcinoma have been analyzed. A block of tissue microarray was produced with the neoplastic mucosa and with the adjacent non-neoplastic mucosa. An immunohistochemichal study was performed with monoclonal antibodies NM23 and CD10 on the neoplastic tissue and non-neoplastic tissue of the adjacent mucosa. The interpretation of the slides was made by a scanner device. The immunoexpression was evaluated by the percentage of colored cells and the obtained intensity scores. The results were related to the morphological and histopathological characteristics of the carcinoma, clinical staging, presence of hepatic metastasis and to the prognostic of the patients. In the statistic study were used the Mann-Whitney test, the Kruskal-Wallis test and Fisher’s exact test. The analysis of survival was conducted with the use of the Kaplan-Meier curve and the comparison conclusion between the curves was calculated through the Longrank test. Results: Both markers CD10 and NM23 presented a higher expression on the carcinoma tissue rather than on the non-neoplastic adjacent mucosa (p<0,0001 for both). The expression of the proteins NM23 and CD10 did not present any relation to the degree of cellular differentiation (p=0,57 and p=0,48, respectively) , vascular invasion (p=0,85 and p=0,67, respectively), lymphatic invasion (p=0,41and 0,73, respectively), perineural infiltration (p=0,46 and p=0,24, respectively) and with the staging by the TNM classification (p=0,19). The immunoexpression of CD10 on the colorectal carcinoma tissue was higher (p=0,15) on the exophytic neoplasias than on the non-exophytic tumors. The expression of the proteins NM23 and CD10 did not present any relation with the incidence of lymphonodal metastasis (p=0,08 and 0,30, respectively). The tissue expression of the markers NM23 and CD10 did not relate to the occurrence of hepatic metastasis (p=0,59 and 0,31 respectively). The disease-free survival disclosed a significant relation (p=0,01) with a higher intensity of immunoexpression of the protein NM23 on the colorectal carcinoma’s tissue. However, the same did not occur with the immunoexpression of the protein CD10 (p=0,18). The global survival did not show any relation with the expression of the proteins NM23 and CD10 (p=0,13 and p=0,24, respectively). Conclusions: The neoplastic tissue of the colorectal carcinoma expresses more intensely the proteins NM23 and CD10 than the adjacent nonneoplastic mucosa. The expression of the proteins NM23 and CD10 does not relate to the presence of lymphonodal metastasis, hepatic metastasis, degree of cellular differentiation, colonic or rectal localization of the neoplasia, presence of vascular and/or lymphatic invasion, presence of neural infiltration and the staging of the colorectal carcinoma. The patients with increased immunoexpression of the protein NM23 presented a disease-free survival significantly higher. The intensity of the tissue immunoexpression of the protein CD10 did not influence the disease-free survival. The global survival does not relate to the immunoexpression of the proteins NM23 and CD10. / TEDE / BV UNIFESP: Teses e dissertações

Dyslipidemia can be a comorbidity of both insulin-resistance and atherosclerosis. Hypertriglyceridemia is common in hyperglycemia and is associated with hypoalphalipoproteinemia (low HDL) and with altered nucleotide or purinergic signaling. We therefore hypothesized that extracellular nucleotides may affect hepatic lipoprotein metabolism. Our studies confirm this view and show that nucleotides regulate cellular proteolytic pathways in liver cells and thereby control lipoprotein secretion and their metabolism by hepatic lipase (HL). Treatment of liver cells with the nucleotide, adenosine diphosphate (ADP), stimulates VLDL-apoB100 and apoE secretion, but blocks HDL-apoA-I and HL secretion. ADP functions like a proteasomal inhibitor to block proteasomal degradation and stimulate apoB100 secretion. Blocking the proteosome is known to activate autophagic pathways. The nucleotide consequently stimulates autophagic degradation in liver cells and increases cellular levels of the autophagic proteins, LC3 and p62. Confocal studies show that ADP increases cellular LC3 levels and promotes co-localization of LC3 and apoA-I in an autophagosomal degradation compartment. ADP acts through the G-protein coupled receptor, P2Y13, to stimulate autophagy and block both HDL and HL secretion. Overexpression of P2Y13 increases cellular LC3 levels and blocks the induction of both HDL and HL secretion, while P2Y13 siRNA reduce LC3 protein levels and cause up to a ten-fold stimulation in HDL and HL secretion. P2Y13 gene expression regulates autophagy through the insulin receptor (IR-β). A reduction in P2Y13 expression increases the phosphorylation of IR-β and protein kinase B (Akt) >3-fold, while increasing P2Y13 expression inhibits the activation of IR-β and Akt. Experiments with epitope-labeled apoA-I and HL show that activation of purinergic pathways has no effect on the internalization and degradation of extracellular apoA-I and HL, which confirms the view that nucleotides primarily impact intracellular protein transport and degradation. In conclusion, elevated blood glucose levels may promote dyslipidemia by stimulating purinergic signaling through P2Y13 and IR-β and perturbing the intracellular degradation and secretion of both HDL and VLDL.

Nucleoside 5'-Diphosphate Kinase (NdPK EC 2.7.4.6) is an enzyme (phosphotransferase) with extraordinary characteristics due to its unique ability to transfer phosphor groups to interconvert all nucleoside di- and triphosphates as a part of the DNA synthesis. Due to Biovica International AB's use of signaling and complementing enzymes in their in vitro diagnostic (IVD) test assays for Thymidine Kinase activity, an investigation was proposed to evaluate NdPK, which is a complementing enzyme in the assay. The aim of the study was to evaluate the enzymatic turnover of the enzyme NdPK with a spectrophotometric assay to obtain the specific activity (Units/mg solid protein). To determine the specific activity, enzyme kinetic methodology was applied, including the Michaelis-Menten model. In this study, the method is proposed as a general internal control procedure for the company, as a tool for function control of the different purchased enzymes used in their products in development. Results from the study reflects the different methods used to gain the specific activity for NdPK, where they were compared with the already specified specific activity from the manufacturing company. The results were auspicious, but before the method's authorization as an internal quality procedure, a few amendments are in mind. For instance, determining a method for the graphical readings, validating the method for quality control, and investigating if the method is applicable to other complementing enzymes. In conclusion, the method for determining the specific activity of the enzyme NdPK can be done, by executing the procedure of colorimetric enzyme assay.

La plupart des traitements anticancéreux, comme la chimiothérapie ou la radiothérapie, sont cytotoxiques et causent des dommages à l'ADN dans le but d’induire la mort des cellules tumorales. Cependant, l’efficacité d’activité de réparation de l'ADN des tumeurs entraine des résistances intrinsèques et acquises aux traitements. L'une des étapes précoces de la réparation de l’ADN est le recrutement de protéines au niveau du site de dommage. Ce recrutement est coordonné par une cascade de modifications et est contrôlé par des protéines senseurs telles que la protéine kinase ADN dépendante (DNA-PK) et / ou la poly (ADP- ribose) polymérase (PARP). Dans ce manuscrit, nous avons identifié et caractérisé le mécanisme d'action de petites molécules d'ADN (les siDNA), mimant des cassures double brin (appelé Dbait) ou simple brin (appelé Pbait), dans l’inhibition des voies de réparation des cassures simple brin (SSBR/BER). Nous démontrons que les molécules Dbait recrutent et activent à la fois PARP et DNA-PK, contrairement aux molécules Pbait qui ne recrutent que la PARP. L'étude comparative de ces deux molécules permet d'analyser les rôles respectifs des deux voies de signalisation: les deux molécules recrutent les protéines impliquées dans la voie de réparation des cassures simple brin (comme PARP, PCNA et XRCC1) et empêchent leurs recrutements aux niveaux des lésions chromosomiques. Les molécules Dbait inhibent par ailleurs le recrutement des protéines impliquées dans la voie de réparation des cassures double brin (NHEJ et HR). Pbait et Dbait désorganisent la réparation de l’ADN et sensibilisent les cellules tumorales aux traitements. L’inhibition de la réparation des cassures simple brin semble dépendre d’un piégeage des protéines directement sur les siDNA ou indirectement sur les polymères PAR. L’inhibition des voies de réparation des cassures double brin (DSB) semble par contre se faire de façon indirecte ; cette inhibition résulterait plutôt de la phosphorylation des protéines de réparation des DSB de part l’activation de DNA-PK. Les molécules Dbait et Pbait induisent un effet de létalité synthétique des cellules tumorales BRCA mutées. Cependant, la mutation BRCA semble être suffisante mais non nécessaire pour induire la sensibilité des cellules tumorales aux traitements Dbait. En effet, nous avons démontré que les molécules Dbait peuvent aussi sensibiliser les cellules ne présentant pas de mutation BRCA mais ayant toutefois une forte instabilité génétique. Nous avons trouvé une corrélation entre le niveau basal de protéines de réparation de l'ADN (ɣH2AX, PARP et PAR), le taux basal de cassures à l’ADN, la présence de micronoyaux (MN) et la sensibilité des cellules tumorales au traitement Dbait. Nous avons émis l’hypothèse que cette instabilité génétique, déterminé par la quantification de MN dans des biopsies tumorales, pourrait être un biomarqueur prédictif de l’effet du Dbait, non seulement dans les cancers du sein, mais aussi dans les glioblastomes, les mélanomes, les mélanomes uvéaux et les cancers du côlon. / Most conventional cancer treatments, such as chemotherapy or radiotherapy, are cytotoxic and cause DNA damages in the tumoral treated cells, which ultimately lead to their death. However, several intrinsic and acquired resistances of tumors to these treatments are due to the tumor efficient DNA repair activities. One of the major early steps of DNA repair is the recruitment of repair proteins at the damage site and this is coordinated by a cascade of modifications controlled by sensor proteins such as DNA-dependent protein kinase (DNA-PK) and/or poly (ADP-ribose) polymerase (PARP). In this manuscript, we identify and characterize the mechanism of action of short interfering DNA molecules (siDNA), mimicking double-strand breaks (called Dbait) or single-strand breaks (called Pbait) in Single Strand Break Repair pathway (SSBR/BER) inhibition. We demonstrate that Dbait bound and induced both PARP and DNA-PK activities, whereas Pbait acts only on PARP. The comparative study of the two molecules allows analysis of the respective roles of the two signaling pathways: both molecules recruit proteins involved in single-strand break repair (such as PARP, XRCC1 and PCNA) and prevent their recruitment at chromosomal damage. Dbait, but not Pbait, also inhibits recruitment of proteins involved in double-strand break (DSB) repair. By these ways, Pbait and Dbait disorganized DNA repair, thereby sensitizing cells to treatments. SSB repair inhibition depends upon a direct trapping of the main proteins on both molecules and an indirect trapping in PAR polymers. DSB repair inhibition may be indirect, resulting from the phosphorylation of DSB repair proteins by activated DNA-PK. The DNA repair inhibition by both molecules is confirmed by their synthetic lethality with BRCA mutations tumoral cell lines. However, BRCA mutation could be sufficient but not necessary to induce breast cancer cell lines and tumors sensitivity to Dbait treatment. In fact, we demonstrate that Dbait molecules could also have a stand-alone effect in BRCA wild type cells with a high genetic instability. We found a correlation between DNA repair proteins basal level (ɣH2AX, PARP and PAR), DNA break basal level, presence of micronucleus (MN) and tumoral cell lines sensitivity to Dbait treatment. We hypothesis that this genetic instability, determined by MN in tumor biopsies, could be a predictive biomarker of Dbait stand-alone effect, not only in breast cancer treatment, but also in glioblastoma, melanoma, uveal melanoma and colon cancer treatment.

Les propriétés physiques d’un matériau sont intimement liées à sa structure cristalline et dans le cas d’ions dopants aux sites qu’ils occupent. La première partie de ce travail est dédiée au matériau diphosphate de sodium et de zinc Na2ZnP2O7, cristallisé out vitreux et ceci pour les ions dopants Co2+, Ni2+, Mn2+ et Eu3+. Les phases cristallisées ont été obtenues par la méthode Czochralski, les verres par trempe à partir de l’état fondu. Un ensemble de caractéristiques physiques ont été mises en jeu (Raman, infrarouge, RPE, absorption optique, luminescence) pour déterminer les sites occupés par les ions dopants et l’influence sur les propriétés optiques. La deuxième partie de cet travail consiste à une meilleure connaissance des matériaux diélectriques sans plomb appartenant à la famille pérovskite et plus particulièrement à la solution solide NaNbO3-BaTiO3. Des monocistaux ont été obtenus par la méthode des flux et caractérisés en utilisant plusieurs techniques : diffraction X, microanalyse, évolution thermique des domaines ferroélectriques-ferroélastiques, mesures diélectriques, piézoélectriques et pyroélectriques. / Abstract The physical properties of a material are intimately related to its crystalline structure and in the case of doped ions they are dependent on to the sites they occupy. The first part of this work is dedicated to the diphosphate material of sodium and zinc Na2ZnP2O7, glassy and crystallized for doped ions of Co2+, Ni2+, Mn2+ and Eu3+. The crystallized phases were obtained by the Czochralski method whereas the glasses were obtained by quenching from the molten state. A set of physical characteristics were studied (Raman, infrared, EPR, optical absorption, luminescence) to determine the sites occupied by the doped ions and its influence on the optical properties. The second part of this work consists of a better knowledge of lead-free dielectric materials belonging to the family of Perovskite, more particularly to NaNbO3-BaTiO3 solid solution. Single crystals were obtained by the flux method and were characterized using several techniques: X-rays diffraction, microanalysis, thermal evolution of ferroelectric and ferroelastic domains; dielectric, piezoelectric and pyroelectric measurements.

No description available.

Living organisms employ different kinds of mechanisms, to regulate the functions of genes or their products, which may help in maintaining homeostasis inside the cell or may help in fighting hostile environment in the case of pathogenic organisms. These mechanisms act at the transcriptional, post-transcriptional, translational, and post-translational levels. In order to understand the physiology of an organism, it is essential to obtain an in-depth knowledge of such mechanisms, in which several proteins participate in interlinked pathways. In this regard, the present study focuses on two such proteins: (i). the newly identified Fic (Filamentation induced by cAMP) protein; and (ii). NDK (Nucleoside Diphosphate Kinase), which had been studied for decades. Fic protein and NDK share several common features: (i). both use nucleoside triphosphate (NTPs) or nucleoside diphosphate (NDPs) or their derivatives as one of their substrates; (ii). they have been found to be involved in diverse cellular pathways, involving different types of substrates that form the second substrate of these proteins; (iii). both are ubiquitously present in all the living organisms - from bacteria to humans to plants. However, there is very little information on these proteins from mycobacterial systems, which include some major human pathogens, Mycobacterium tuberculosis and Mycobacterium leprae, which are the causative agents of Tuberculosis and Leprosy, respectively. In view of these reasons, in the present study, the structural and/or functional features of the Fic and NDK proteins from Mycobacterium tuberculosis, were analysed, as it might be of medical significance for effectively combating the pathogen. The Chapter 1 of the thesis contains the Introduction to the research work and Chapter 2 is on the overall Materials and Methods. The remaining chapters pertain to the data obtained on the structural and/or functional features of the Fic and NDK proteins from Mycobacterium tuberculosis. Chapter 3. Cloning, Expression and Purification of Mycobacterium tuberculosis Fic The role of FIC (Filamentation induced by cAMP) domain containing proteins in the regulation of many vital pathways, mostly through the transfer of NMPs from NTPs to specific target proteins (NMPylylation), in microorganisms, higher eukaryotes, and plants is emerging. In order to understand the biological role of FIC domain containing proteins in mycobacteria, the gene for the FIC domain containing protein of the human pathogen, Mycobacterium tuberculosis, MtuFic, was cloned, overexpressed, purified to homogeneity, and biochemically characterised. Neither the His-tagged nor the GST-tagged MtuFic protein, overexpressed in Escherichia coli, nor expression of Mtufic in Mycobacterium smegmatis, yielded the protein in the soluble fraction. However, the maltose binding protein (MBP) tagged MtuFic (MBP-MtuFic) could be obtained partly in the soluble fraction. Denatured-refolded protein was used for the antibody generation in mice and rabbit. The cellular localisation and secretion of MtuFic were characterised using the antibody. Chapter 4. Biochemical Characterisation of Mycobacterium tuberculosis Fic Sequence alignment with several FIC motif containing proteins, complemented with homology modeling on the FIC motif containing protein, VbhT of Bartonella schoenbuchensis as the template, showed conservation and interaction of residues constituting the FIC domain. MtuFic, possesses the critical His144 residue, in the characteristic FIC Motif, HPFREGNGRSTR (HPFxxGNGRxxR), spanning 144th to 155th residue. Site-specific mutagenesis of the His144, or Glu148, or Asn150 of the FIC motif, or of Arg87 residue that constitutes the FIC domain, or complete deletion of the FIC motif, abolished the NTP to NMP conversion activity. The activity of MtuFic was consistent with the biochemical activities hitherto reported for a variety of bacterial FIC domain containing proteins. Studies were also carried out on NMPylylation in the presence of eukaryotic proteins and eukaryotic and mycobacterial cell lysates. Although formation of NMPs from NTPs mediated by MBP-MtuFic could be detected, we could not identify any protein as the target substrate either in the human macrophage (THP1) cells or in the M. tuberculosis cells. VopSΔ30 (kind gift from Dr. Kim Orth), along with human G proteins as targets, were used as the positive controls. Various possibilities for the inability to detect a protein target substrate are discussed. Chapter 5. Transcriptional Analysis of Mycobacterium tuberculosis fic Gene (Mtufic) In parallel, in order to understand the transcriptional regulation of Mtufic, primer extension analysis was carried out. The Transcription Start Site (TSS; +1 site) of Mtufic were mapped under different growth/stress conditions, which tubercle bacilli encounter in human host. Mtufic got expressed mainly through two transcripts, T1 and T2, arising from two different transcription start sites (TSS). Putative promoter regions were cloned in a promoter probe vector, which expresses a GFP protein of very high intensity, in order to qualitatively detect the activity of the promoters. The half-life of the gfp mRNA was determined to be 4 min and therefore justifiably quantitated the Mtufic promoter activity by determining the gfp mRNA levels. The levels of Mtufic mRNA were two-fold higher under nutrient-depleted stationary phase of growth, as compared to the levels at mid-log phase. The activity of P1 and P2, as quantitated real-time using the short half-life gfpm2+ mRNA levels in Mycobacterium smegmatis transformants, showed that the activity of P2 was upregulated two-fold under nutrient-depleted stationary phase of growth, while that of P1 remained unaltered while of P1 and P2 were low under hypoxia. Co-transcription of Mtufic, with the immediate upstream gene, Rv3642c, of unknown function, was observed. Taken together, the data strongly indicated that the expression of Mtufic gets altered under nutrient-depleted and hypoxic conditions, which are the stress conditions experienced by tubercle bacilli in granuloma in tuberculosis patients. Chapter 6. Functional Characterisation of Mycobacterial FtsZ-NDK Interaction During the past few decades, our laboratory has been carrying out extensive molecular and functional studies on the cytokinetic protein, FtsZ, of different mycobacterial species, and of a variety of other mycobacterial proteins that are believed to be interacting with the cell division machinery. In this regard, in parallel to the work on MtuFic, we carried out work on the identification and characterisation of the proteins that interact with mycobacterial FtsZ. In this context, we found for the first time that the nucleoside diphosphate kinase (NDK), which can generate NTPs from ATP/GTP and NDPs, interacts with FtsZ and that the interaction was conserved across several mycobacterial species. Therefore, the FtsZ-NDK interaction was extensively characterised in vitro, using the recombinant, purified FtsZ and NDK proteins from different mycobacterial species. This novel finding on the interaction of NDK with FtsZ adds another role to NDK, namely in bacterial cell division.