Immunosenescence results in reduced immune response to infections with Streptococcus pneumoniae as well as to pneumococcal polysaccharide vaccines. The antibody response to the capsular polysaccharide (CPS) provides protection against S. pneumoniae infection. CPS immunoresponse is T cell independent and needs the macrophage-derived cytokines such as IL-12, IL-6 and IL-1β to elicit an antibody response. We showed a cytokine dysregulation, i.e. a decrease in IL-12, IL-6 and TNF-α but an increase in IL-10, in the aged (18-24 months old comparable to >65 years in human) compared to young adult mouse (8-12 weeks less than 65 years old) splenic macrophages (SM) or bone marrow derived macrophages (BMDM) activated via TLR4, TLR2 or TLR9 as well as heat killed Streptococcus pneumoniae (HKSP). There is also an age-associated defect in splenic B cells in the production of IgG3 upon stimulation with these ligands. A microarray analysis in SM followed by validation by both qt-RTPCR and western blots indicated that this age-associated defect in aged SM, BMDM and B cells was due to a heightened activity of the PI3K-Akt signaling pathway. We hypothesized that the senescence of immune responses in macrophages and B cells is due to an increase in activity of PI3K/Akt and decrease in the activity of GSK-3, the downstream kinase. Inhibition of the PI3-kinase with either LY294002 or Wortmannin restored the TLR2, 4, 9 and HKSP induced cytokine phenotype of the aged to that of the young adult in both the SM and BMDM and an enhanced IgG3 production in aged mice. We also showed that inhibition of glycogen synthase kinase-3 (GSK-3) the downstream target of the PI3K-Akt signaling pathway with SB216763 in SM, BMDM and B cells resulted in an enhancement in production of IL-10, IL-6 and IL-1β by macrophages and in B cell activation. Treatment of B cells with SB216763 in the presence of ligands for TLR-1/2, 4 or 9 as well as HKSP under in vitro conditions led to enhanced production of IgG3 and IgA, plasma cell formation and a slight increase in the proliferation of the B-cells with no adverse effects on the viability of the cells. Therefore, targeting the PI3K-AKT-GKS-3 signaling pathway could rescue the intrinsic signaling defect in the aged macrophages, increase IL-12 and IL-6, and enhance anti-CPS antibody responses.

Mitochondria are essential for promoting cell survival and growth through aerobic metabolism and energy production. Mitochondrial function is typically analyzed using mitochondria freshly isolated from tissues and cells because they yield tightly coupled mitochondria, whereas those from frozen tissue can consist of broken mitochondria and membrane fragments. A method, utilizing a well-characterized cryoprotectant such as dimethyl sulfoxide (DMSO), is described. Such mitochondria show preserved structure and function that presents us with a possible strategy to considerably expand the time-frame and the range of biochemical, molecular and metabolic studies that can be performed without the constraints of mitochondrial longevity ex vivo. Mitochondrial dysfunction is implicated in Alzheimer’s disease (AD) mainly through oxidative stress and altered metabolism. Mitochondria are isolated from post-mortem brain samples from selective regions of AD and control patients and, utilizing the cryopreservation strategy, analyzed for respiration and oxidative damage. While we did not observe increases in free radicals, we did observe decreased respiration and increases in oxidative damage markers in AD patients, suggesting a role for oxidative stress in mitochondrial dysfunction. While in the mitochondria, calcium (Ca2+) increases free radical generation by processes not completely understood. A new isoform of nitric oxide synthase (mtNOS) has been isolated and localized to mitochondria; though its existence and physiological role is debated. Nitric oxide synthase (NOS), when activated by Ca2+, produces nitric oxide (NO•) that can interact with ROS producing various reactive nitrogen species (RNS). These highly reactive radical species can damage DNA, proteins and lipids, ultimately resulting in cell death via apoptosis or necrosis. The current research is aimed at understanding the role of Ca2+ and NOS in oxidative stress leading to mitochondrial dysfunction. We observed a significant reduction in mitochondrial respiration with increasing doses of calcium. We also observed NOS enzyme activity and detected NOS protein in the purified mitochondrial fraction. Lastly, we were also able to show that Ca2+ increased the levels of free radicals and changes in oxidative damage markers. These results suggest the presence of NOS in mitochondria that could play a role in Ca2+ induced mitochondrial dysfunction and potentially leading to cell death as relevant to aging and neurodegenerative diseases.

α-Isopropylmalate synthase (α-IPMS) catalyses the first committed step in leucine biosynthesis in bacteria, including Neisseria meningitidis and Mycobacterium tuberculosis. It catalyses the condensation of α-ketoisovalerate (α-KIV) and acetyl coenzyme A (AcCoA) to form α-isopropylmalate (α-IPM). Like many key enzymes in biosynthesis, α-IPMS is inhibited by the end-product of the biosynthetic pathway, in this case leucine. α-IPMS is homodimeric, with monomers consisting of a (β/α)8-barrel catalytic domain, two subdomains and a C-terminal regulatory domain, responsible for binding leucine and providing feedback inhibition for leucine biosynthesis. The exact mechanism of feedback inhibition in this enzyme is unknown, despite the elucidation of crystal structures with and without leucine bound. This thesis explores the nature of allosteric regulation in α-IPMS, including the effects of the regulatory domain and the importance of structural asymmetry on catalytic activity. Chapter 2 details the characterisation of wild-type α-IPMS from N. meningitidis (NmeIPMS). This protein was successfully cloned, expressed and purified by metal-affinity and size-exclusion chromatography. NmeIPMS has similar characteristics to previously characterised α-IPMSs, being a dimer and demonstrating substrate binding affinities in the micromolar range. This enzyme has a turnover number of 13s⁻¹ and is sensitive to mixed, non-competitive inhibition by the amino acid leucine. Small angle X-ray scattering experiments reveal that the solution-phase structure of this protein is likely similar to existing crystal structures of other α-IPMSs. In Chapter 3, substitutions of residues potentially involved in the binding and transmission of the leucine regulatory mechanism are described. Most of these amino acid substituted variants reduce enzyme sensitivity to leucine, and one variant is almost entirely insensitive to this inhibitor. Another of these variants demonstrates an unexpected decrease in substrate affinity, despite the substituted residue being located far from the active site. The independence of α-IPMS domains is investigated in Chapter 4. The catalytic domains were isolated from NmeIPMS and the α-IPMS from M. tuberculosis (MtuIPMS), and found to be unable to catalyse the condensation of substrates, despite maintaining the wild-type structural fold. Complementation studies with Escherichia coli cells lacking the gene for α-IPMS show that the truncated variants are unable to rescue growth in these cells. Binding of α-KIV in the truncated NmeIPMS variant is much stronger than in the wild-type, and this may be the reason for lack of competent catalysis. A crystal structure was solved for the truncated variant of NmeIPMS and indicates that the regulatory domain is required for proper positioning of large regions of the protein. Two isolated regulatory domains from NmeIPMS were cloned, but with limited success in characterisation. Finally, Chapter 5 describes substitutions made in MtuIPMS to affect relative domain orientations within the protein. Dimer asymmetry is investigated by substituting residues at the domain interfaces. These substitutions did have some effect on catalysis and inhibition, but did not show any change in average solution-phase structure. These results are drawn together in the greater context of allostery in general in Chapter 6, along with ideas for future research in this field. This chapter reviews the insights gained into protein structure from this thesis, particularly the importance of residues at protein domain interfaces. The asymmetry in the α-IPMS structure is discussed, along with small-molecule binding regulatory domains.

Borage (Borago officinalis) is an annual herb plant for culinary and medicinal uses. Due to a high level of gamma-linolenic acid (GLA) in its seed oil and the health-related benefits of GLA, borage is commercially cultivated. However, a herbicide-resistant variety has not yet been developed for effective weed management in borage farming. Thus, this thesis aimed to create, identify and characterize ethyl methanesulfonate (EMS) induced borage mutants for herbicide imidazolinone resistance. An EMS-mutagenized borage population was generated by using a series of concentrations of EMS to treat M1 seeds. After screening M2 borage plants with the herbicide, tolerant plants were selected, self-pollinated and grown to their maturity. The offsprings were subjected to herbicide screening again to confirm the phenotype, resulting in identification of two genetically stable imidazolinone-resistant lines. Two acetohydroxyacid synthase (AHAS) genes, AHAS1 and AHAS2, involved in the imidazolinone resistance were isolated and sequenced from both mutant (resistant) and wild type (susceptible) borage plants. Comparison of these AHAS sequences revealed that a single nucleotide substitution occurred in the AHAS1 resulting in an amino acid change from serine (S) in the susceptible plant to asparagine (N) in the first resistant line. The similar substitution was later found in the AHAS2 of the second resistant line. A KASP marker was developed for the AHAS1 mutation to differentiate the homozygous susceptible, homozygous and heterozygous resistant borage plants for the breeding purpose. The in vitro assay showed homozygous resistant borage containing the AHAS1 mutation could retain significantly higher AHAS activity than susceptible borage across different imazamox concentrations. The herbicide dose response test showed that the resistant line with the AHAS1 mutation was tolerant to four times the field applied concentration of the “Solo” herbicide.

Les résultats obtenus dans ce travail ont permis de démontrer que chez les deux bactéries lactiques modèles, L. lactis subsp. cremoris et O. oeni, la transcription du gène cfa, codant pour la Cfa synthase, est stimulée lorsque les cellules entrent en phase stationnaire de croissance ou lorque que les cellules sont cultivées en conditions de stress (milieu acide ou présence d'éthanol dans le milieu). Le rôle des CFA a pû être appréhendé par l'analyse physiologique comparative de la souche parentale L. lactis subsp. cremoris et du mutant Δcfa généré chez cette souche. Les forts pourcentages de survie obtenus chez les souches cultivées à pH 5,0, puis subissant un stress acide (pH 3,0), prouvent que la cyclopropanation des acides gras insaturés n'est pas indispensable à la survie de L. lactis subsp. cremoris en conditions de stress acide. En outre, les données d'anisotropie de fluorescence démontrent que la présence de CFAs membranaires ne permet pas de réguler le niveau de fluidité membranaire, en particulier avec les souches cultivées en présence d'éthanol, où une fluidification membranaire est observée dans tous les cas. L'hypothèse d'un équilibre entre les acides palmitoléique / cis-vaccénique / lactobacillique pour réguler la fluidité membranaire chez L. lactis subsp. cremoris est avancée. L'étude du rôle physiologique des CFA chez O. oeni nécessite l'expression du gène cfa de la bactérie en système hétérologue. Les résultats obtenus confirment la fonctionnalité du gène chez la bactérie hôte L. lactis subsp. cremoris, cependant la cyclisation du précurseur acide cis-vaccénique, reste partielle chez la souche mutante complémentée. Un travail de caractérisation biochimique in vitro de l'enzyme Cfa synthase de O. oeni a donc été entrepris afin d'expliquer la faible efficacité de l'enzyme de O. oeni chez la bactérie hôte. Les travaux réalisés ont permis la surproduction et la purification de l'enzyme. Une technique de mesure d'activité in vitro a été également développée. Une stratégie d'expression du gène cfa de O. oeni en système hétérologue dans la bactérie hôte E. coli BL21 Star a permis la surpoduction d'une protéine d'environ 45 kDa, en accord avec la masse moléculaire théorique de la Cfa synthase de O. oeni. A partir de l'extrait protéique, une purification a été réalisée par chromatographie d'affinité sur colonne d'agarose nickel. Les tests d'activité enzymatique in vitro ont pu été réalisés. La température optimale de l'enzyme est de 35,8°C. Son pH optimal est de 5,6. Même en se plaçant dans les conditions physico-chimiques optimales de l'enzyme, nous constatons que la cyclisation in vitro de l'acide cis-vaccénique issu des phospholipides membranaires du mutant L. lactis subsp. cremoris Δcfa reste partielle. Ceci induit une détermination expérimentale de valeurs de KM et Vmax largement supérieures aux données citées dans la littérature. La différence, entre les deux bactéries modèles, de nature et de répartition des phospholipides membranaires pourrait expliquer l'action partielle de la Cfa synthase de O. oeni.

Systemic fungal infection is a life-threatening problem. Anti-fungal drugs are the most effective clinical strategy to cure such infections. However, most current anti-fungal drugs either have high toxicity or have a narrow spectrum of effect. Meanwhile, anti-fungal drugs are losing their clinical efficacy due to emerging drug resistance. To protect us from these deadly pathogenic fungi, scientists need to study new drug targets and to solve problems related to drug resistance. The cell wall is essential for fungal cell survival and is absent from animal cells, so it is a promising reservoir for screening safe and effective drug targets. Alpha-1,3-glucan is one of the major cell wall carbohydrates and is important for the virulence of several pathogenic fungi. In this thesis, molecular biology and microscopy techniques were used to investigate the function and the synthesis process of α-1,3-glucan in the model fungus A. nidulans. My results showed that α-1,3-glucan comprises about 15% of A. nidulans cell wall dry weight, but also that α-1,3-glucan does not have an important role in cell wall formation and cell morphology. Deletion of α-1,3-glucan only affects conidial adhesion and cell sensitivity to calcofluor white. In contast, elevated α-1,3-glucan content can cause severe phenotypic defects. To study the α-1,3-glucan synthesis process, I systematically characterized four proteins, including two α-1,3-glucan synthases (AgsA and AgsB) and two amylase-like proteins (AmyD and AmyG). Results showed AgsA and AgsB are both functional synthases. AgsB is the major synthase due to its constant expression. AgsA mainly functions in conidiation stages. AmyG is a cytoplasmic protein that is critical for α-1,3-glucan synthesis, likely being required for an earlier step in the synthesis process. In contrast to the other three proteins, AmyD has a repressive effect on α-1,3-glucan accumulation. These results shed light on therapeutic strategies that might be developed against α-1,3-glucan. I also developed a strategy to investigate drug resistance mutations. The tractability of A. nidulans and the power of next generation sequencing enabled an easy approach to isolate single mutation strains and to identify the causal mutations from a genome scale efficiently. I suggest this strategy has applications to study the drug resistance mechanisms of current anti-fungal drugs and even possibly future ones.

Fungal polyketides are synthesized by polyketide synthases (PKS) encoded by PKS genes. The function of many PKS genes is unknown and the number of PKS genes exceeds the number of polyketides in many genomes. The lichen-forming fungi, Cladonia and Ramalina have chemical variants separated by habitat suggesting that environmental conditions may influence polyketide production. The goal of this thesis was to examine evolutionary relationships as a framework to investigate PKS gene function in the lichen-forming fungal families Cladoniaceae and Ramalinaceae. A phylogenetic analysis of the genus Ramalina (Chapter 2) using nuclear and mitochondrial ribosomal DNA sequences showed monophyly for seven species and included three species, which were not examined in phylogenies prior to this study. One monophyletic species, R. dilacerata was chosen for further tests of the effect of growing conditions on PKS gene expression (Chapter 3). Growth media containing yeast extracts produced the largest colony diameters and the fewest number of polyketides. A significant negative relationship occurred between colony diameter and number of secondary metabolites. Expression of two types of PKS genes was correlated with pH-level and media conditions that produced larger numbers of secondary products in R. dilacerata. A PKS gene phylogeny was constructed for 12 paralogs detected in members of the C. chlorophaea complex (Chapter 4) and gene selection was inferred using dN/dS estimations. The gene phylogeny provided evidence for independent origins and purifying and positive selection of PKS paralogs. This research provided insight into the evolution of PKS genes in the C. chlorophaea complex and identified potential genes that produce non-reduced polyketides present in C. chlorophaea. This thesis provided evidence for diversification of both morphological and chemical species and monophyly of previously unstudied Ramalina species. This research also supported theories of secondary metabolite synthesis based on growing conditions of R. dilacerata, and it revealed that PKS genes under selection in the Cladonia chlorophaea group provide the lichen with the adaptive capacity to survive under variable conditions. Knowledge of the ecological function of fungal polyketides can be valuable for conservation management and policy makers; and for understanding the potential pharmaceutical roles of these natural products.

Increasingly, cell-based therapy with autologous progenitor populations, such as endothelial progenitor cells (EPC), are being utilized for treatment of vascular diseases. However, both the number and functional capacity are diminished when cells are derived from patients with established risk factors for coronary artery disease (CAD). Herein, we report that inhibition of glycogen synthase kinase 3 (GSK) can improve both the number and function of endothelial progenitor cells in patients with CAD or diabetes mellitus (DM) leading to greater therapeutic benefit. Specifically, use of various small molecule inhibitors of GSK (GSKi) results in a 4-fold increased number of EPCs. Moreover, GSKi treatment improves the functional profile of EPCs through reductions in apoptosis, improvements in cell adhesion through up-regulation of very-late antigen-4 (VLA-4), and by increasing paracrine efficacy by increasing vascular endothelial growth factor (VEGF)secretion. Therapeutic improvement was confirmed in vivo by increased reendothelialization(RE) and reductions of neointima (NI) formation achieved when GSKi-treated cells were administered following vascular injury to CD-1 nude mice. Because cell-based therapy is technically challenging, we also tested a strategy of local delivery of GSKi at the site of arterial injury through GSKi-eluting stents. In vitro, GSKi elution increased EPC attachment to stent struts. In vivo, GSKi-eluting stents deployed in rabbit carotid arteries resulted in systemic mobilization of EPCs, improved local RE, and important reductions in in-stent NI formation. Finally, we tested the ability of GSKi to improve EPC-mediated arterial repair in patients with DM. As in patients with CAD, GSKi treatment improved EPC yield and diminished in vitro apoptosis. Utilizing a proteomics approach, we identified Cathepsin B (catB) as a differentially regulated protein necessary for reductions in apoptosis. Indeed, antagonism of catB prevented GSKi improvements in GSKi treated EPC mediated arterial repair in a xenotransplant wire injury model. Thus, our data demonstrates that GSKi treatment results in improvements in EPC number and function in vitro and in vivo resulting in enhanced arterial repair following mechanical injury. Accordingly, GSK antagonism is an effective cell enhancement strategy for autologous cell-based therapy with EPCs from high risk patients such as CAD or DM.

No description available.

Fatty acid synthase (FAS) is over-expressed in many human cancers and its activity is required for cancer cell survival. To understand why FAS is over-expressed, we compared in breast cancer cells the utilization of fatty acids synthesized endogenously by FAS to those supplied exogenously in the culture medium. We found that endogenously synthesized fatty acids are esterified to the same lipid and phospholipid classes in the same proportions as those derived exogenously and that some endogenous fatty acids are excreted. Thus, FAS over-expression in cancer does not fulfill a specific requirement for endogenously synthesized fatty acids. We next investigated whether lipogenic activity mediated by FAS was, instead, involved in the maintenance of high glycolytic activity in cancer cells. By culturing breast cancer and non-cancer cells in anoxic conditions, we increased glycolysis 2-3 fold but observed no concomitant increase in lipogenesis. More research is needed to understand why FAS is over-expressed in cancer.