Helicoverpa armigera is an insect, causes important economical losses in crops. To reduce this loss, chemical insecticides such as pyrethroids have been commonly used against H. armigera in farming areas all over the world. However, excess and continuous usages of them cause resistance development in H. armigera. Insects develop resistance against applied insecticides by following three main mechanisms / by reducing the amount of insecticide entering into the insect body, developing insensitivity of the insecticide effective site and increasing detoxification metabolism of insecticides such as increased metabolism of them in midgut tissue of H. armigera. Therefore, changes in differentially expressed midgut proteins were analysed at protein level with two-dimensional gel electrophoresis (2D-PAGE) and matrix-assisted laser desorption/ionization-time-of-flight-mass spectrometry (MALDI-TOF-MS) together with examine biochemical activity changes of certain detoxification enzymes such as esterases (EST) and glutathione S-transferases (GST). Moreover, transcriptional level analysis of certain genes from EST and GST systems together with cytochrome P450 monooxygenases (CYP450) system were done with quantitative real-time PCR method, too. According to the comparative proteome analysis, it was found that H. armigera field samples overcome pyrethroid stress mainly by increasing energy metabolism related proteins expressions such as ATP synthase, Vacuolar ATPase A and B and arginine kinase proteins. Furthermore, certain detoxification enzymes such as thioredoxin peroxidase and NADPH cytochrome P450 reductase were up-regulated in Mardin population, suggesting that they were actively participating in response to pyrethroid stress. NADPH cytochrome P450 reductase could play a role in detoxification of toxic pyrethroid metabolites such as 3-phenoxybenzaldehyde. However, while glutathione S-transferases (GSTs) were not found up-regulated in the comparative proteome analysis, biochemical assays (GST-CDNB, GST-DCNB and GST-PNBC) showed significant increases in enzyme activities in the Adana and in the Mardin field population, as compared to the susceptible strain. Furthermore, GST-DCNB and GST-PNBC activities showed significant increase in &Ccedil / anakkale population. As overcoming energy crisis may lead to an increase in oxidative stress, detoxification enzymes (GSTs and thioredoxin peroxidase) might be involved in pathways for eliminating toxic reactive oxygen species such as H2O2. Similarly, although esterases (EST) were not found as differentially expressed, biochemical assays for ESTs showed significant increases in enzymatic activities in the Adana and the Mardin field populations. Thus, ESTs are also proposed to be involved in developing resistance as an initiator of pyrethroid metabolism in H. armigera from Turkey. Quantitative real-time PCR results showed that while CYP9A14 gene expression was up-regulated in all analyzed field populations, CYP9A12 gene expression was up-regulated in both &Ccedil / anakkale and Mardin populations. CYP4S1 gene expression was also up-regulated only in Mardin field population. However, while CYP6B7 gene expression together with CYP9A12 and CYP4S1 genes expressions were down-regulated in Adana population, CYP6B7 gene expression was not significantly changed in both &Ccedil / anakkale and Mardin populations. In addition, GST, GSTX01 and ESTX018 gene expressions were not significantly changed in all field populations in comparison to susceptible population. Therefore, CYP9A14, CYP9A12 and CYP4S1 genes proposed to be involved in detoxification of toxic pyrethroid metabolites possibly through regulation of NADPH cytochrome P450 reductase. In conclusion, it is suggested that one of the main mechanisms of resistance development is increased energy metabolism in the midgut tissue of H. armigera which may be a general prerequisite for compensating the costs of energy-consuming detoxification processes.

Dissertation (Ph.D.)--University of South Florida, 2008. / Includes vita. Also available online. Includes bibliographical references.

Aescin is a mixture of several acidic triterpenoid saponin glycosides found in the extracts of the horse chestnut tree. Horse chestnut, Aesculus Hipoocastanum, is one of the 25 domestic species of Aesculus that are mostly large, ornamental shade trees. Although known to be poisonous, the nuts of the horse chestnut are used by Amerindians, after detoxification. Horse chestnuts are said to have several traditional medicinal usages including even cancer. In this study the biochemical, genotoxic, and cytotoxic effects of aescin was studied using isolated lymphocytes, whole blood lymphocytes and HL-60 promyeloid leukemia cell lines. Cytotoxicity of aescin was examined by trypan blue viability staining of the cells in culture treated with varying aescin concentrations. It was observed that aescin was cytotoxic at all concentrations, for all cell types studied, except whole blood lymphocytes, where it was not cytotoxic at 10-9 and 10-10 M concentrations. Genotoxicity of aescin was examined by sister chromatid exchange and micronucleus. The genotoxic effect of Aescin was observed to be more significant over isolated lymphocytes compared to other cell lines. On the otherhand, aescin at 10-8 M and lower concentrations were observed to be non-genotoxic over whole blood lymphocytes whereas this concentration was considerably toxic for isolated lymphocytes and for HL-60 cell lines. Apoptotic properties of aescin were determined by DNA fragmentation, cytochrome c release and negative NAPO staining. All the Aescin concentrations tested resulted in apoptosis over HL-60 cell lines, whereas necrosis was not observed. However, isolated lymphocytes showed both apoptosis and necrosis upon treatment with 10-6 M to 10-8 M aescin, exhibiting apoptosis only at 10-9 M and 10-10 M. Biochemical effects of aescin were investigated by following GST and NAT enzyme activities. An increase in GST enzyme activity was observed over all cell lines treated with increasing aescin concentrations for 72 hours. Whereas NAT activity was decreased upon treatment with aescin in similar manner.

Diss. (sammanfattning) Umeå : Univ., 2007. / Härtill 4 uppsatser.

Dissertation (Ph.D.)--University of South Florida, 2008. / Title from PDF of title page. Document formatted into pages; contains 187 pages. Includes vita. Includes bibliographical references.

La détoxication est un processus biochimique présent chez tous les organismes biologiques et qui leur permet d’assurer leur survie face aux xénobiotiques provenant de leur environnement. Les glutathion transférases (GST) représentent une large famille d’enzymes participant à la phase II de détoxication en conjuguant le glutathion au composé à éliminer. Par ailleurs, certaines GST ont un rôle non catalytique et assurent la séquestration ou le transport de molécules d’un compartiment cellulaire à un autre. Alors que l’activité catalytique des GST est étudiée depuis plusieurs décades, l’identification précise des molécules physiologiques ciblées par les GST reste un défi. Chez les organismes fongiques dégradeurs de bois, certaines classes de GST se sont multipliées au niveau génomique. Cette redondance serait le reflet de la diversité des molécules chimiques libérées lors de la dégradation du bois. Dans cette thèse, des approches biochimiques et structurales ont été employées pour caractériser onze isoformes de GST du basidiomycète Trametes versicolor. De plus, une approche utilisant des librairies de molécules a permis d’identifier une famille de ligands reconnus par ces GST : les polyphénols. Les modes d’interaction de ces ligands ont été décrits précisément à partir de la résolution de nombreuses structures cristallographiques. L’identification d’un flavonoïde à partir d’un extrait de bois de merisier (Prunus avium), arbre sur lequel croît T. versicolor, a été permise par une approche de cristallographie d’affinité. Ces données suggèrent que les GST d’organismes fongiques saprotrophes pourraient prendre en charge les polyphénols libérés lors de la décomposition du bois / The ubiquitous biochemical process that enables each organism to cope with xenobiotics from its environment and thus ensures its survival is called detoxification. Glutathione transferases (GSTs) form a large family of enzymes divided into several classes. These enzymes participate in the detoxification phase II by conjugating the tripeptide glutathione to the molecule to be eliminated. Moreover, some GSTs are involved in non-catalytic processes such as sequestration or transport of molecules from one cellular compartment to another. Studies dedicated to the catalytic activity of GSTs have been ongoing for decades, yet precise identification of molecules targeted by GSTs remains challenging. In wood-decaying organisms, some of the GST classes have expanded with an increase of the number of isoforms encoded at the genomic level. This redundancy would reflect the diversity of the small molecules released upon wood enzymatic degradation. Through this thesis work, biochemical and structural approaches were used in order to characterize eleven GST isoforms from the saprotrophic fungus Trametes versicolor. In addition, the use of libraries of molecules helped in identifying polyphenols as a family of ligands that bind these GSTs. The molecular interaction modes were described precisely based on the resolution of numerous crystal structures. The identification of a flavonoid from an extract of the wild-cherry tree (Prunus avium) on which T. versicolor grows, was enabled by using an affinity crystallography approach. These data suggest that fungal GSTs could interact with plant polyphenols released during wood degradation

Arabinogalactan proteins (AGPs) are proteoglycans heavily substituted by arabinogalactan polysaccharides. These are composed of arabinose and galactose, and minor sugars such as glucuronic acid (GlcA), fucose and xylose. The arabinogalactan polysaccharides do not decorate classical AGPs exclusively, but they can also be found decorating a wide range of proteins. Arabinogalactan proteins have been implicated in many processes of plant development. Recently, AGPs were proposed to bind and store calcium at the plasma membrane. They are extracellular, and are localised mainly at the plasma membrane via a GPI-anchor. They can also be soluble in the apoplast. Their low abundance, chemical similarity and high functional redundancy have hindered their study. My strategy to overcome these difficulties was to study knock-out Arabidopsis thaliana plants of glycosyltransferases that transfer sugars specifically onto AG-polysaccharides. Glucuronic acid makes up about 10% of the arabinogalactan polysaccharide structure in Arabidopsis thaliana cell culture AGPs. Previously, the glucuronic acid transferase A TGLCA T14A, a member of the CAZy Glycosyl Transferase 14 family, was shown to transfer GlcA specifically onto AGPs, and knock-out Arabidopsis plants showed a 30% reduction in [Me]GlcA substitution in AGP-enriched preparations. However, no clear growth phenotype was observed. The characterisation of knock-out plants of other GT14 family members and combinations thereof is described here. Based on previous studies (Lamport and Várnai, 2013), I assayed in vitro the calcium binding capacity of AGP extracts from WT and knock-out plants. The results showed that AGP extracts from knock-out plants can hold less calcium than WT plants in vitro. A wide range of plant growth phenotypes were identified. Growth phenotypes can be explained by changes in the cytoskeleton and deficiencies in calcium signaling. Our evidence suggests links between structural deficiencies of extracellular proteoglycans to extracellular calcium and cytoskeleton. This research has the potential to create a new model system for the study of molecular mechanisms dependent on calcium that drive cell expansion, division and differentiation in plants.

Heavy chain isotype switch recombination is preceded by the appearance of RNA initiating 5' of the specific switch region which will undergo recombination. In an effort to understand the potential function of germline transcripts in switch recombination and the degree to which the regulation of germline transcripts correlates with the regulation of switching, we studied this process in the murine B-lymphoma cell line I.29μ, which in the presence of bacterial lipopolysaccharide (LPS) switches primarily to IgA and less frequently to IgE. Levels of α-germline transcripts initiating upstream of α switch (Sα) sequences are elevated in clones of this line which switch well as compared to clones which switch less frequently. TGFβ1 has been shown to increase α-germline transcripts and switching to IgA expression in LPS-stimulated murine splenic B-cells. We now demonstrate in I.29μ cells that TGFβ also increases switching to IgA and increases the level of α-germline transcripts 5 to 9 fold. Nuclear run-on analysis shows that this increase is at the level of transcription. Thus, TGFβ appears to direct switching to IgA by inducing transcription from the unrearranged Sα- CαDNA segment. Germline α RNA is quite stable in I.29μ cells, having a half life of about 3 to 5 hours, and we find only slight stabilization in the presence of TGFβ. Levels of ε-germline transcripts are not increased by TGFβ . IL-4, which modestly increases switching to IgA in I.29μ cells, slightly increases trancription of α-germline RNA. However, we present evidence suggesting that endogenously produced IL-4 may also act at additional levels to increase switching to IgA. IFNγ, which reduces IgA expression in these cells, also reduces the level of α-germline transcripts. IFNγ also reduces the level of ε-germline transcripts induced by IL-4. Our results support the hypothesis that the regulation of transcription of particular switch sequences by cytokines in turn regulates the specificity of recombination. In studies aimed at identifying other signalling pathways that promote class switching, we discovered that inhibitors of the nuclear enzyme poly(ADP-ribose) polymerase (PARP) increase lipopolysaccharide (LPS)-induced switching to IgA in the B cell lymphoma I.29μ and to IgG1 in LPS + IL-4-treated splenic B cells. PARP, which binds to and is activated by DNA strand breaks, catalyzes the removal of ADP-ribose from NAD+ and poly(ADP-ribosylation) of chromatin-associated acceptor proteins. This enzyme is believed to function in cellular processes involving DNA strand breaks as well as in modulating chromatin structure. In I.29μ cells, PARP inhibitors increase IgA switching by day 2 and cause a 5-fold average increase in switching on day 3 as assayed by immunofluorescence microscopy. The PARP inhibitor, nicotinamide, also causes a reduced intensity of hybridization of Cμ and Cα specific probes to genomic DNA fragments containing the expressed VDJ-Cμ and the unrearranged Sα - Cα segments, respectively, indicating that PARP inhibition increases rearrangment of these fragments. Induction of switching by PARP inhibitors is not mimicked by treatment with cAMP analogs or reduced by inhibitors of protein kinase A (PKA). Induction of switching by PARP inhibitors does not appear to involve increased levels of transcription of the unrearranged Cα gene, although TGFβ is required for optimal induction by PARP inhibitors, consistent with a requirement for transcription of the unrearranged CH gene. PARP inhibitors do not overcome the requirement for endogenously produced IL-4.

The glycophosphatidylinositol(GPI)-linked membrane protein ART2 is an antigenic determinant for T lymphocytes that regulate the expression of diabetes in the BB/W rat model. Though little is understood of the physiologic role of ART2 on T lymphocytes, ART2 is a member of the mono-ADP-ribosyl transferase subgroup ofthe ADP-ribosyl transferase (ART) protein family. The ART protein family, which traditionally has been divided into mono-ADP-ribosyl transferases (mono-ARTs), poly(ADP)-ribose polymerases (PARPs), and ADP-ribosyl cyclases, influences various aspects of cellular physiology including: apoptosis, DNA damage repair, chromatin remodeling, telomere replication, cellular transport, immune regulation, neuronal function, and bacterial virulence. A structural alignment of ART2.2 with chicken PARP indicated the potential for ART2.2 to catalyze ADP-ribose polymers in an activity thought to be specific to the PARP subgroup and important for their regulation of nuclear processes. Kinetic studies determined that the auto-ADP-ribosyl transferase activity of ART2.2 is multitmeric and heterogeneous in nature. Hydroxylamine-cleaved ADP-ribose moieties from the ART2.2 multimers ran as polymers on a modified sequencing gel, and digestion of the polymers with snake-venom phosphodiesterase produced AMP and the poly(ADP)ribose-specific product, PR-AMP, which was resolved by analytical HPLC and structurally confirmed by ESI-MS. The ratio of AMP to PR-AMP was higher than that of PARP raising the possibility that the ART2.2 polymers had a different branching structure than those of PARP. This alternative branching was confirmed by the presence of ribose phosphate polymers in the snake venom phophodiesterase treated samples. The site of the auto-poly(ADP)-ribose modification was determined to be R185, a residue previously proposed to influence the level of auto-ADP ribosylation of ART2.2 by mutational analysis. These data provide the first demonstration of a hybrid between mono-ARTs and PARPs and are the earliest indication that PARP-like enzymes can exist outside the nucleus and on the cell surface.

The "RNA world" hypothesis makes two predictions that RNA should have been able both to catalyze RNA replication and to direct protein synthesis. The evolution of RNA-catalyzed protein synthesis should be critical in the transition from the RNA world to the modem biological systems. Peptide bond formation is a fundamental step in modem protein biosynthesis. Although many evidence suggests that the ribosome is a ribozyme, peptide bond formation has not been achieved with ribosomal RNAs only. The goal of this thesis is to investigate whether RNA could catalyze peptide bond formation and how RNA catalyzes peptide bond formation. Two systems have been employed to approach these questions, the ribozyme system and the ribosome system. Ribozymes have been isolated by in vitro selection that can catalyze peptide bond formation using the aminoacyl-adenylate as the substrate. The isolation of such peptide-synthesizing ribozymes suggests that RNA of antiquity might have directed protein synthesis and bolsters the "RNA world" hypothesis. In the other approach, a novel assay has been established to probe the ribosomal peptidyltransferase reaction in the presence of intact ribosome, ribosomal subunit, or ribosomal RNA alone. Several aspects of the peptidyltransfer reaction have been examined in both systems including metal ion requirement, pH dependence and substrate specificity. The coherence between the two systems is discussed and their potential applications are explored. Although the ribozyme system might not be a reminiscence of the ribosome catalysis, it is still unique in other studies. The newly established assay for ribosomal peptidyltransferase reaction provides a good system to investigate the mechanism of ribosomal reaction and may have potential application in drug screening to search for the specific peptidyltransferase inhibitors.