Hannover, Universiẗat, Diss., 1998.

Hannover, Universiẗat, Diss., 2004.

In the quest to understand the physiological basis of postharvest needle loss in balsam fir, we hypothesized that, volatile terpene compounds (VTCs) have a role in needle abscission. This study focused on understanding the role of VTC’s in postharvest needle abscission. We demonstrated that balsam fir contains twelve VTCs with varying concentrations depending on whether it is a seedling or a clonal tree branch. Total VTC concentration consistently increased prior to needle loss. Five specific VTCs (?-Pinene, ?-Terpinene, Fenchyl acetate, Camphene and 3-Carene) have been identified as possible key signal molecules in needle abscission. VTCs were synthesized independently of ethylene, thus VTCs can be a possible signal molecule to needle abscission. Exposure of branches to ethylene showed an increase in both ethylene and VTC however, total VTC concentration was below the threshold required to cause needle abscission.

Techn. Universiẗat, Diss., 2006--München.

Zugl.: München, Techn. Universiẗat, Diss., 2008.

Universiẗat, Diss., 2001--Jena.

No description available.

Plant sesquiterpene synthases catalyze the conversion of the linear substrate farnesyl diphosphate, FPP, into a remarkable array of secondary metabolites. These secondary metabolites in turn mediate a number of important interactions between plants and their environment, such as plant-plant, plant-insect and plant-pathogen interactions. Given the relative biological importance of sesquiterpenes and their use in numerous practical applications, the current thesis was directed towards developing a better understanding of the mechanisms employed by sesquiterpene synthases in the biosynthesis of such a diverse class of compounds. Substrate preference for sesquiterpene synthases initially isolated from Nicotiana tabacum (TEAS), Hyoscyamus muticus (HPS) and Artemisia annuna (ADS) were optimized with regards to a divalent metal ion requirement. Surprisingly, careful titration with manganese stimulated bona fide synthase activity with the native 15-carbon substrate farnesyl diphopshate (FPP) as well as with the 10-carbon substrate geranyl diphosphate (GPP). Reaction product analysis suggested that the GPP could be used to investigate early steps in the catalytic cascade of these enzymes. To investigate how structural features of the sesquiterpene synthases translate into enzymatic traits, a series of substrate and active site residue contacts maps were developed and used in a comparative approach to identify residues that might direct product specificity. The role and contribution of several of these residues to catalysis and product specificity were subsequently tested by the creation of site-directed mutants. One series of mutants was demonstrated to change the reaction product to a novel sesquiterpene, 4-epi-eremophilene, and while another series successfully transmutated TEAS into a HPS-like enzyme. This is the first report of a rational redesign of product specificity for any terpene synthase. The contact map provides a basis for the prediction of specific configurations of amino acids that might be necessary for as yet uncharacterized sesquiterpene synthases from natural sources. This prediction was tested by the subsequent isolation and validation that valencene synthase, a synthase from citrus, did indeed have the amino acid configuration as predicted. Lastly, an in vitro system was developed for analyzing the interaction between sesquiterpene synthases and the corresponding terpene hydroxylase. Development of this in vitro system is presented as a new important tool in further defining those biochemical features giving rise to the biological diversity of sesquiterpenes.

Specialized metabolites have important roles as infochemicals in inter- and intraspecific interactions of insects. A particularly abundant class of specialized metabolites are terpenes, which are released by many members of taxonomically diverse insect lineages as pheromone and defense compounds. Despite the broad occurrence of terpenes in insects, knowledge of their biosynthesis remains limited compared to that in other forms of life. Terpenes are biosynthetically produced by the action of terpene synthase (TPS) enzymes. While insects lack TPS enzymes found in plants and microbes, there is growing evidence that insect TPS proteins have evolved independently from isoprenyl diphosphate synthase (IDS) enzymes in core terpene metabolism. To gain deeper insight into the transition from IDS to TPS function, I have explored the genomic and functional evolution of TPS enzymes in representatives of major insect lineages. First, I investigated evolutionary and functional relationships of TPS enzymes with roles in pheromone biosynthesis in pentatomids (stink bugs) including the invasive and economically critical pests Nezara viridula (Southern green stink bug) and Halyomorpha halys (brown marmorated stink bug). I also performed a comprehensive phylogenetic analysis of TPS genes in species across the broader order of piercing-sucking insects (Hemiptera), which provided evidence for an ancient emergence of TPS function in this group of insects. To gain a better understanding of core structural determinants of insect TPS evolution, we next defined distinct IDS catalytic motifs that are consistently substituted in enzymes with TPS function. These sequence characteristics were used to make predictions of TPS functionality in a large dataset of insect proteins. I determined the evolutionary dynamics of predicted and known TPS and IDS enzymes through extensive phylogenetic analysis to make top-level inferences about the distribution and evolution of TPS function in insects. Using this knowledge, I further explored functional transitions and subfunctionalization of TPS genes in the large order of beetles (Coleoptera), and more specifically, in species of the lady beetle family (Cocinellidae) including the globally invasive pest, Harmonia axyridis. Comparative genome analyses and IDS/TPS gene functional characterizations revealed gene duplication patterns and enzyme transitions that suggest TPS function evolved in part through processes of subfunctionalization and bifunctional enzymatic states. Additionally, this study provided the first experimental evidence for the mitochondrial localization of terpene metabolism in insects. Lastly, I identified putative TPS enzymes in the American cockroach, Periplaneta americana, and conducted an investigation into their catalytic activity. I found first evidence for TPS enzymatic activity in Blattodea as the most anciently diverging order of terpene-emitting insects and made inferences on the relationship of these enzymes to characterized IDS and TPS proteins in other insects. Our findings in the American cockroach point to the potential independent evolution of TPS function in blattodean cockroaches and termites in types of IDS ancestors. This work significantly advances our understanding of the evolution, functional diversity, and biochemical properties of TPS enzymes in insects, highlighting their recurring pattern of parallel evolution from IDS ancestors and its significance as a model for the emergence of novel specialized functions in core metabolic enzymes. / Doctor of Philosophy / Insects use many types of chemicals for purposes of communication and defense. Terpenes represent a common and diverse class of natural chemicals, which are used by insects to send pheromone signals and to protect themselves from predators. Terpenes also occur in other kingdoms of life. For example, in plants, they are especially widespread, forming a large portion of their essential oil and floral scent compounds. In contrast to plants and other organisms, not much is known about how insects produce terpenes. Terpenes are made by proteins called terpene synthase (TPS) enzymes. TPS enzymes have been traditionally associated with plants and microbes but have not been found in any insect species. Instead, there is growing evidence that insects have developed their own versions of these enzymes, known as isoprenyl diphosphate synthase (IDS)-type TPS enzymes, from proteins with essential functions in metabolism. To learn more about these unique insect enzymes, we explored their evolution and activity in species of several different groups of insects. First, we investigated TPS enzymes that are required for the biosynthesis of pheromones in stink bugs including two agriculturally important pests, the Southern green stink bug and the brown marmorated stink bug. This research showed that the ability to make terpenes might be quite ancient in this group of insects compared to TPS enzymes in other insects. Next, we examined the protein structure of insect TPS enzymes and determined features that are characteristic for these types of enzymes. This information was used to predict the occurrence of TPS proteins and their evolution across many different groups of insects. In particular, I found evidence for the emergence of TPS enzymes in lady beetles, with a focus on the invasive Asian lady beetle, which emits a terpene pheromone for aggregation. My research suggested that lady beetle TPS enzymes evolved through a process called subfunctionalization, where genes duplicate and progressively split their ancestral functions with new features to evolve novel functions. This study also provided the first evidence that insects might produce terpenes in their mitochondria, a part of the cell known for energy production. Finally, I discovered potential TPS enzymes in the American cockroach. My investigation showed that cockroaches and termites, both part of the oldest-diverging group of terpene-releasing insects, may have independently developed their own TPS enzymes from different ancestor proteins. Overall, this research helps us understand how insects produce chemical compounds important to their biology and ecology and how these abilities have evolved over time. This knowledge can be useful in agriculture, pest control, and for our understanding of insect biology.

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / O Acetato de lupeol (FAL), isolado do lÃtex extraÃdo do caule de Himatanthus drasticus (APOCYNCEAE), à quimicamente classificado como sendo um triterpeno pentacÃclico pertencente à classe do lupano, foi avaliado em modelos de nocicepÃÃo e inflamaÃÃo. No teste das contorÃÃes abdominais induzidas por Ãcido acÃtico (10 ml/kg, i.p.) em camundongos, a FAL (50 e 100 mg/kg, i.p.) reduziu de forma significativa o nÃmero de contorÃÃes abdominais em 56 e 61%, respectivamente, e a indometacina (10 mg/kg, i.p.) reduziu em 66%. No teste da formalina, a FAL (25 e 50 mg/kg, i.p.) reduziu de forma significativa o tempo gasto pelo animal lambendo a pata, tanto na fase inicial (21 e 46,5%, respectivamente) quanto na fase tardia (57,6 e 61,3%, respectivamente) e a morfina (7,5 mg/kg, i.p.) reduziu em 62 e 91%, respectivamente. O prÃ-tratamento com Naloxona (2 mg/kg, i.p) reverteu de modo significativo, os efeitos da FAL e da morfina tanto na fase inicial quanto na tardia do teste da formalina. No edema de pata induzido por carragenina, a FAL (10, 25 e 50 mg/kg,i.p.) reduziu de modo significativo, o volume do edema na 1Â, 2 e 3 hora apÃs a aplicaÃÃo da carragenina (1%, 50μl, s.p.). AnÃlise histopatolÃgica do tecido de pata de camundongo submetido à carragenina, demonstrou reduÃÃes significativas no edema e do infiltrado celular. Na marcaÃÃo imunohistoquÃmica, em tecido de pata de camundongo submetida ao estÃmulo da carragenina, a FAL (50mg/kg, i.p.) promoveu uma discreta reduÃÃo na expressÃo de TNF- α, porÃm causou uma significante reduÃÃo dos nÃveis de iNOS teciduais. No edema de pata induzido por dextrano, a FAL (12,5 e 25 mg/kg, i.p.) reduziu de modo significativo, o volume do edema na 2 e 3 hora apÃs a aplicaÃÃo de dextrano (12%, 50μl, s.p.). Na peritonite induzida por carragenina, a FAL (1, 10 e 20 mg/kg, i.p.) diminuiu de forma significativa, o nÃmero de leucÃcitos em 56, 80 e 92%, respectivamente. A Pentoxifilina (1 e 25mg/kg, i.p.) inibiu em 39 e 68%, respectivamente o nÃmero de leucÃcitos. No teste da inibiÃÃo da atividade da enzima mieloperoxidase (MPO), a FAL (10, 25, 50 e 100 μg/ml) reduziu a atividade da MPO em 36, 80, 79 e 74 %, respectivamente. A FAL nÃo demonstrou atividade antioxidante no teste do DPPH. Em conjunto, esses dados revelam que a FAL apresenta atividade antinociceptiva, que pode ser explicada pela habilidade deste composto em mimetizar efeitos de opiÃides endÃgenos, e antiinflamatÃria, explicada pela diminuiÃÃo da expressÃo de TNF-α e iNOS, bem como pela diminuiÃÃo da atividades da mieloperoxidase, resultando na inibiÃÃo da migraÃÃo de leucocitÃria para o foco da inflamaÃÃo. / The lupeol acetate (FAL), isolated from the latex extracted of the stem of Himatanthus drasticus (APOCYNCEAE) is chemically classified as a pentacyclic triterpene belonging to the lupane class, was evaluated in nociception and inflammation models. In the writhing test induced by acetic acid (10 ml/kg, i.p.) in mice, FAL (50 and 100 mg/kg, i.p.) significantly reduced the number of writhing in 56 and 61%, respectively, and indomethacin (10 mg/kg, i.p.) reduced by 66%. In the formalin test, FAL (25 and 50 mg/kg, i.p.) significantly reduced the time spent by the animal licking the paw, both in the initial phase (21 and 46.5%, respectively) and in the late phase (57 , 6, and 61.3%, respectively) and morphine (7.5 mg/kg, i.p.) reduced by 62 and 91%, respectively. Pretreatment with naloxone (2 mg/kg, i.p.) significantly reversed the effects of FAL and morphine in both the early and in late phase in formalin test. In the carrageenan induced paw oedema, FAL (10, 25 and 50 mg/kg, i.p.) reduced in significant way the oedema volume in the 1st, 2nd and 3rd hour after carrageenan application (1%, 50μl, s.p.). Histopathologic analysis of mice paw tissue subjected to carrageenan, showed significant reductions in oedema and cellular infiltration. In the immunohistochemical staining in mice paw tissue subjected to carrageenan stimulus, the FAL (50mg/kg, i.p.) induced a slight reduction in the expression of TNF-α, but caused a significant reduction of tissue iNOS levels. In the dextran induced paw oedema, FAL (12.5 and 25 mg/kg, i.p.) significantly reduced the oedema volume in the 2nd and 3rd hour after dextran application (12%, 50μl, s.p.). In carrageenan-induced peritonitis, FAL (1, 10 and 20 mg/kg, i.p.) significantly reduced the number of leukocytes at 56, 80 and 92%, respectively. Pentoxifylline (1 and 25mg/kg, i.p.) inhibited by 39 and 68%, respectively, the number of leukocytes. In the myeloperoxidase (MPO) enzyme inhibition test, FAL (10, 25, 50 and 100 μg/ml) reduced the activity of MPO at 36, 80, 79 and 74%, respectively. The FAL showed no antioxidant activity in DPPH test. Together, these data reveal that the FAL has antinociceptive activity, which can be explained by its ability in mimicking the endogenous opioids effects, and antiinflammatory, explained by TNF-α and iNOS expression decreased, as well as by the myeloperoxidase activity decreasing, resulting in inhibition of leukocyte migration to the focus of inflammation.