Adenine derivatives called cytokinins (CK) are a group of plant hormones, which in cooperation with other plant hormones orchestrates almost every aspect of plant growth and development. Despite the rapid progress in plant hormone research, there are still many aspects we may shed light on due to the metabolic and signalling pathways redundancy and the network complexity with crosstalk hubs. CK N-glucosides (in this case trans-zeatin-7-glucoside, tZ7G, and trans-zeatin-9-glucoside, tZ9G) have been traditionally viewed as irreversibly deactivated products of CK active form metabolism (in this regard trans-zeatin, tZ). Nevertheless, the tZ9G antisenescent activity was shown in oat leaf senescence bioassays (Avena sativa cv. Abel) and the possibility of metabolic conversion to O-glucosides was hypothesized. The aim of this work was to test the hypothesis on the close model of oat (Avena sativa cv. Patrik) and to examine also the metabolic conversion of related substances - N 6 -(Δ2 -isopentenyl)adenine (iP) and its N7- and N9- glucosides (iP7G and iP9G). While the senescence retardation caused by exogenous tZ9G application was confirmed in the Avena sativa cv. Patrik, metabolic conversions to O-glucosides remain to be verified. Besides the effects of above-mentioned substances on the oat leaf...

Skin sensitization resulting for allergic contact dermatitis (ACD) is an occupational and environmental health issue. The allergic hazard for workers and consumers is a serious problem for individuals, employers and marketing certain products. Consequently, it is necessary to accurately identify chemicals skin sensitization potential. According to the new EU chemical regulation REACH (Registration, Evaluation, Authorization and Restriction of Chemicals), information of skin sensitization of chemicals manufactured or imported at or above 1 ton/year should be available. Currently, valid approaches assessing skin sensitization rely on animal testing, such as local lymph node assay (LLNA). However, it now ultimately eliminates using animals for this purpose. Based on the fact that a key step in the skin sensitization process is formatting a covalent adduct between allergic sensitizers and proteins and/or peptides in skin, a lot of additional approaches are proposed and developed for replacing or reducing animal used. In this research, three bioassays, 24 h growth inhibition toward Tetrahymena pyriformis, long term (24 h) and short term (30 min) bacterial toxicity (to Vibrio fischeri), and a kinetic glutathione chemoassay are applied for predicting the organic chemicals’ skin sensitization potential. The major results and conclusions obtained are listed as follows: 1. Toxicity enhancement (Te) of 55 chemicals comprising different sensitization potencies were determined and compared with their narcotic toxicity to predict their skin sensitization. Three linear regressions yielded for all allergic sensitizer without nonsensitizers for each bioassay. The linear regressions are improved after classifying sensitizers into five different reaction mechanistic domains. Correspondingly, five different slopes from various reaction mechanisms indicate a decreased sensitivity of toxicity enhancement to skin sensitization potential with order SNAr > SN2 > acylation ≈ Schiff base > aromatic Michael addition. Based on the fact that a key step in the skin sensitization process is forming a covalent adduct between allergic sensitizers and proteins and/or peptides, Te > 10 as a threshold is applied to discriminate these allergic sensitizers, with 100% accuracy for strong (with extreme) and weaker sensitizers, up to 72% accuracy for moderate sensitizers and less than 69% accuracy for nonsensitizers. Compared with these bioassays, a decreasing order of sensitivities is 24 h growth inhibition (Tetrahymena pyriformis) > 24 h growth inhibition (Vibrio fischeri) > 30 min bioluminescence inhibition (Vibrio fischeri). These three bioassays are useful tools for screening sensitization potency of allergic chemicals, and the toxicity enhancement (Te) can be used to discriminate sensitizers from weak or nonsensitizers. However, in this context we should separate aromatic from aliphatic Mas (Michael acceptors). Moreover, metabolic biotransformation should be considered during predicting nonsensitizers’ skin sensitization. 2. Chemical reactivity of selected 55 compounds measuring through kinetic glutathione chemoassay applies to predict their skin sensitization. This chemoassay confirms the fact that the key step of sensitizers eliciting skin sensitization is formatting a covalent adduct between sensitizers and skin proteins or peptides. The chemical reactivity of tested sensitizers strongly relates with their sensitization potential, with strong (extreme) sensitizers presenting the highest reactivity as followed with moderate sensitizers, weak sensitizers as well as nonsensitizers. Moreover, an integrated platform of this chemoassay data and three bioassays data is performed, and this performance shows good sensitivity for monitoring skin sensitization potency, with more rational accuracy for each sensitizing classifications. 3. Thiol reactivity (kGSH) as well as toxicity enhancement (Te) of additional 21 aliphatic α,β-unsaturated compounds are determined for predicting their skin sensitization potential. The linear regressions of skin sensitization versus thiol reactivity and skin sensitization versus toxicity enhancement are significantly improved after classifying these 21 compounds to four chemical subgroups (acrylates, other esters, ketones and aldehydes). Thiol reactivity of these subgroups presented different sensitivity to skin sensitization, with a decreasing order as acrylates (－2.05) > other esters (－1.26) > ketones (－0.43) > aldehydes (－0.21). Moreover, thiol reactivity is confirmed to be a more sensitive tool for predicting skin sensitization, compared with toxicity enhancement. Although the datasets are probably too small to give a definite decision, hydrophobicity reveals contribution to skin sensitization for aliphatic MAs, which is different with literature report. This study suggests that aliphatic MAs should be treated separately into different chemical subgroups for analysis, and their skin sensitization potency can be predicted using kinetic glutathione chemoassay as well as toxicity enhancement bioassay.

Thesis (MSc (Botany and Zoology))--Stellenbosch University, 2011. / ENGLISH ABSTRACT: See full text for abstract / AFRIKAANSE OPSOMMING: sien volteks vir opsomming

Aufgrund ihrer gut dokumentierten, umfangreichen gesundheitsfördernden biologischen Aktivitäten wird den Flavonoiden eine große Bedeutung zugeordnet. Die Ergebnisse von in vitro- und Tierstudien deuten zudem darauf hin, dass diese Verbindungen bei der Prävention und Therapie von Erkrankungen wie Krebs oder Alzheimer Krankheit (AD) positive Effekte zeigen. Zur besseren Charakterisierung der Interaktion von Flavonoiden mit Krebszellen wurden von uns die Cytotoxizität verschiedener Flavonoide auf T-Lymphoblastomzellen untersucht und Strukturelemente identifiziert, welche für einen Flavonoid-induzierten Zelltod relevant sind. Weitere Studien waren der potentiell neuroprotektiven Wirkung von Flavonoiden gewidmet. Die sowohl in neuronalen Zellkulturen als auch in transgenen Alzheimer-Mäusen (TgAPPsw) festgestellte Erhöhung der sAPPα Produktion und Reduktion von Aβ-Bildung wurden mit Aktivitäts- und Expressionssteigerung der α-Sekretase ADAM-10 assoziiert. Um herauszufinden, ob Flavonoide eine neuroprotektive Wirkung zeigen, wurden erste Vorbereitungen für ein Flavonoid-Screening mit einer sowohl hAPP alsauch ADAM-10 stabil transfizierten HT1080 Zellen getroffen. Dies beinhaltete die Suche nach einer potenten siRNA/shRNA und einem effektiven Flavonoid. Im zweiten Teil der Arbeit wurden Experimente durchgeführt, um die Rolle von Heparansulfat (HS) bei der foamyviralen Anbindung an die Wirtszelle zu untersuchen. Foamyviren (FV) sind Spumaviren und gehören zur Familie der Retroviren. Bei unseren Studien wurde die Bindung von FV an Heparin, die Korrelation der Suszeptibilität verschiedener Zellen mit zellulärem HS und die Reduktion der Infektion durch lösliches Heparin sowie durch enzymatischen HS-Abbau festgestellt. / Flavonoids have well-documented, beneficial biological effects. Furthermore different in vitro- and animal-experiments indicate that these compounds demonstrate positive effects in prevention and therapy of diseases, like cancer or neurodegenerative diseases. In order to characterize the interactionsbetween flavonoids and cancer cells, we examined the cytotoxicity of different flavonoids on a human leukemia cell line and identified structure elements that could be associated to flavonoid-induced cell death. Our further studies were dedicated to the potentially neuroprotective activity of flavonoids. It has been described that in neuronal cells as well as in transgenic AD mice EGCG increases levels of sAPPα- and reduces Aβ-production. This influence of EGCG was associated with enhanced activity and expression of the α-secretase, ADAM-10. To find out, if flavonoids showed neuroprotective activity, preliminary work for a later flavonoid screening on hAPP and ADAM-10 wit stably-transfected HT1080 cells was done. This includes the determination of flavonoid candidates and research of effective siRNAs towards ADAM-10. Furtherrmore we investigated the role of heparansulfat (HS) in attachment of FV to host cell. Foamyviruses (FV) are retroviruses and belong to the subfamily of spumaretroviruses. In our studies we discovered the binding of FV on heparin, the correlation between susceptibility of different cells and cellular HS and the reduction of infectivity through soluble heparin and through enzymatic HS-degradation.

In der vorliegenden Arbeit werden Studien zur Identifizierung und Charakterisierung von Lebensmittelinhaltsstoffen als natürliche Liganden des Ah Rezeptors („aryl hydrocarbon receptor“) vorgestellt. Der Ah Rezeptor ist ein liganden-abhängiger Transkriptionsfaktor, der an der Expression zahlreicher Metabolismusenzyme beteiligt ist. Im Mittelpunkt unserer Untersuchungen anhand von funktionellen AhR-abhängigen Bioassays stand die Klasse der ß-Carboline und ihrer Derivate, deren natürliches Vorkommen bereits in zahlreichen Lebensmitteln und im menschlichen Organismus beschrieben ist. Die ß-Carboline wurden für die Untersuchung auf ihr mögliches AhR Ligandenpotential ausgewählt, da sie von der Aminosäure Tryptophan abgeleitet sind, die selbst als schwacher AhR Agonist identifiziert wurde, und weil das trizyklische 9H-Pyridol[3,4-b]-Ringsystem der ß-Carboline eine strukturelle Ähnlichkeit zum prototypischen AhR Liganden 2,3,7,8-Tetrachlordibenzo-p-dioxin (TCDD) aufweist. Die Schwerpunkte der Untersuchungen zur AhR Ligandenaktivität von ß-Carbolinen lagen auf den Aspekten: 1) Etablierung von rekombinanten, zellbasierten funktionellen Reportergenassays; 2) Identifizierung und Charakterisierung von ß-Carbolinderivaten als Liganden des Ah Rezeptors mittels funktioneller Reportergenassays; 3) Charakterisierung mechanistischer Aspekte im AhR vermittelten Signaltransduktionsweg mittels in vitro und ex vivo Gelretardation Assays am Beispiel ausgewählter ß-Carbolinderivate mit AhR Ligandenaktivität und 4) Beschreibung der AhR Ligandenaktivität von Soja- und Würzsaucen als Modellsysteme für komplexe natürliche Lebensmittel. / This research summarizes studies on the identification and characterization of compounds present in food samples, which can act as ligands of the aryl hydrocarbon receptor (Ah receptor, AhR). The Ah receptor represents a ligand-dependent transcription factor and is involved in the expression of various enzymes of the xenobiotic metabolism. Our analyses using functional AhR-dependent bioassays were focusing on ß-carbolines and their derivatives. These compounds have been described to be naturally occurring in foods and in the human body. The ß-carbolines were chosen to be evaluated for their potential to act as AhR ligands because they are derived from the amino acid tryptophan, which is a weak AhR agonist itself, and due to their structural resemblance to the tricyclic 9H-pyridol[3,4-b] ring system of the ß-carbolines to the prototypic AhR ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). As the main focus of the studies was to evaluate the capacity of ß-carbolines to act as AhR ligands, the studies covered the following aspects: 1) Establishment of recombinant, cell-based functional reporter gene assays; 2) Identification and characterization of ß-carboline derivatives as ligands of the AhR by the means of functional reporter gene assays; 3) Characterization of mechanistic aspects in the AhR-mediated signal transduction pathway of distinct ß-carboline derivatives acting as AhR ligands using in vitro and ex vivo gelretardation assays; and 4) Description of the AhR ligand activities of soy and seasoning sauces, representing model systems for complex and natural food products.

Neste trabalho foi empregada uma metodologia rápida e eficiente para a identificação de metabólitos fitotóxicos produzidos por microrganismos. O isolamento do composto bioativo foi guiado através de bioensaio com Lemna minor. A espectrometria de massas, em especial o LC-MS, foi utilizada para acelerar o processo de identificação do composto ativo. As bactérias estudadas eram simbióticas do fungo fitopatogênico Sclerotium rolfsii. Seus respectivos extratos orgânicos obtidos de culturas em meio BD (batata dextrose) foram submetidos ao ensaio de fitotoxicidade com Lemna minor. Entre cinco bactérias foi selecionada a bactéria Burkholderia sp, a qual apresentou maior atividade no ensaio de fitotoxicidade. O fracionamento por cromatografia em coluna de sílica propiciou a identificação de uma fração ativa. A fitotoxina foi caracterizada como sendo um macropentólido de 20 membros. O composto pertence à classe dos polihidroxibutiratos (PHBs). Sua estrutura foi determinada por RMN 1H, RMN 13C, HMQC, HMBC, IV, ESI-MS/MS e também por comparação com dados da literatura. Esse composto nunca foi isolado de fontes naturais. Foi descrito na literatura uma rota sintética para sua obtenção, porém esta é a primeira vez que sua atividade fitotóxica é relatada. Este trabalho mostra uma nova perspectiva para o emprego de PHBs de baixo peso molecular e apresenta uma proposta de estrutura de composto fitotóxico que pode servir de modelo para a síntese de novos herbicidas. / In this work a quick and efficient methodology was employed for the identification of phytotoxic metabolites produced by microorganisms. The isolation of the bioactive compound was guided by Lemna minor bioassay. Mass spectrometry, especially LC-MS, was used to accelerate the process of identification of the phytotoxin. All bacteria were symbiotic to the phytopatogenic fungi Sclerotium rolfsii.. The bacterium Burkholderia sp was selected among the five bacteria analyzed, due to its greater phytotoxic activity in the bioassay. The phytotoxin was characterized as a 20 member macropentolide. This compound belongs to the polyhidroxybutirates (PHBs) chemical class. Its structure was determined by NMR1H, NMR 13C, HMQC, HMBC, IV, ESI-MS/MS and HRMS. It has never been isolated from natural sources before. Although a synthetic route has been proposed in the literature this is the first time that its phytotoxic activity is reported. This work leads to a new perspective for the application of low molecular weight PHBs and propose a phytotoxic structure that can be used as a model for the synthesis of new herbicide class.

A biota nos ecossistemas aquáticos é geralmente exposta a diversas condições de estresse, como variações ambientais naturais e distúrbios antropológicos, incluindo as descargas de poluentes nos recursos hídricos. Sendo assim importante a realização de estudos que avaliem os efeitos desses poluentes sobre os ecossistemas. O malathion é um exemplo de poluente, pois, é um inseticida comumente utilizado como defensivo agrícola em culturas ao redor do mundo e, embora apresente baixa toxicidade para espécies de mamíferos, espécies aquáticas podem ser sensíveis mesmo a pequenas concentrações. Dentre os diversos organismos aquáticos os crustáceos tem se destacado como organismos sensíveis para avaliar a poluição do ecossistema aquático. Nesse sentido, o objetivo do presente trabalho foi avaliar os efeitos do malathion sobre camarões Macrobrachium amazonicum através da obtenção da DL50 e da análise morfológica de suas brânquias. Para isso, camarões nativos do rio Grande foram capturados e aclimatados ao ambiente laboratorial para a realização de bioensaios. Os camarões foram divididos em grupos de dez animais cada e expostos a concentrações crescentes do inseticida por 48 horas. Nossos achados mostraram que a DL50 foi atingida a 0,73 mg/L de malathion, e que alterações relevantes na arquitetura das brânquias podem ser observadas em camarões expostos a concentrações de 1,25 a 1,5 mg/L do inseticida. Este é o primeiro estudo demonstrando que mesmo em curto prazo, o malathion interfere na estrutura microscópica das brânquias de camarões do gênero Macrobrachium, e vem contribuir para uma melhor compreensão dos efeitos promovidos por determinados defensivos agrícolas sobre organismos aquáticos que, comumente ficam expostos a estes. / Biota in aquatic ecosystems is usually exposed to various stress conditions, like natural environmental variations and anthropological disturbs including pollutant discharges into water resources. Malathion is an insecticide commonly used as an agricultural pesticide in crops around the world, and although it presents low toxicity to mammalian species, aquatic species may be sensitive even at small concentrations. Among the various aquatic organisms, crustaceans have distinguished themselves as sensitive organisms to evaluate the pollution of the aquatic ecosystem. In this sense, the objective of the present work was to evaluate the effects of malathion on Macrobrachium amazonicum shrimps by obtaining LD50 and morphological analysis of the gills. For this, native prawns of the Rio Grande were captured and acclimated to the laboratory environment for the realization of bioassays. The shrimp were divided into groups of ten animals each and exposed to increasing concentrations of the malathion for 48 hours. Our findings showed that the LD50 was reached at 0.73 mg/L malathion, and that relevant changes in the architecture of the gills can be observed in shrimps exposed to concentrations of 1.25 and 1.5 mg/L of the insecticide. This is the first report demonstrating that even in the short term, malathion interferes in the microscopic structure of Macrobrachium shrimp gills, and contributes to a better understanding of the effects promoted by certain pesticides on aquatic organisms that are commonly exposed to these.

Made available in DSpace on 2017-07-25T19:30:57Z (GMT). No. of bitstreams: 1 Juliane Aparecida Schneider.pdf: 1640887 bytes, checksum: 9c49a377ab710c7f2b18f803f5812aab (MD5) Previous issue date: 2015-12-11 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Noctuids stand out among the main groups of Lepidoptera that cause damage to crops. They exhibit great diversity of species of high economic important pests. Among them, we can highlight the occurrence of Soybean Looper, Chrysodeixis includens (Walker), species from the genus Spodoptera such as S. cosmioides (Walker), S. eridania (Cramer) and S. frugiperda (Walker) and even species of the subfamily Heliothinae, such as Helicoverpa armigera (Hübner). The search for alternatives to control these species has enabled the introduction of more environmentally safe insecticides and high insecticidal activity, such as the group of diamides and oxadiazines. Our objective was quantify the susceptibility of C. includens, H. armigera, S. frugiperda, S. cosmioides and S. eridania to flubendiamide, chlorantraniliprole and indoxacarb, through bioassays with application of insecticide incorporated in the artificial diet. Samples were collected during the seasons 2013/2014 and 2014/2015, in different regions of Brazil. Bioassays were developed at the Entomology Laboratory of Embrapa-Soja in Londrina. The mortality evaluation and weighing of caterpillars survivors were held on the seventh day. Mortality data were subjected to Probit’s analysis to estimate LC50 and LC99. Larval weight data were subjected to analysis of variance and means compared by Skott-Knott test at 5% probability. The LC50 of the C. includens population ranged from 0.861 to 187.28 μg of a.i./mL of diet (flubendiamide), 0.025 to 5.331 (chlotantraniliprole) and 1.345 to 2.94 (indoxacarb). For H. armigera ranged from 0.076 to 0.093 (flubendiamide), 0.050 to 0.051 (chlorantraniliprole) and 1.581 (indoxacarb). The LC50 for S. frugiperda, S. cosmioides and S. eridania ranged from 0.255 to 2.73 (flubendiamide), 0.032 to 0.104 (chlorantraniliprole), 0.834 to 3.0886 (indoxacarbe). Discriminating concentrations suggested for monitoring resistance of C. includens were 12 and 120 μg of flubendiamide/ml of diet, 9 and 90 μg of chlorantraniliprole/ml of diet and 5 and 50 μg of indoxacarb/ml of diet. The high variations found in susceptibility to diamides in populations of C. includens indicate resistance of Montividiu-GO and Campo Verde-MT populations. Among the insecticides studied, chlorantraniliprole have greater potential for controlling H. armigera, S. frugiperda, S. eridania and S. cosmioides. / Os Noctuídeos se destacam entre os principais grupos de lepidópteros que causam prejuízos às lavouras. Apresentam grande diversidade de espécies pragas de elevada importância econômica. Dentre elas, podemos destacar a ocorrência da lagarta falsa-medideira, Chrysodeixis includens (Walker), espécies do gênero Spodoptera, tais como S. cosmioides (Walker), S. eridania (Cramer) e S. frugiperda (Walker) e ainda espécies da subfamília Heliothinae, como a Helicoverpa armigera (Hübner). A busca de alternativas para o controle dessas espécies tem possibilitado a introdução de inseticidas mais seguros ambientalmente e de alta atividade inseticida, como o grupo das diamidas e oxadiazinas. Buscou-se, com a realização deste trabalho, quantificar a suscetibilidade de C. includens, H. armigera, S. frugiperda, S. cosmioides e S. eridania aos inseticidas flubendiamida, clorantraniliprole e indoxacarbe, por meio de bioensaios com aplicação de inseticida incorporado na dieta artificial. As coletas foram realizadas durante as safras 2013/2014 e 2014/2015, em diferentes regiões do Brasil. Os bioensaios foram desenvolvidos no Laboratório de Entomologia da Embrapa-Soja em Londrina-PR. A avaliação da mortalidade e as pesagens das lagartas sobreviventes foram realizadas ao sétimo dia. Os dados de mortalidade de cada população testada foram submetidos à análise de Probit para estimativa de CL50 e CL99. Os dados de peso foram submetidos à análise de variância e as médias comparadas pelo teste de Skott-Knott a 5% de probabilidade. As CL50 das populações de C. includens variaram de 0,861 a 187,28 μg de i.a/mL de dieta (flubendiamida), 0,025 a 5,331 (clorantraniliprole) e 1,345 a 2,94 (indoxacarbe); de H. armigera 0,076 a 0,093 (flubendiamida), 0,050 e 0,051 (clorantraniliprole) e 1,581 (indoxacarbe); das espécies S. frugiperda, S. cosmioides e S. eridania variaram de 0,255 a 2,73 (flubendiamida), 0,032 a 0,104 (clorantraniliprole), 0,834 a 3,0886 (indoxacarbe). As concentrações diagnósticas encontradas e sugeridas para o monitoramento da resistência de C. includens foram de 12 e 120 μg de flubendiamida/mL de dieta, 10 e 100 μg de clorantraniliprole/mL de dieta e 5 e 50 μg de indoxacarbe/mL de dieta. As variações encontradas para as diamidas nas populações de C. includens indicam resistência das populações de Montividiu-GO e Campo Verde-MT. Entre os inseticidas estudados, o clorantraniliprole apresenta maior potencial de controle para H. armigera, S. frugiperda, S. eridania e S. cosmioides.

Along with an increasing understanding of the harmful effects on the environment of a wide range of pollutants has come the need for more sensitive, faster and less expensive detection methods of identification and quantitation. Many environmental pollutants occur in low levels and often in complex matrices thus analysis can be difficult, time consuming and costly. Because of the availability and easy cultivation of the microorganisms with potentially high specificity, there is considerable interest in the use of living microorganisms as the analytical component (the biocomponent) of sensors for pollutants. While a number of biosensors using bacteria have been developed, yeast has been comparatively rarely used as the biocomponent. Yeast are attractive because they are easy to culture and they are eukaryotes which means their biochemistry is in many respects closer to that of higher organisms. This thesis describes the development of whole cell bioassays that use yeast cells as a sensing element and redox mediators to probe the intracellular redox reactions to monitor the catabolic activity of the yeast resulting from the external substrate, steady-state voltammetry is utilised as the electrochemical detection technique. The isogenic differential enzyme analysis (IDEA) concept of Lincoln Ventures Limited, lead NERF funded research consortium uses bacteria that have been cultured using specific organic pollutants as the carbon source which are the biocomponent in sensors. The use of wild type yeast Arxula adeninivorans that has the ability to use a very wide variety of substrates as sources of carbon and nitrogen was used as an alternative to bacteria to validate the “IDEA” concept. Naphthalene and di-butyl phthalate were chosen as model target contaminant molecules. The performance, detection limits and the usefulness of yeast based biosensor applications for environmental analysis are discussed. This thesis also describes the development and optimisation of a simple, cost effective in vivo estrogens bioassay for the detection of estrogens using either genetically modified or a wild type yeast Saccharomyces cerevisiae. In this study, catabolic repression by glucose was exploited to achieve specificity to estrogens in complex environmental samples that eliminates the requirement for conventional sample preparation. This is the first time that the use of wild type yeast to quantify estrogens has been reported. The attractive features of the bioassay are its use of a non-GMO organism, its speed, its high specificity and sensitivity with a detection limit of 10-15 M. The similarity of binding affinities for major estrogens to those of human estrogens receptors makes this in vivo estrogen bioassay very useful for analytical/screening procedures. The electrochemical detection method also makes it easy to interface with a variety of electronic devices.

Thyrotropin (TSH) is a glycoprotein hormone released from the pituitary gland to promote the synthesis and secretion of thyroid hormone. The existence of well-established peripheral mechanisms for regulation of thyroid hormone delivery to targets has called into question the significance of TSH as a primary regulator of circulating thyroid hormone concentrations in fish. However, relatively little is known about the regulation or action of endogenously secreted teleost TSH, largely due to lack of purified TSH suitable for biological testing and immunoassay development. I developed a red drum in vivo bioassay to aid in the production and purification of recombinant TSH from the red drum, a perciform fish demonstrating dynamic daily thyroxine (T4) cycles hypothesized to be driven by TSH. Exogenous bovine TSH injection resulted in a time and dose-dependent increase in circulating TSH and T4 in red drum. However, the sensitivity of the red drum thyroid gland to stimulation by bovine TSH was lost during growth under controlled laboratory conditions, even when circulating levels of exogenously-administered mammalian TSH remained elevated. The insensitivity of the thyroid was not due to prior TSH injection or feed source. Because insensitivity of the Thyrotropin (TSH) is a glycoprotein hormone released from the pituitary gland to promote the synthesis and secretion of thyroid hormone. The existence of well-established peripheral mechanisms for regulation of thyroid hormone delivery to targets has called into question the significance of TSH as a primary regulator of circulating thyroid hormone concentrations in fish. However, relatively little is known about the regulation or action of endogenously secreted teleost TSH, largely due to lack of purified TSH suitable for biological testing and immunoassay development. I developed a red drum in vivo bioassay to aid in the production and purification of recombinant TSH from the red drum, a perciform fish demonstrating dynamic daily thyroxine (T4) cycles hypothesized to be driven by TSH. Exogenous bovine TSH injection resulted in a time and dose-dependent increase in circulating TSH and T4 in red drum. However, the sensitivity of the red drum thyroid gland to stimulation by bovine TSH was lost during growth under controlled laboratory conditions, even when circulating levels of exogenously-administered mammalian TSH remained elevated. The insensitivity of the thyroid was not due to prior TSH injection or feed source. Because insensitivity of the red drum thyroid precluded their use as a bioassay species, the plasma TSH and T4 response to exogenous TSH was next characterized in goldfish. The T4 response in goldfish was stable and repeatable, with T4 levels peaking at 5 hours and remaining elevated for more than 11 hours after bovine TSH injection. Plasma TSH peaked from 2-5 hours following TSH injection with more than 90 percent cleared by 11 hours. The goldfish bioassay was further utilized to evaluate the effects of structural modifications on TSH biological activity. Substitution of four positively charged amino acids at the n-recombinant human TSH, had the same effect in goldfish. The heterothyrotropic potency of mammalian follicle stimulating hormone in goldfish was also enhanced by the same amino acid substitutions. Finally, the importance of oligosaccharides to TSH bioactivity was also examined in goldfish. Deglycosylation abolished TSH bioactivity, even when immunoreactivity persisted in circulation. Furthermore, recombinant canine TSH was less potent when produced in cell lines generating insect-type glycosylation than when produced in a cell line capable of mammalian-type glycosylation. These studies utilizing recombinant mammalian demonstrated conservation of mammalian TSH hormone-receptor interactions in goldfish, suggesting TSH function might likewise be conserved. Thus, I have established goldfish as a sensitive and stable bioassay which can now be utilized to monitor the biological activity of teleost TSH expressed in vitro as well as to evaluate how structural modifications of the TSH molecule influence its vivo biological activity.