No description available.

Contamination of seafood by marine toxins has been a consistent public health problem. Gymnodimine (GYM) is a member of a family of spirocyclic imine containing marine natural products which was shown to be highly toxic (LD50 96 mg/kg, intraperitoneal injection); thus ensuring public safety requires stringent monitoring of gymnodimine. Current detection methods for GYM and spirolides include the mouse bioassay and LC-MS-based detection techniques which, however, have significant limitations. Therefore, more efficient and convenient detection methods are required. Building on our recently completed total synthesis of (-)-gymnodimine, the synthesis of two haptens were targeted for eventual production of monoclonal antibodies (mAb) to be used in an eventual Enzyme-Linked Immunosorbent Assay (ELISA) for gymnodimine. As an extension of the intramolecular nucleophilic catalyzed aldol lactonization (NCAL) process from aldehyde acid to keto acid substrates, carbocyclic and nitrogen heterocyclic B-lactones were synthesized. Demonstration of the utility of the NCAL process for keto acids was applied to the synthesis of dihydroplakevulin A and the core of tussilagine. In addition, although initial attempts to develop guanidine catalysts for the asymmetric NCAL process were unsuccessful, homobenzotetramisole (HBTM) was found to be a suitable asymmetric catalyst for keto acid substrates. Finally, synthetic studies toward the proposed structure of thiolyngbyan are described. Thiolyngbyan was isolated from a blue-green algae and it exhibited antifungal activity.

<p>Contact allergy is one of the most common skin diseases with great social and economical impact. The origin and nature of contact allergens (haptens) capable of inducing T-cell mediated allergic reactions are diverse, ranging from organic molecules to metal ions. Most of the current knowledge on T-cell responses to haptens in humans with contact allergy have been established by studies on the metal ion nickel (Ni), the most common cause of contact allergy, whereas reactivity to the large group of organic haptens has been less studied.</p><p>Haptens are not immunogenic by themselves but must bind carrier molecules prior to their presentation on MHC class I or II molecules and subsequent recognition by T cells. Due to differences in their chemical nature, haptens interact with host molecules by different mechanisms and differences in their solubility can influence their access to different antigen-presenting pathways.</p><p>The aim of the present study was to define immune responses elicited by haptens of different chemical nature including Ni (hydrophilic metal ion), methylisothiazolinones (hydrophilic organic molecule) and parthenolide (lipophilic organic molecule). The immune response displayed by subjects with allergy to these substances, and non-allergic control subjects, was assessed by measuring hapten-induced cytokine production in peripheral blood mononuclear cells (PBMC) with a focus on ELISpot analysis of T-cell type 1 (e.g. IFN-g and IL-2) and type 2 (e.g. IL-4, IL-5 and IL-13) cytokines. For Ni and parthenolide, the phenotype of the hapten-reactive T cells was determined. The allergic status of subjects was defined by clinical history and patch testing. The latter is the established diagnostic method for contact allergy, based on applying various haptens to the subjects’ back and grading the skin reaction after 2-3 days.</p><p>All three haptens elicited a concomitant T-cell type 1 and 2 response in subjects with contact allergy to the corresponding hapten, suggesting the induction of a functionally related cytokine profile, irrespective of the chemical character of the hapten. The cytokine response was related to the degree of the subjects’ patch test reactivity; PBMC from a vast majority of subjects with strong and moderate patch test reactivity displayed detectable cytokine responses to the corresponding haptens, whereas subjects with weak or no (controls) patch test reactivity did not. Despite the similar cytokine profile induced, the phenotype of the reactive T cells was found to differ between haptens with Ni eliciting CD4+ T cells and parthenolide eliciting CD8+ T cells. This difference may be explained by a better ability of a lipophilic hapten to gain access to the MHC class I-restricted antigen-presentation pathway. Moreover, the data suggest that analysis of cytokine responses to haptens may facilitate future development of <i>in vitro</i>-based diagnostics assay for contact allergy.</p><p>Finally, the relationship between the variation over time in patch test reactivity and systemic reactivity to Ni, in terms of cytokine responses to Ni <i>in vitro</i>, was investigated. The degree of patch test reactivity is known to vary over time, in particular in subjects with weak reactivity. Ni-allergic subjects were patch tested three times with three month intervals and PBMC obtained at the same time points were assessed for<i> in vitro</i> reactivity to Ni. The overall reactivity in the patch test and the <i>in vitro</i> test was well correlated confirming that both methods provide a good and comparable estimate of the systemic reactivity to Ni. However, fluctuations in the patch test reactivity over time were not well correlated with variations in the cytokine response elicited <i>in vitro</i> suggesting that other parameters besides changes in the systemic reactivity could significantly contribute to the variation in patch test reaction over time.</p>

Contact allergy is one of the most common skin diseases with great social and economical impact. The origin and nature of contact allergens (haptens) capable of inducing T-cell mediated allergic reactions are diverse, ranging from organic molecules to metal ions. Most of the current knowledge on T-cell responses to haptens in humans with contact allergy have been established by studies on the metal ion nickel (Ni), the most common cause of contact allergy, whereas reactivity to the large group of organic haptens has been less studied. Haptens are not immunogenic by themselves but must bind carrier molecules prior to their presentation on MHC class I or II molecules and subsequent recognition by T cells. Due to differences in their chemical nature, haptens interact with host molecules by different mechanisms and differences in their solubility can influence their access to different antigen-presenting pathways. The aim of the present study was to define immune responses elicited by haptens of different chemical nature including Ni (hydrophilic metal ion), methylisothiazolinones (hydrophilic organic molecule) and parthenolide (lipophilic organic molecule). The immune response displayed by subjects with allergy to these substances, and non-allergic control subjects, was assessed by measuring hapten-induced cytokine production in peripheral blood mononuclear cells (PBMC) with a focus on ELISpot analysis of T-cell type 1 (e.g. IFN-g and IL-2) and type 2 (e.g. IL-4, IL-5 and IL-13) cytokines. For Ni and parthenolide, the phenotype of the hapten-reactive T cells was determined. The allergic status of subjects was defined by clinical history and patch testing. The latter is the established diagnostic method for contact allergy, based on applying various haptens to the subjects’ back and grading the skin reaction after 2-3 days. All three haptens elicited a concomitant T-cell type 1 and 2 response in subjects with contact allergy to the corresponding hapten, suggesting the induction of a functionally related cytokine profile, irrespective of the chemical character of the hapten. The cytokine response was related to the degree of the subjects’ patch test reactivity; PBMC from a vast majority of subjects with strong and moderate patch test reactivity displayed detectable cytokine responses to the corresponding haptens, whereas subjects with weak or no (controls) patch test reactivity did not. Despite the similar cytokine profile induced, the phenotype of the reactive T cells was found to differ between haptens with Ni eliciting CD4+ T cells and parthenolide eliciting CD8+ T cells. This difference may be explained by a better ability of a lipophilic hapten to gain access to the MHC class I-restricted antigen-presentation pathway. Moreover, the data suggest that analysis of cytokine responses to haptens may facilitate future development of in vitro-based diagnostics assay for contact allergy. Finally, the relationship between the variation over time in patch test reactivity and systemic reactivity to Ni, in terms of cytokine responses to Ni in vitro, was investigated. The degree of patch test reactivity is known to vary over time, in particular in subjects with weak reactivity. Ni-allergic subjects were patch tested three times with three month intervals and PBMC obtained at the same time points were assessed for in vitro reactivity to Ni. The overall reactivity in the patch test and the in vitro test was well correlated confirming that both methods provide a good and comparable estimate of the systemic reactivity to Ni. However, fluctuations in the patch test reactivity over time were not well correlated with variations in the cytokine response elicited in vitro suggesting that other parameters besides changes in the systemic reactivity could significantly contribute to the variation in patch test reaction over time.

No description available.

Les analyses physico-chimiques des arômes du vin prennent aujourd’hui un essor considérable pour faciliter la prise de décision des professionnels de la vigne et du vin. Des analyses performantes, pour un certain nombre de molécules parmi les plus pertinentes, ont été développées (GC-MS, LC-MS). Cependant les techniques utilisées ne sont pas facilement transposables au sein des exploitations. L’objectif de cette thèse est donc de proposer un nouveau type de dosage peu onéreux et simple à mettre en œuvre. La technique envisagée est le dosage immunologique (tests ELISA) qui permet, pour quelques Euros, de doser directement sur le terrain les molécules pertinentes sans préparation préalable des échantillons. Les molécules visées (alkylméthoxypyrazines et précurseurs cystéinylés de thiols volatiles) sont présentes dans les vins de Cabernet Sauvignon et de Sauvignon blanc. / Analytical methods for the detection and quantification of wines aroma typically utilise HPLC-MS or GC-MS. The methods require some isolation and concentration step preceding the analysis. Enzyme-linked immunosorbent assays (ELISAs) are becoming either alternative complementary analytical tools to conventional methods because of their rapidity, sensitivity, selectivity, and low cost. In this Thesis, the applicability of ELISAs for detection and quantification of precursors of volatile thiols and alkylmethoxypyrazines, which have been isolated from wines, made from Cabernet Sauvignon or Sauvignon Blanc, are described.

Die vorliegende Arbeit beschreibt die Herstellung und Charakterisierung der ersten Antikörper gegen Triacetontriperoxid (TATP), einem hoch empfindlichen und unkonventionellen (nicht-kommerziellen) Initialsprengstoff. Entscheidend dafür war die Synthese eines TATP-imitierenden Haptens, welches die typische nonagonale Struktur des TATP mit seinen drei Peroxid- und sechs Methylgruppen nahezu perfekt nachbildet, aber den Vorzug einer zusätzlichen Carboxygruppe zur kovalenten Kopplung an Proteine aufweist. Dadurch konnte das TATP-Hapten an Rinderserumalbumin (BSA) gebunden werden, um ein immunogenes Konjugat zu erzeugen, welches die erfolgreiche Immunisierung zweier Säugetierarten, Maus und Kaninchen, ermöglichte. Der Verlauf der In vivo-Immunisierungen wurde durch die Analyse der Tierseren in regelmäßigen Abständen mittels enzymgekoppeltem Immunoassay (ELISA) verfolgt. Die polyklonalen Antikörper beider Spezies waren ungewöhnlich selektiv gegenüber TATP. Jedoch unterschied sich die Affinität der Antikörper der zwei Spezies um das 5000-fache, wobei die Kaninchenseren den Mausseren überlegen waren. Entsprechend war auch die mit Kaninchenserum erreichbare TATP-Nachweisgrenze von 0.01 µg/L deutlich besser im Vergleich zu 50 µg/L, die mit Mausserum erzielt wurden. Der Messbereich des TATP-ELISA mit Kaninchenserum deckte zudem mehr als vier Zehnerpotenzen ab, wie mittels Präzisionsprofil bestimmt wurde. Die erhaltenen TATP-Antikörper aus Kaninchen stehen damit Anwendungen in Nachweissystemen für die sehr empfindliche Detektion von TATP zur Verfügung, die u. a. in sicherheitsrelevanten Bereichen zum Einsatz kommen könnten. Als erste Anwendung wurde ein TATP-ELISA realisiert, der im Rahmen dieser Arbeit ausführlich optimiert wurde. Außerdem wurden erste Schritte zur Entwicklung eines TATP-Schnelltests (LFA) unternommen. Weitere Biosensoren auf Grundlage der neu entwickelten TATP-Antikörper sind denkbar. / The present work decribes the production and characterization of the first antibodies against triacetone triperoxide (TATP), a highly sensitive and improvised (non-commercial) primary explosive. Crucial to this work was the synthesis of a TATP-related hapten that mimics almost perfectly the typical nonagonal structure of TATP with its three peroxide and six methyl groups. Advantageously, it has an additional carboxylic acid group, which provides a conjugation site for covalent attachment to proteins. Thus, the TATP hapten could be linked to bovine serum albumin (BSA) to produce an immunogenic conjugate, allowing the successful immunization of two different mammalian species, mouse and rabbit. The in vivo immunization progress was followed by periodically analyzing the animals’ sera using enzyme-linked immunosorbent assay (ELISA). The polyclonal antibodies of both species were remarkably selective to TATP. The affinity of these TATP-antibodies was, however, different between the two species, with the rabbit sera showing an affinity about 5000-fold superior than the murine one. Consequently, the TATP detection limit of 0.01 µg/L was considerably better using the sera from rabbit in contrast to 50 µg/L when mouse serum was used. The working range of the TATP-ELISA with rabbit sera covers more than four decades, calculated from a precision profile. The obtained TATP antibodies from rabbit are now available for applications in highly sensitive detection systems for TATP, which could be employed, among others, in security-relevant areas. The first application was the realization of a TATP-ELISA, which was extensively optimized within the course of this work. Furthermore, the first steps towards the development of a lateral flow assay (LFA) targeting TATP were taken, making conceivable further biosensor platforms based on the newly developed TATP antibodies.

Protein farnesylation is an essential post-translational modification required for the function of numerous cellular proteins including the oncoprotein Ras. The farnesyl transferase (FTase) catalyzed reaction is unique because farnesyl diphosphate (FPP), the farnesyl group donor for the reaction, forms a significant portion of a target protein binding site. The major goal of this research was to exploit this unique property of the FTase reaction and determine if changing the structure of the farnesyl donor group would affect FTase protein targeting. A small library of structural analogues of FPP was synthesized. Michelis-Menten steady-state kinetic analyses and competition reactions were used to determine the effect of these structural modifications on FTase targeting. We found that the analogues did affect FTase protein selectivity and that this could be exploited to induce unnatural target selectivity into the enzyme. The second goal of this research was to determine the effect of FPP analogues on the function of FTase target proteins. To test the effect of these analogues we determined whether the unnatural lipid could ablate oncogenic H-Ras biological function in a Xenopus laevis model system. Several analogues were able to disrupt oncogenic H-Ras function while others mimicked the activity of FPP. These results indicated that some of the FPP analogues may act a prenyl group function inhibitors that could lead to an important new class of anti-cancer therapeutics. Another major goal of this research was to use the FPP analogues as unnatural probes for the endogenous cellular activity of FTase target proteins. We developed antibodies to two of the unnatural FPP analogues to study their activity in cell cultureUtilizing these antibodies we found that alcohol prodrugs of the FPP analogues could be incorporated into cellular proteins in an FTase dependent manner. The ability of cell permeant analogues to be incorporated into live cells enhances the chances that such a molecule could be used to modify oncogenic cellular proteins with a prenyl group function inhibitor.

Universiẗat, Diss., 2000--Wuppertal.

No description available.