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The relationships between caterpillars of Pierisbrassicae L. and Pierisrapae L. (Lepidoptera: Pieridae) and a common host plant Brassicaoleracea L. were studied using chemosensory and nutritional techniques. Attention was focussed on amino acids, which are in part essential nutrients, and on phenolic and flavonoid derivatives of two aromatic amino acids, that are products of the secondary metabolism in the host plant.An electrophysiological study of amino acid gustation showed that in both species 14 out of 22 amino acids were stimulants to a receptor cell in a maxillary sensillum. The nutritionally essential amino acids were generally stronger stimuli than dispensable ones. A correlation analysis provided indirect evidence that the amino acid receptor possessed four sites, one less specific and three or possibly four specific ones. A comparison of data on free amino acid concentrations in B.oleracea with dose-response relations of the amino acid cell showed that this cell can quantitatively sense foliar amino acids.Phenolic acids and an anthocyanin that naturally occur in B.oleracea elicited neural responses from two to three maxillary gustatory cells. Chlorogenic and protocatechuic acids, both carrying ortho-substituted hydroxyl groups on the aromatic ring, were the most effective stimulants. A steep increase in responsiveness was occurring with increasing concentrations in the range 0.2 - 5.0 mM. P.rapae was the less sensitive of both species. Flavonols were ineffective. The predominant anthocyanin in B.oleracea , cyanin, evoked neural activity in some cells but inhibited the activity in gustatory cells sensitive to sugars, amino acids and glucosinolates in P.brassicae . Chemosensory responsiveness was reflected in preference behaviour. Naturally occurring levels of phenolic acids in B.oleracea as found in phytochemical studies are able to affect sensory processes in the caterpillars (Chapter 3).Assessment of possible metabolic effects of dietary phenolics and some other dietary variations was performed using a flow-through respirometer. This was designed to monitor continuously the gas exchange of feeding caterpillars during the complete final instar. The results of these measurements were compared to the results obtained using standard gravimetric techniques that make use of the measurement of food intake to calculate metabolic efficiency. Respirometric results yielded small effects on the energetic efficiency of growth, which was in contrast to gravimetric results. The causes of the discrepancies between both methods and the consequences of these findings for studies on insect food utilization in general are discussed (Chapter 4).The nutritional utilization of amino acids and nitrogen was studied comparatively for caterpillars of both species on an artificial diet and on B.oleracea . Food consumption in the final instar was lower on the artificial diet. More food was consumed when leaf amino acid content was lower. Relationships were found between food consumption and the absorption efficiencies of most of the essential amino acids. Absorption efficiencies for glycine, cystein and serine were lower on the artificial diet, differences for other amino acids were small between the diets. Amino acid utilization patterns were similar for both species. Balance sheet calculations showed that an extensive conversion from phenylalanine to tyrosine occurred. For both species indications were obtained that tyrosine and cystein may become limiting for growth when dietary protein levels are low (Chapter 5).Phenolic acids (caffeic and chlorogenic acids) and flavonoids (oenin and quercetin-3-rutinoside) inhibited survival, development and growth when larvae of both species were continuously exposed to these compounds present in an artificial diet. P.brassicae was distinctly more sensitive at lower levels of the compounds (0.4 and 1.0 mM). Final instars of both species were much less sensitive than earlier instars. Growth inhibition in final instars was primarily due to reduced food consumption. The results suggest a potential role of phenolic acids and flavonoids, normal constituents of leaves of B.oleracea , in defence against Pieris caterpillars (Chapter 6).Seven cultivars of B.oleracea were offered as food to study their suitability as a host plant for larvae of both caterpillar species. Parameters of larval performance showed differences between cultivars. Highperformance liquid chromatography was used to analyse leaf tissues of five cultivars with respect to concentrations of phenolic acids and flavonoids. Each of the cultivars was found to have its own quantitative pattern of these compounds. Unidentified polar flavonoid components were detected in highly variable amounts. These preliminary results further support a potential role of phenolic and flavonoid compounds in resistance of B.oleracea against Pieris (Chapter 7).

32 - Ö Z E T - Bronş kanserinde, bronş sekresyonu laktik dehidregenaz (LDH) enzim düzeyinin, hastalığın tanısındaki önemini saptamak amacıyla, 26 bronş kanserli ve 15 kontrol gurubu hasta da serum ve bronş sekresyonu LDH enzim düzeyleri saptandı. Kanser gurubunu oluşturan hastaların bronş sekresyonu LDH enzim düzeyi, kontrol gurubu hastalarınkinden, anlamlı olarak yüksek bulunmuştur. Ancak, bu yüksekliği, kanserin varlığına bağlamamız doğru olmaz. Bronş sekresyonu LDH enzim aktivasyonu, izoenzimlerine göre saptanıldığında daha anlamlı sonuçların elde edilebilineceğine inanıyoruz. 36

This thesis describes investigations into the total synthesis of momilactones, germination inhibitors and phytoalexins isolated from rice. These compounds possess a Δ 7,8 -pimaradiene type skeleton with an unusual trans-syn ring-arrangement (figure 1).In chapter 1 a survey is given of the momilactones and the other hitherto known trans-syn pimaranelactones, with emphasis on their structures, biosynthesis and physiological activities.Chapter 2 is devoted to a literature survey of synthetic studies towards trans-syn(-cis) perhydrophenanthrene systems. The chemical reactivity of trans-syn pimaranelactones is also discussed.In chapter 3 the results are presented of a synthetic investigation, based on the Diels-Alder reaction depicted in scheme 1.This approach provided a total synthesis of Δ 8,9 -pimaradiene and Δ 8,9 -sandaracopimaradiene. However, attempts to isomerize the double bond to the desired Δ 7,8 -position met with little success. We therefore turned our attention towards starting compounds bearing an oxo group on C-7, in order to utilize this group for the introduction of the Δ 7,8 -double bond at a later stage.Using the work of W.L.Meyer and coworkers as a starting point, we stereospecifically synthesized a trans-syn-cis perhydrophenanthrene system as outlined in scheme 2. This part of the investigations is described in chapter 4. Several ways for the stereospecific introduction of a second substituent on C-13 were investigated. our synthesis of compound 159 could probably have been elaborated further, but we chose to focus our attention on a more promising approach which is described in chapter 5.Our stereospecific synthesis of trans-syn-cis perhydrophenanthrene systems, which forms the subject of chapter 5, is based on the stereospecific Diels-Alder reaction depicted in scheme 3 and culminates in the succesful synthesis of model compound 122. Initially we used 2- trimethylsilyloxybutadienes as diene components, but severe hydrolysis problems were encountered with the resulting adducts. These problems were effectively overcome by using diene 190. The adduct possesses a regiospecific silylenolether system which can be alkylated at C-13. Adduct 191 could be deformylated and stereoselectively reduced to the alcohol 197 leaving the t-butyldimethylsilylenolether intact. Two possible synthetic routes were then investigated.Alkylation of compound 199 with 2-ethoxy-1,3-dithiolan surprisingly only gave one thiolanyl compound (200) which proved to have the thiolanylgroup in the α-position. Reduction and hydrolysis of this compound gave the hydroxyaldehyde 205. However, during the Wittig reaction of the latter compound, equilibration occurred via (retro-)aldol reaction, resulting in considerable epimerization at C-13. Only a small amount of α-vinylproduct was found. Oxidation and Wolff-Kishner reduction finally afforded the model compound 122.Alkylation of compound 201 with 2-ethoxy-1,3-dithiolan yielded stereospecifically the β-thiolanyl product, as could be expected for steric reasons. This product was elaborated further as shown. Here, too, a (retro-)aldol reaction occurred during the Wittig reaction of compound 225, resulting in both hydroxy-epimers of the β-vinyl alcohol. No α-vinylproduct could be detected in this case. This concluded the stereospecific synthesis of compound 122.X-ray crystallography of thiolanyl-compounds 200 and 219 and 13 C-NMR spectroscopy were used to establish the stereochemistry of a number of reaction products, especially concerning the configuration at C-13. Details of these measurements can be found in chapter 6.Finally, in chapter 7, the results of the investigations are summarized and evaluated in relation to the total synthesis of momilactones.

This thesis describes investigations into the total synthesis of momilactones, germination inhibitors and phytoalexins isolated from rice. These compounds possess a Δ 7,8 -pimaradiene type skeleton with an unusual trans-syn ring-arrangement (figure 1).In chapter 1 a survey is given of the momilactones and the other hitherto known trans-syn pimaranelactones, with emphasis on their structures, biosynthesis and physiological activities.Chapter 2 is devoted to a literature survey of synthetic studies towards trans-syn(-cis) perhydrophenanthrene systems. The chemical reactivity of trans-syn pimaranelactones is also discussed.In chapter 3 the results are presented of a synthetic investigation, based on the Diels-Alder reaction depicted in scheme 1.This approach provided a total synthesis of Δ 8,9 -pimaradiene and Δ 8,9 -sandaracopimaradiene. However, attempts to isomerize the double bond to the desired Δ 7,8 -position met with little success. We therefore turned our attention towards starting compounds bearing an oxo group on C-7, in order to utilize this group for the introduction of the Δ 7,8 -double bond at a later stage.Using the work of W.L.Meyer and coworkers as a starting point, we stereospecifically synthesized a trans-syn-cis perhydrophenanthrene system as outlined in scheme 2. This part of the investigations is described in chapter 4. Several ways for the stereospecific introduction of a second substituent on C-13 were investigated. our synthesis of compound 159 could probably have been elaborated further, but we chose to focus our attention on a more promising approach which is described in chapter 5.Our stereospecific synthesis of trans-syn-cis perhydrophenanthrene systems, which forms the subject of chapter 5, is based on the stereospecific Diels-Alder reaction depicted in scheme 3 and culminates in the succesful synthesis of model compound 122. Initially we used 2- trimethylsilyloxybutadienes as diene components, but severe hydrolysis problems were encountered with the resulting adducts. These problems were effectively overcome by using diene 190. The adduct possesses a regiospecific silylenolether system which can be alkylated at C-13. Adduct 191 could be deformylated and stereoselectively reduced to the alcohol 197 leaving the t-butyldimethylsilylenolether intact. Two possible synthetic routes were then investigated.Alkylation of compound 199 with 2-ethoxy-1,3-dithiolan surprisingly only gave one thiolanyl compound (200) which proved to have the thiolanylgroup in the α-position. Reduction and hydrolysis of this compound gave the hydroxyaldehyde 205. However, during the Wittig reaction of the latter compound, equilibration occurred via (retro-)aldol reaction, resulting in considerable epimerization at C-13. Only a small amount of α-vinylproduct was found. Oxidation and Wolff-Kishner reduction finally afforded the model compound 122.Alkylation of compound 201 with 2-ethoxy-1,3-dithiolan yielded stereospecifically the β-thiolanyl product, as could be expected for steric reasons. This product was elaborated further as shown. Here, too, a (retro-)aldol reaction occurred during the Wittig reaction of compound 225, resulting in both hydroxy-epimers of the β-vinyl alcohol. No α-vinylproduct could be detected in this case. This concluded the stereospecific synthesis of compound 122.X-ray crystallography of thiolanyl-compounds 200 and 219 and 13 C-NMR spectroscopy were used to establish the stereochemistry of a number of reaction products, especially concerning the configuration at C-13. Details of these measurements can be found in chapter 6.Finally, in chapter 7, the results of the investigations are summarized and evaluated in relation to the total synthesis of momilactones.

The production of thiophene-biocides by cell cultures invitro was simultaneously investigated with Tageteserecta , T.patula and T.minuta . The calli from which the liquid cultures had to be derived differed between species in the appearance of organoid structure, texture, and colour, Independently of the nutrition of the plants and explants. In particular, the difference between the friability of calli of different species Is obviously related to the expression of the activity of silent genes In only a late phase of the callus andlor the cell suspension culture. Therefore 'origin effects' may eventually determine the suitability of calli to Initiate liquid cultures (Chapter 1). The differences between calli, however, showed that the production of thiophenes In the calli was positively related with the measure of differentiation. Rapidly growing and fine granular cell sus~ pensions, for Instance obtained from smooth calli of T.minuta , did not produce thiophenes (Chapters II and III).Differentiated calli of T.erecta did not provide suitable material to initiate cell cultures In liquid medium. However, minced cauliflower-like calli of T.patula with irregularly occurring small root- or shoot-like differentiations, formed large cell aggregates (3- 8 mm) in liquid media. These cell aggregates accumulated non-polar thiophenes and released spontaneously relatively high amounts of a water-soluble thiophene (BBTOH) in to the medium (Chapter VII). Apparently, the Increased morphological dedifferentation of calli runs parallel with a decreased production of thiophenes In the cell suspensions derived from them. The long-term accumulation of thiophenes In cell aggregates and the release of such compounds into the mediom open perspectives for the commercial production of such compounds under fermentor conditions.Embedding of the fine granular suspension cells of T.minuta in alginate resulted in the release of secondary metabolites into the liquid medium, but did not provide adequate conditions to reinitiate the production of thiophenes (Chapter V). In contrast, naturally formed cell aggregates which can be considered as a natural system of entrapment, as formed by T.patula cells, obviously provide suitable conditions for the production of thiophenes.Genetic transformation of intact Tagetes by means of infection with wild-type and mutant strains of Agrobacteriumtumefaciens and A . rhizogenes , induced neoplastic outgrowth of various organized and unorganized tissues without added growth regulators (Chapter VI). The change in this potential may be related to an altered synthesis of endogenously formed phytohormones. The species-dependent relationship between morphological differentiation and thiophene production persisted In all transformed tissues examined.In conclusion, the results of the present experiments on thiophene production in cell cultures of Tagetes species support the view that, despite the totipotency of plant cells (Chapter IV), major differences exist between closely related species in the ability to serve as a biotechnological unit invitro . Consequently, extensive research to adapt a certain recalcitrant plant species for plant cell biotechnology should be avoided by looking for a better producing species.

The relationships between caterpillars of Pierisbrassicae L. and Pierisrapae L. (Lepidoptera: Pieridae) and a common host plant Brassicaoleracea L. were studied using chemosensory and nutritional techniques. Attention was focussed on amino acids, which are in part essential nutrients, and on phenolic and flavonoid derivatives of two aromatic amino acids, that are products of the secondary metabolism in the host plant.An electrophysiological study of amino acid gustation showed that in both species 14 out of 22 amino acids were stimulants to a receptor cell in a maxillary sensillum. The nutritionally essential amino acids were generally stronger stimuli than dispensable ones. A correlation analysis provided indirect evidence that the amino acid receptor possessed four sites, one less specific and three or possibly four specific ones. A comparison of data on free amino acid concentrations in B.oleracea with dose-response relations of the amino acid cell showed that this cell can quantitatively sense foliar amino acids.Phenolic acids and an anthocyanin that naturally occur in B.oleracea elicited neural responses from two to three maxillary gustatory cells. Chlorogenic and protocatechuic acids, both carrying ortho-substituted hydroxyl groups on the aromatic ring, were the most effective stimulants. A steep increase in responsiveness was occurring with increasing concentrations in the range 0.2 - 5.0 mM. P.rapae was the less sensitive of both species. Flavonols were ineffective. The predominant anthocyanin in B.oleracea , cyanin, evoked neural activity in some cells but inhibited the activity in gustatory cells sensitive to sugars, amino acids and glucosinolates in P.brassicae . Chemosensory responsiveness was reflected in preference behaviour. Naturally occurring levels of phenolic acids in B.oleracea as found in phytochemical studies are able to affect sensory processes in the caterpillars (Chapter 3).Assessment of possible metabolic effects of dietary phenolics and some other dietary variations was performed using a flow-through respirometer. This was designed to monitor continuously the gas exchange of feeding caterpillars during the complete final instar. The results of these measurements were compared to the results obtained using standard gravimetric techniques that make use of the measurement of food intake to calculate metabolic efficiency. Respirometric results yielded small effects on the energetic efficiency of growth, which was in contrast to gravimetric results. The causes of the discrepancies between both methods and the consequences of these findings for studies on insect food utilization in general are discussed (Chapter 4).The nutritional utilization of amino acids and nitrogen was studied comparatively for caterpillars of both species on an artificial diet and on B.oleracea . Food consumption in the final instar was lower on the artificial diet. More food was consumed when leaf amino acid content was lower. Relationships were found between food consumption and the absorption efficiencies of most of the essential amino acids. Absorption efficiencies for glycine, cystein and serine were lower on the artificial diet, differences for other amino acids were small between the diets. Amino acid utilization patterns were similar for both species. Balance sheet calculations showed that an extensive conversion from phenylalanine to tyrosine occurred. For both species indications were obtained that tyrosine and cystein may become limiting for growth when dietary protein levels are low (Chapter 5).Phenolic acids (caffeic and chlorogenic acids) and flavonoids (oenin and quercetin-3-rutinoside) inhibited survival, development and growth when larvae of both species were continuously exposed to these compounds present in an artificial diet. P.brassicae was distinctly more sensitive at lower levels of the compounds (0.4 and 1.0 mM). Final instars of both species were much less sensitive than earlier instars. Growth inhibition in final instars was primarily due to reduced food consumption. The results suggest a potential role of phenolic acids and flavonoids, normal constituents of leaves of B.oleracea , in defence against Pieris caterpillars (Chapter 6).Seven cultivars of B.oleracea were offered as food to study their suitability as a host plant for larvae of both caterpillar species. Parameters of larval performance showed differences between cultivars. Highperformance liquid chromatography was used to analyse leaf tissues of five cultivars with respect to concentrations of phenolic acids and flavonoids. Each of the cultivars was found to have its own quantitative pattern of these compounds. Unidentified polar flavonoid components were detected in highly variable amounts. These preliminary results further support a potential role of phenolic and flavonoid compounds in resistance of B.oleracea against Pieris (Chapter 7).

The production of thiophene-biocides by cell cultures invitro was simultaneously investigated with Tageteserecta , T.patula and T.minuta . The calli from which the liquid cultures had to be derived differed between species in the appearance of organoid structure, texture, and colour, Independently of the nutrition of the plants and explants. In particular, the difference between the friability of calli of different species Is obviously related to the expression of the activity of silent genes In only a late phase of the callus andlor the cell suspension culture. Therefore 'origin effects' may eventually determine the suitability of calli to Initiate liquid cultures (Chapter 1). The differences between calli, however, showed that the production of thiophenes In the calli was positively related with the measure of differentiation. Rapidly growing and fine granular cell sus~ pensions, for Instance obtained from smooth calli of T.minuta , did not produce thiophenes (Chapters II and III).Differentiated calli of T.erecta did not provide suitable material to initiate cell cultures In liquid medium. However, minced cauliflower-like calli of T.patula with irregularly occurring small root- or shoot-like differentiations, formed large cell aggregates (3- 8 mm) in liquid media. These cell aggregates accumulated non-polar thiophenes and released spontaneously relatively high amounts of a water-soluble thiophene (BBTOH) in to the medium (Chapter VII). Apparently, the Increased morphological dedifferentation of calli runs parallel with a decreased production of thiophenes In the cell suspensions derived from them. The long-term accumulation of thiophenes In cell aggregates and the release of such compounds into the mediom open perspectives for the commercial production of such compounds under fermentor conditions.Embedding of the fine granular suspension cells of T.minuta in alginate resulted in the release of secondary metabolites into the liquid medium, but did not provide adequate conditions to reinitiate the production of thiophenes (Chapter V). In contrast, naturally formed cell aggregates which can be considered as a natural system of entrapment, as formed by T.patula cells, obviously provide suitable conditions for the production of thiophenes.Genetic transformation of intact Tagetes by means of infection with wild-type and mutant strains of Agrobacteriumtumefaciens and A . rhizogenes , induced neoplastic outgrowth of various organized and unorganized tissues without added growth regulators (Chapter VI). The change in this potential may be related to an altered synthesis of endogenously formed phytohormones. The species-dependent relationship between morphological differentiation and thiophene production persisted In all transformed tissues examined.In conclusion, the results of the present experiments on thiophene production in cell cultures of Tagetes species support the view that, despite the totipotency of plant cells (Chapter IV), major differences exist between closely related species in the ability to serve as a biotechnological unit invitro . Consequently, extensive research to adapt a certain recalcitrant plant species for plant cell biotechnology should be avoided by looking for a better producing species.

Nippostrongylus brasiliensis is a rodent intestinal nematode with an important pulmonary migrating stage. Previous studies have observed a lack of TNF-α production and minimal recruitment of neutrophils, which led us to the belief that anti-inflammatory mechanisms could be active in the lung stage. In this study, lipopolysaccharide (LPS) stimulated alveolar macrophages (NR8383) or rat lungs were used as in vitro or in vivo inflammation models respectively.Both live N.brasiliensis larvae and NES significantly reduced the production of LPS-induced pro-inflammatory mediators, TNF-α and NO, but not IL-1β, in NR8383 cells. The inhibition of TNF-α production was related to the heatlabile and trypsin-sensitive fraction of NES concentrate. 1-D protein gel of NES concentrate revealed that the molecular weights of proteins are between 6kDa and 100kDa. Glycoproteins were found abundant in NES concentrate. The inflammatory processes, including NF-κB translocation and TNF-α gene transcription were significantly inhibited by NES and/or NES concentrate. In vivo, we observed a significant reduction of neutrophil recruitment (≈ 40%) by NES on a background of LPS (100ng/ml) induced inflammation. This reduction was associated with the significant inhibition in gene transcriptions of proinflammatory mediators TNF-α, IL-1β, iNOS, ICAM-1 and MIP-2 in bronchoalveolar lavage (BAL) cells. The down-regulation of pro-inflammatory mediators and inflammatory processes observed in this study suggests that N.brasiliensis larvae and/or NES are capable of modifying the normally potent LPS inflammatory response, both in vitro and in vivo. This study and planned future studies could be fundamental in developing anti-inflammatory agents with immune-active molecules in N.brasiliensis-derived products.

Background α1-Antitrypsin deficiency (AATD) is associated with a high risk of developing lung and liver disease. Despite being one of the most common hereditary disorders worldwide, AATD remains under-diagnosed and prolonged delays in diagnosis are usual. The aim of this study was to validate the use of buccal swab samples and serum circulating DNA for the complete laboratory study of AATD. Methods Sixteen buccal swab samples from previously characterized AATD patients were analyzed using an allele-specific genotyping assay and sequencing method. In addition, 19 patients were characterized by quantification, phenotyping and genotyping using only serum samples. Results The 16 buccal swab samples were correctly characterized by genotyping. Definitive results were obtained in the 19 serum samples analyzed by quantification, phenotyping and genotyping, thereby performing the complete AATD diagnostic algorithm. Conclusions Buccal swab samples may be useful to expand AATD screening programs and family studies. Genotyping using DNA from serum samples permits the application of the complete diagnostic algorithm without delay. These two methods will be useful for obtaining more in depth knowledge of the real prevalence of patients with AATD.