A herança autossômica recessiva pode ser responsável por aproximadamente 77% dos casos de surdez hereditária. Em 1996, Chaib e col. mapearam o loco responsável por surdez profunda neurossensorial de herança recessiva na região cromossômica 2p22-23 (DFNB9). Em 1999, Yasunaga e col. identificaram esse gene como o que codifica a proteína OTOFerlina (OTOF) nessa região. Até hoje, já foram descritas 31 mutações patogênicas diferentes no gene OTOF em populações de várias origens, com destaque a mutação Q829X que foi encontrada em ~3% dos casos de surdez na Espanha (Migliosi e col., 2002; Rodríguez-Ballesteros e col., 2003). Alguns pacientes com mutações no gene OTOF apresentavam neuropatia auditiva, um tipo de deficiência auditiva neurossensorial caracterizada pela ausência ou anomalia das ondas no exame dos Potenciais Evocados Auditivos do Tronco Encefálico ou BERA com a presença das emissões otoacústicas e/ou microfonismo coclear. O objetivo desse projeto foi investigar a contribuição relativa das mutações no gene OTOF ao casos de neuropatia auditiva e de outros tipos surdez em famílias brasileiras. Uma amostra de 343 propósitos portadores de deficiência auditiva foi submetida ao estudo da mutação Q829X. Não foi identificada em nenhum caso. Dessa casuística foram selecionados 48 propósitos de famílias com consangüinidade ou com 2 ou mais afetados na irmandade e quatro pacientes com neuropatia auditiva e com consangüinidade parental ou com dois ou mais afetados na irmandade. Além disso, foram também selecionados 7 casos isolados com neuropatia auditiva e 5 casos de portadores de alterações no tronco encefálico. Essa amostra totalizou 64 propósitos. Propósitos dessas 64 famílias foram genotipados em relação a cinco marcadores de microssatélites ligados ao gene OTOF. A análise dos haplótipos excluiu ligação ao gene OTOF em 34 casos, 19 não eram conclusivos e 11 indicaram possibilidade de ligação ao gene OTOF (incluindo uma família com pais consangüíneos e neuropatia auditiva e três propósitos com neuropatia auditiva). Simultaneamente, os 64 propósitos foram triados para mutações em oito exons do gene OTOF, nos quais mutações já haviam sido descritas, por meio de SSCP seguido de seqüenciamento. Os 11 casos com resultados compatíveis com ligação ao gene OTOF (4 com neuropatia auditiva) e os sete casos de neuropatia auditiva foram selecionados para o seqüenciamento de todos os exons (total de 18 propósitos). Identificamos no total 58 alterações diferentes. Onze variantes eram potencialmentes patogênicas, encontradas em sete dos propósitos, todos pertencentes ao grupo dos 18 selecionados. Quatro casos eram heterozigotos compostos [98G>A (R33Q) e 2401G>T e 2402A>T (E801L)]; [1841G>A (G614E) e 3239G>C (R1080P)], [3751T>G (C1251G) e 5431A>T (K1811X)] e [2348delG (G783fs) e 5800-5801insC (L1934fs)], dois eram heterozigotos [1552-1567del16 (R518fs); 2905- 2923del19ins11 (A969fs)] sem que uma segunda mutação fosse detectada e um apresentava a mutação em homozigose [3400C>T (R1134X)]. Desses sete propósitos com mutações patogênicas, somente um paciente com mutação em heterozigose não apresentava neuropatia auditiva. Dentre os 11 casos com neuropatia auditiva, seis tinham pelo menos uma mutação no gene OTOF que poderia ser a causa de surdez. Esse achado reforça a associação entre o fenótipo da neuropatia auditiva e mutações no gene OTOF. A variante Q829X não foi encontrada nenhuma vez em nossa amostra, portanto, não deve ser causa importante de surdez na nossa população. Porém, nosso estudo mostra que mutações no gene OTOF são causas freqüentes de neuropatia auditiva no Brasil (mais de 50% dos casos). Nossos resultados reforçam a hipótese que pacientes com neuropatia auditiva devem ser selecionados para pesquisa de mutações no gene OTOF e que talvez mais de 50% dos casos de neuropatia auditiva tenham causa genética. / 77% of nonsyndromic prelingual deafness have an autosomal recessive inheritance. In 1996, Chaib et al. mapped a locus associated with sensorineural nonsyndromic recessive deafness to chromosome region 2p22-23 (DFNB9) by linkage studies. In 1999, Yasunaga et al. identified the OTOF gene encoding OTOFerlin, in this region. To date, there are 31 different pathogenic mutations described in the OTOF gene, from populations of variable origins. A Q829X mutation was found at a frequency of ~3% of deafness in Spain (Migliosi e col., 2002; Rodríguez-Ballesteros e col., 2003). Some affected individuals with mutations in the OTOF gene were reported to present auditory neuropathy, a type of deafness characterized by an absent or severely abnormal auditory brainstem response, with preservation of otoacoustic emissions and/or cochlear microphonics. The main purpose of this project was to investigate the relative contribution of OTOF mutations to auditory neuropathy and other type of deafness, amongst Brazilian families. We enrolled 343 Brazilian unrelated subjects with nonsyndromic hearing loss. A specific test for the Q829X mutation was performed first. We failed to find any subjects carrying this mutation. From this group, we selected 48 probands from families with consanguinity or with two or more affected sibs and four probands with diagnosis of auditory neuropathy and from consanguineous unions or with two or more affected sibs. In addition, we selected 7 isolated subjects with auditory neuropathy and 5 cases with diagnosis of brainstem alteration. This gave a total of 64 probands. Subjects from the 64 families were genotyped for five microsatellites markers, linked to the OTOF gene. The analysis of the haplotype excluded linkage to the OTOF gene in 34 families, it was inconclusive in 19 families and it showed compatibility with linkage in the remaining 11 families (including one with consanguineous parents and auditory neuropathy and three with diagnosis of auditory neuropathy). Simultaneously, the 64 subjects were screened for mutations in 8 exons previously identified to other mutations using the SSCP technique. In positive cases, DNA sequencing was carried out. In the 11 subjects consistent with putative linkage to OTOF gene and the 7 isolated cases of auditory neuropathy, an exon by exon screening for mutations in the OTOF gene was performed using DNA sequencing (Total of 18 subjects). We found a total of 58 different variants. Eleven were possibly causative mutations and were found in seven of the 18 subjects. Amongst them, four cases were compound heterozygotes R33Q with E801L, G614E with E1080P, 2348delG with 5800-5801insC and K1811X with C1251G, two cases were heterozygotes [1552- 1567del16 and 2905-2923del19in11] without a second mutation and one presented a mutation in homozygous form [3400C>T (R1134X)]. Among these seven probands, only one patient with a heterozygote mutation did not have a diagnosis of auditory neuropathy. In the 11 cases of auditory neuropathy, six had at least one mutation in the OTOF gene that is the probable cause of their deafness. These findings support the association between auditory neuropathy and mutations in the OTOF gene. While we failed to confirm the high frequency of Q829X mutation found in Spain, our study shows that mutations in the OTOF gene are frequent causes of auditory neuropathy in Brazil (more than 50%). Our results reinforced that patients with auditory neuropathy must be selected for mutation detection in the OTOF gene and that more than 50% of cases of auditory neuropathy have a defined genetic etiology.

A herança autossômica recessiva pode ser responsável por aproximadamente 77% dos casos de surdez hereditária. Em 1996, Chaib e col. mapearam o loco responsável por surdez profunda neurossensorial de herança recessiva na região cromossômica 2p22-23 (DFNB9). Em 1999, Yasunaga e col. identificaram esse gene como o que codifica a proteína OTOFerlina (OTOF) nessa região. Até hoje, já foram descritas 31 mutações patogênicas diferentes no gene OTOF em populações de várias origens, com destaque a mutação Q829X que foi encontrada em ~3% dos casos de surdez na Espanha (Migliosi e col., 2002; Rodríguez-Ballesteros e col., 2003). Alguns pacientes com mutações no gene OTOF apresentavam neuropatia auditiva, um tipo de deficiência auditiva neurossensorial caracterizada pela ausência ou anomalia das ondas no exame dos Potenciais Evocados Auditivos do Tronco Encefálico ou BERA com a presença das emissões otoacústicas e/ou microfonismo coclear. O objetivo desse projeto foi investigar a contribuição relativa das mutações no gene OTOF ao casos de neuropatia auditiva e de outros tipos surdez em famílias brasileiras. Uma amostra de 343 propósitos portadores de deficiência auditiva foi submetida ao estudo da mutação Q829X. Não foi identificada em nenhum caso. Dessa casuística foram selecionados 48 propósitos de famílias com consangüinidade ou com 2 ou mais afetados na irmandade e quatro pacientes com neuropatia auditiva e com consangüinidade parental ou com dois ou mais afetados na irmandade. Além disso, foram também selecionados 7 casos isolados com neuropatia auditiva e 5 casos de portadores de alterações no tronco encefálico. Essa amostra totalizou 64 propósitos. Propósitos dessas 64 famílias foram genotipados em relação a cinco marcadores de microssatélites ligados ao gene OTOF. A análise dos haplótipos excluiu ligação ao gene OTOF em 34 casos, 19 não eram conclusivos e 11 indicaram possibilidade de ligação ao gene OTOF (incluindo uma família com pais consangüíneos e neuropatia auditiva e três propósitos com neuropatia auditiva). Simultaneamente, os 64 propósitos foram triados para mutações em oito exons do gene OTOF, nos quais mutações já haviam sido descritas, por meio de SSCP seguido de seqüenciamento. Os 11 casos com resultados compatíveis com ligação ao gene OTOF (4 com neuropatia auditiva) e os sete casos de neuropatia auditiva foram selecionados para o seqüenciamento de todos os exons (total de 18 propósitos). Identificamos no total 58 alterações diferentes. Onze variantes eram potencialmentes patogênicas, encontradas em sete dos propósitos, todos pertencentes ao grupo dos 18 selecionados. Quatro casos eram heterozigotos compostos [98G>A (R33Q) e 2401G>T e 2402A>T (E801L)]; [1841G>A (G614E) e 3239G>C (R1080P)], [3751T>G (C1251G) e 5431A>T (K1811X)] e [2348delG (G783fs) e 5800-5801insC (L1934fs)], dois eram heterozigotos [1552-1567del16 (R518fs); 2905- 2923del19ins11 (A969fs)] sem que uma segunda mutação fosse detectada e um apresentava a mutação em homozigose [3400C>T (R1134X)]. Desses sete propósitos com mutações patogênicas, somente um paciente com mutação em heterozigose não apresentava neuropatia auditiva. Dentre os 11 casos com neuropatia auditiva, seis tinham pelo menos uma mutação no gene OTOF que poderia ser a causa de surdez. Esse achado reforça a associação entre o fenótipo da neuropatia auditiva e mutações no gene OTOF. A variante Q829X não foi encontrada nenhuma vez em nossa amostra, portanto, não deve ser causa importante de surdez na nossa população. Porém, nosso estudo mostra que mutações no gene OTOF são causas freqüentes de neuropatia auditiva no Brasil (mais de 50% dos casos). Nossos resultados reforçam a hipótese que pacientes com neuropatia auditiva devem ser selecionados para pesquisa de mutações no gene OTOF e que talvez mais de 50% dos casos de neuropatia auditiva tenham causa genética. / 77% of nonsyndromic prelingual deafness have an autosomal recessive inheritance. In 1996, Chaib et al. mapped a locus associated with sensorineural nonsyndromic recessive deafness to chromosome region 2p22-23 (DFNB9) by linkage studies. In 1999, Yasunaga et al. identified the OTOF gene encoding OTOFerlin, in this region. To date, there are 31 different pathogenic mutations described in the OTOF gene, from populations of variable origins. A Q829X mutation was found at a frequency of ~3% of deafness in Spain (Migliosi e col., 2002; Rodríguez-Ballesteros e col., 2003). Some affected individuals with mutations in the OTOF gene were reported to present auditory neuropathy, a type of deafness characterized by an absent or severely abnormal auditory brainstem response, with preservation of otoacoustic emissions and/or cochlear microphonics. The main purpose of this project was to investigate the relative contribution of OTOF mutations to auditory neuropathy and other type of deafness, amongst Brazilian families. We enrolled 343 Brazilian unrelated subjects with nonsyndromic hearing loss. A specific test for the Q829X mutation was performed first. We failed to find any subjects carrying this mutation. From this group, we selected 48 probands from families with consanguinity or with two or more affected sibs and four probands with diagnosis of auditory neuropathy and from consanguineous unions or with two or more affected sibs. In addition, we selected 7 isolated subjects with auditory neuropathy and 5 cases with diagnosis of brainstem alteration. This gave a total of 64 probands. Subjects from the 64 families were genotyped for five microsatellites markers, linked to the OTOF gene. The analysis of the haplotype excluded linkage to the OTOF gene in 34 families, it was inconclusive in 19 families and it showed compatibility with linkage in the remaining 11 families (including one with consanguineous parents and auditory neuropathy and three with diagnosis of auditory neuropathy). Simultaneously, the 64 subjects were screened for mutations in 8 exons previously identified to other mutations using the SSCP technique. In positive cases, DNA sequencing was carried out. In the 11 subjects consistent with putative linkage to OTOF gene and the 7 isolated cases of auditory neuropathy, an exon by exon screening for mutations in the OTOF gene was performed using DNA sequencing (Total of 18 subjects). We found a total of 58 different variants. Eleven were possibly causative mutations and were found in seven of the 18 subjects. Amongst them, four cases were compound heterozygotes R33Q with E801L, G614E with E1080P, 2348delG with 5800-5801insC and K1811X with C1251G, two cases were heterozygotes [1552- 1567del16 and 2905-2923del19in11] without a second mutation and one presented a mutation in homozygous form [3400C>T (R1134X)]. Among these seven probands, only one patient with a heterozygote mutation did not have a diagnosis of auditory neuropathy. In the 11 cases of auditory neuropathy, six had at least one mutation in the OTOF gene that is the probable cause of their deafness. These findings support the association between auditory neuropathy and mutations in the OTOF gene. While we failed to confirm the high frequency of Q829X mutation found in Spain, our study shows that mutations in the OTOF gene are frequent causes of auditory neuropathy in Brazil (more than 50%). Our results reinforced that patients with auditory neuropathy must be selected for mutation detection in the OTOF gene and that more than 50% of cases of auditory neuropathy have a defined genetic etiology.

Le projet ANDROMEDE a pour but de créer un nouvel instrument d’imagerie ionique sub-micrométrique et d’analyse par spectrométrie de masse, en utilisant l’impact d’ions sur des nano-objets présents à la surface des échantillons solides et plus particulièrement sur les échantillons biologiques. L’étude de ces échantillons avec l’objectif d’analyse in vitro et in vivo nécessite une préparation complexe et requiert une expérimentation à la pression atmosphérique. Cet instrument unique ouvre une nouvelle voie dans l’analyse de surfaces, complémentaire aux méthodes utilisées de nos jours.Au sein du projet ANDROMEDE, deux éléments ont été développés dans le cadre de notre étude. La source NAPIS qui délivre les nanoparticules permettant d’augmenter le rendement d’éjection des ions secondaires, et le spectromètre de masse PSI-TOF pour l’analyse chimique des éléments émis depuis la surface de l’échantillon.Le faisceau primaire de nanoparticules de la source NAPIS est accéléré dans un accélérateur de type Pelletron 4MeV et amené sur une cible. La source de nanoparticules NAPIS a été développée et validée indépendamment au sein de la société ORSAY PHYSICS, avant son couplage sur l’accélérateur.Une nouvelle optique d’extraction appelée ExOTOF ainsi que le spectromètre de masse à extraction orthogonale PSI-TOF ont été développés pour permettre l’analyse des ions secondaires et augmenter la résolution en masse du système. Ces ensembles ont été spécialement dessinés pour ce projet. Ils permettront une extraction et une analyse efficace des ions secondaires émis depuis la surface de l’échantillon en utilisant des faisceaux continus et auront leur application pour les analyses à la pression atmosphérique. L’ensemble a été validé et les premiers tests de sortie du faisceau primaire ont été réalisés avec succès. / The goal of the ANDROMEDE project is to create a new instrument for sub-micrometric ion imaging and analysis by mass spectrometry, using ion impacts on nano-objects present in the solid sample surface and more particularly on biological samples. In-vitro and in-vivo analysis of these types of samples require mostly complex preparation and even atmospheric pressure experimentation. This unique instrument opens a new path for surface analysis characterization, which is complementary to the standard methods and technics used today.In the ANDROMEDE project, two elements have been developed in our study. The NAPIS source which delivers the nanoparticles allowing the increase of the secondary ion yield and the PSI-TOF mass spectrometer for the chemical analysis of the elements emitted from the sample surface.The NAPIS source delivers a primary beam of accelerated nanoparticles in a Pelletron 4MeV accelerator which is driven to a target. The NAPIS nanoparticles source has been developed and validated independently in the ORSAY PHYSICS Company firstly before its coupling on the accelerator. The new extraction optics called ExOTOF as well as the PSI-TOF orthogonal extraction mass spectrometer have been developed for the reliable secondary ions study and the increase of the mass resolution.These instruments have been specially designed for this project. This development will allow an efficient extraction and analysis of the secondary ions emitted from the sample surface using continuous primary beams and will have applications for atmospheric pressure studies. The assembly has been completely validated and the first tests of the output beam have been successfully carried out.

Im Rahmen dieser Disseration wurden im Einzelnen folgende Ergebnisse erzielt: A. Isolierung und Strukturaufklärung von Naphthylisochinolin-Alkaloiden aus verschiedenen Ancistrocladus-Spezies: • Die bisher noch nicht phytochemisch untersuchten Rindenextrakte der vietnamesischen Unterart Ancistrocladus tectorius ssp. cochinchinensis wurden im Hinblick auf ihre Sekundärmetabolite analysiert. Dabei identifizierte man vier bereits bekannte und drei neuartige Naphthylisochinoline-Alkaloide. Die Strukturen dieser drei Metabolite wurden nach Isolierung unter Verwendung diverser 2D-NMR-Techniken aufgeklärt. Die entdeckten Substanzen – Ancistrocladinium A (30) und seine beiden O-Demethylderivate 31 und 32 – waren die drei ersten Vertreter des neuartigen N,8'-Naphthyldihydroisochinolin-Kupplungstyps. Diese Naturstoffe verfügen über vielversprechende pharmakologische Wirkungen – vor allem gegen den Erreger der Leishmaniose. • Die botanisch noch nicht vollständig charakterisierte Lianenart ''A. ikela'', die aus der Demokratischen Republik Kongo stammt, wurde im Laufe der Arbeit morphologisch und phytochemisch untersucht und beschrieben. Neben den beiden N,C-verknüpften Naphthylisochinolinen Ancistrocladinium A (30) und Ancistrocladinium B [(M/P)-39] wurde bei der phytochemischen Analyse ein neuartiges C,C-gekuppeltes Alkaloid – 8-O-Methylancistrogriffin C (40) – isoliert. Des weiteren wurde ein Gradient entwickelt, der die vollständige Trennung der beiden Atrop-Diastereomere von 39 und dadurch HPLC-NMR- und HPLC-CD-Analysen der einzelen Epimere ermöglichte, so dass die Rotationsbarriere der bei Raumtemperatur langsam drehenden Biarylachse bestimmt werden konnte. • Aus Blättern der bereits gut untersuchten indischen Ancistrocladus-Art A. heyneanus wurde mit 6-O-Methyl-8,4'-O-didemethylancistrocladin (42) ein weiteres neues Naphthylisochinolin-Alkaloid isoliert. • Eine phytochemische Untersuchung der Familie der Ancistrocladaceae auf das Vorkommen von N,C-verküpften Naphtylisochinolinen ergab, dass diese strukturell außergewöhnlichen Alkaloide in diesen Lianen weit verbreitet sind. B: Die Rolle des Phloems bei der Pathogen-vermittelten Ausbreitung von Signalen: • Im Rahmen des Teilprojektes B8 des SFBs 567 wurden Untersuchungen zur Rolle des Phloems bei der Weiterleitung von Langstreckensignalen nach Infektion von Arabidopsis-thaliana-Pflanzen mit virulenten oder avirulenten Stämmen von Pseudomonas syringae pv. tomato durchgeführt. Zunächst wurde dazu eine im nL-Maßstab anwendbare Analysenmethode für die Hauptmetabolite von A. thaliana – die Glucosinolate – entwickelt. Mit Hilfe dieser empfindlichen Methode wurden in Pflanzenextrakten von A. thaliana viele bekannte und einige neue Glucosinolate (8-Methylsulfonyl-n-octyl-, 2-Hydroxy-4-methylsulfinyl-n-butyl-, 2-Hydroxy-4-methylsulfonyl-n-butyl- und 4-Hydroxy- benzoyloxymethylglucosinolat) identifiziert. Des weiteren wurden MS/MS-Analysen der Glucosinolate durchgeführt, bei denen neben mehreren typischen Fragmenten für die Thiozucker-Einheit auch einige charakteristische Fragmente für die unterschiedlichen Seitenketten (z.B. Methylsulfinyl-n-alkyl- oder Methylthio-n-alkyl-Struktur) detektiert wurden. Leider ergaben vor allem die aromatischen und heteroaromatischen Seitenketten-Typen kein typisches Fragmentierungs-muster. • Bei der Analyse der Phloemexsudate konnte in Phloemsäften von unbehandelten Pflanzen neben Methoxyglucobrassicin (73) ein für Pflanzen neuartiges Phosphat 87 (1-Glycero-1-myo-inositolphosphat) identifiziert werden. In den Phloemsäften der unterschiedlich behandelten Pflanzen (infiltriert mit MgCl2, einem virulentem oder einem avirulentem Pseudomonas-Stamm) kamen sämtliche Hauptmetabolite der Blätter vor. Lediglich ein leichter, nicht signifikanter Konzentrationsanstieg von Methoxyglucobrassicin (73) wurde im Phloemsaft von mit avirulenten Pathogenen infizierten Pflanzen festgestellt. Dieser Anstieg muss aber kritisch betrachtet werden, da er auch ein Artefakt des starken mechanischen Reizes des Infiltrationsprozesses sein könnte. Andere kleine Konzentrationsänderungen könnten außerdem durch das starke ''Grundrauschen'' der Infiltration überlagert werden. C: Strukturaufklärung polyketidischer Sekundärmetabolite aus Mikroorganismen: • Zwei niedermolekulare Naturstoffe aus dem extremophilen Streptomyceten-Stamm KC 1030, die in der Arbeitsgruppe von Prof. H.-P. Fiedler (Universität Tübingen) isoliert worden waren, wurden strukturell aufgeklärt. Bei dem einen handelt es sich um das bereits bekannte Frigocyclinon (89), bei dem anderen um ein neues Angucyclinon 88 mit Fridamycin-E-Grundkörper. Darüber hinaus wurden aus einem weiteren Streptomyces-Stamm (AK 671) zwei neue (97, 98) und drei (96, 99, 100) bekannte biosynthetisch interessante Sekundärmetabolite isoliert. / In detail, the following results were achieved during this doctoral thesis: A: Isolation and structural elucidation of naphthylisoquinoline alkaloids from Ancistrocladus species: • The not yet phytochemically investigated bark extracts of the Vietnamese subspecies Ancistrocladus tectorius ssp. cochinchinensis were analyzed. Four already known and three unknown naphthylisoquinoline alkaloids were identified. The three new metabolites were isolated and structurally elucidated by the use of different 2D NMR techniques. The defined structures – ancistrocladinium A (30) and its two O-demethyl derivatives 31 and 32 – are the first representatives of the new N,8'-naphthyl dihydroisoquinoline coupling type. These natural products exhibit promising biological activities – above all against the pathogen of leishmaniasis. • The botanically not yet fully characterized liana species ''A. ikela'', which was collected in the Congo Basin (Democratic Republic Congo), was morphologically and phytochemically investigated and described botanically. Besides the two N,C-coupled naphthylisoquinolines, ancistrocladinium A (30) and ancistrocladinium B [(M/P)-39], a new C,C-coupled alkaloid – 8-O-methylancistrogriffine C (40) – was isolated. Furthermore, by establishing a gradient system for the baseline separation of both atropodiastereomers of 39 HPLC-NMR and HPLC-CD analyses of the individual two purified epimers were performed to define the rotational barrier of the biaryl axis, which slowly rotates at room temperature. • From the leaves of the already well-investigated Indian Ancistrocladus species A. heyneanus, 6-O-methyl-8,4'-O-didemethylancistrocladine (42), a hitherto unknown naphthylisoquinoline alkaloid, was isolated. • A phytochemical investigation of the plant family of the Ancistrocladaceae with respect to the occurrence of N,C-coupled naphtylisoquinolines proved that these structurally exceptional alkaloids are quite common in this liana family. B: The role of the phloem during the propagation of pathogen-mediated signals: • Within the scope of the project B8 of the SFB 567, investigations on the role of the phloem during the propagation of long-distance signals after infection of plants of Arabidopsis thaliana with virulental or avirulental strains of Pseudomonas syringae pv. tomato were carried out. For this purpose initially an analytical method for the main metabolites – the glucosinolates – of A. thaliana applicable at a nL-scale was developed. Using this sensitive method, many known but also some new glucosinolates (8-methylsulfonyl-n-octyl-, 2-hydroxy-4-methylsulfinyl-n-butyl-, 2-hydroxy-4-methylsulfonyl-n-butyl-, and 4-hydroxy-benzoyloxymethyl-glucosinolate) were identified in plant extracts of A. thaliana. Furthermore, MS/MS analyses of the glucosinolates were carried out to verify their structures. Next to some typical fragments of the thiosugar moiety a few characteristic fragments of the different side chain structures (e.g. methylsulfinyl-n-alkyl- or methylthio-n-alkyl structure) were detected. Unfortunately, not all of the different types of side chaines led to typical fragmentation pattern. • In the phloem sap of entirely untreated plants, only methoxyglucobrassicin (73) and a phosphate 87 (1-glycero-1-myo-inositolphosphate), which was not kown so far to be located in plants, were identified in the course of the analysis of the phloem exudates. In the phloem saps of all the differently treated plants (infiltrated with MgCl2, virulental or avirulental Pseudomonas strain) all of the main leaf metabolites occurred. Solely a slight but not significant increase of the concentration of methoxyglucobrassicin (73) was measured in the phloem exudates of plants infected with avirulental pathogens. One has to be extremely careful with respect to the observed increase since it is possible that the enhanced glucosinolate concentration was the result of the strong mechanical stimulus of the infiltration procedure. Furthermore, slight changes in the concentration of other metabolites could be superimposed by signals caused by the infiltration procedure. C: Structural elucidation of polyketidic secondary metabolites from microorganisms • The structures of two low-molecular natural products isolated in the working group of Prof. H.-P. Fiedler (University of Tübingen) from the extremophilic Streptomyces strain KC 1030 were elucidated. One of them was the already known frigocyclinone (89) and the other one was the new angucyclinone 88 with a fridamycine E core structure. Moreover, from the Streptomyces strain AK 671, two new (97, 98) and three known (96, 99, 100) biosynthetically interesting secondary metabolites were isolated.