<p>PCBs are well known environmental pollutants. They are also precursors to metabolites, as the hydroxy-PCBs and the methylsulfonyl-PCBs. This thesis presents a validated methodology for analysis of PCB metabolites and a structural identification of 38 hydroxy-PCBs in human blood. Further methodological development resulted in an identification of a similar number of methylsulfonyl-PCBs.</p><p>The analytical method has been applied in two extensive studies of humans, consisting of maternal and cord blood from Dutch women and of blood from humans living in Slovakia. The Dutch shows that the relative transfer of hydroxy-PCBs from the mother to the foetus is higher compared to the PCBs. Even though the chemical plant in Michalovce in Slovakia had been shut down for over 20 years, the concentrations of PCB and its metabolites were among the highest detected in European human blood.</p>

<p>Metabolites, small endogenous molecules existing in every living cell, tissue or organism, play a vital role for maintaining life. The collective group of all metabolites, the metabolome, is a consequence of the biochemistry and biochemical pathways that a cell or tissue uses to promote survival. Analysis of the metabolome can be done to reveal changes of specific metabolites which can be a manifestation, a reason or a consequence of for example a disease. The physical chemical diversity amongst these components is tremendous and it poses a large analytical challenge to measure and quantify all of them. Targeting sub groups of the meta­bolome such as specific functional classes has shown potential for increasing metabolite coverage. Group selective labeling with biotin-tags followed by high affinity avidin capture is a well established purification strategy for protein purification.</p><p>The purpose with this project is to explore if it is possible to transfer the avidin biotin approach to metabolomics and use this method for small mole­cules purification. Specifically, this investigation aims to see if it is achievable to make a bio­tinylation of specific functional groups, to increase the sensitivity through reduction of sample complexity in liquid chromatography mass spectrometry metabolomics analyses after high affinity avidin capture. By purifying the analyte of interest and thereby reducing the sample complexity there will be a reduction in ion suppression. The aim is to increase the analytical sensitivity through a reduction in ion suppression during liquid chromatography mass spectrometry analysis.</p><p>Delimitations have been done to only investigate the possibility to obtain a biotinylation of primary amines and amides. As model compounds phenylalanine, spermi­dine, histamine and nicotinamide have been selected.</p><p>The result from this study indicates that it is possible to increase metabolite coverage through biotin labeling followed by high affinity avidin capture. It is a gain in analytical sensitivity of selected model compounds when comparing biotinylation strategy with a control non­biotinylation approach in a complex sample. A broader study of additional model compounds and a method development of this strategy are necessary to optimize a potential future method.</p>

Molecular genetic and enzymological techniques have been employed to study secondary metabolite biosynthesis. These investigations have focused on two projects: the cloning and heterologous expression of biosynthetic gene clusters from unculturable marine organisms and the characterization of individual enzymes involved in the biosynthesis of the antifungal agent blasticidin S. The marine environment is proving to be a valuable source of biologically active compounds, but problems associated with sustainable harvest, laboratory culture, and organic synthesis make obtaining sufficient quantities of compounds for drug development both difficult and expensive. A method has been developed for the isolation of biosynthetic gene clusters from complex marine microbe/invertebrate associations. Using this method a mixed polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS) gene cluster has been cloned from the marine sponge Jaspis splendens. The cloned gene cluster was found to code for a PKS with three extension modules and an NRPS with three extension modules. In addition, several open reading frames (ORFs) were identified that may be involved in the biosynthesis of the PKS starter molecule. Partial characterization of catalytic domains from the NRPS was also completed. The second project centers on the characterization of enzymes involved in blasticidin S (BS) biosynthesis. Two ORFs were identified in the BS gene cluster encoding gene products predicted to be involved in the early steps of BS biosynthesis. The blsG gene product has sequence similarity to lysine 2,3-aminomutase and is believed to be involved in the formation of the β-arginine moiety of BS. A series of heterologous expression studies were undertaken to determine the function of B1sG. The product of blsM exhibits sequence homology with several nucleosidetransferases. blsM was cloned from the BS gene cluster, heterologously expressed in E. coli, and shown to catalyze the formation of cytosine using cytidine 5'- monophosphate as the preferred substrate. Point mutations were introduced in blsM to generate three B1sM mutant enzymes: S92D, E98A, and E98D. All three mutants lost cytidine 5'-monophosphate hydrolysis activity. Surprisingly, the B1sM S92D mutant exhibits cytidine deaminase activity when incubated with cytidine or deoxycytidine, resulting in the formation of uridine and deoxyuridine, respectively. / Graduation date: 2005

Graduation date: 2003

PART I. Asymmetric syntheses of both natural (+)- and nonnatural (-)- byssochlamic acid via a [2+2] photoaddition-cycloreversion strategy are described. X-ray crystallographic analysis of the cyclohexylamine salt 99 showed that the structure of the monomethyl ester 100 from esterase hydrolysis of 44 was originally misassigned as 56. The enantiomeric relationship of the two diolides 106 and 70 permitted syntheses of nonnatural byssochlamic acid (-)-3 and natural byssochiamic acid (+)-3 from enantiopure alcohol (+)-64 and from its enantiomer (-)-110, respectively. Through the use of (��)-103 to reach both enantiomers of byssochlamic acid (3) and subsequent epimerization of the npropyl chain, it was proved that the cis configuration of the two alkyl substituents is strongly preferred in the natural product. PART II. An asymmetric approach towards the nootropic agent huperzine A is described. Formation of cyclobutane 122 with the desired stereochemistry was accomplished using intramolecular [2+2] photoaddition of the enantiopure enone 121. Attempts to prepare the methoxypyridine system via an azadiene Diels- Alder reaction were unsuccessful. However, intramolecular Michael addition of 181 produced silyl ether 182 which was converted into the pyridone 187 by treatment with hydrogen fluoride followed by selenoxide elimination. Attempts to effect the key sigmatropic rearrangement of ketone 197 into a direct precursor of huperzine A were unsuccessful. / Graduation date: 2001

Graduation date: 1993

PCBs are well known environmental pollutants. They are also precursors to metabolites, as the hydroxy-PCBs and the methylsulfonyl-PCBs. This thesis presents a validated methodology for analysis of PCB metabolites and a structural identification of 38 hydroxy-PCBs in human blood. Further methodological development resulted in an identification of a similar number of methylsulfonyl-PCBs. The analytical method has been applied in two extensive studies of humans, consisting of maternal and cord blood from Dutch women and of blood from humans living in Slovakia. The Dutch shows that the relative transfer of hydroxy-PCBs from the mother to the foetus is higher compared to the PCBs. Even though the chemical plant in Michalovce in Slovakia had been shut down for over 20 years, the concentrations of PCB and its metabolites were among the highest detected in European human blood.

Background: Several neuropsychiatrie disorders have shown season of birth associations. Low cerebrospinal fluid (CSF) levels of the serotonin metabolite 5-HIAA and the dopamine metabolite HVA have been associated with suicidal behaviour, impulsivity, and aggression. This thesis investigated associations between the season of birth, the CSF levels of three monoamine metabolites (including MHPG of norepinephrine), the scales of the diagnostic interview for borderline patients (DIB), and psychiatric diagnoses. Also, the methods of suicide were investigated in relation to the season of birth. Methods: We studied a clinical sample of 241 patients in Stockholm with mood, anxiety and adjustment disorders with respect to the CSF levels of monoamine metabolites in relation to the season of birth, and in relation to the DIB in an overlapping sample. We also analysed all completed suicides during the 42 years 1952- 1993inVästerbottenin northern Sweden (1466 cases) by multiple logistic regressions to relate suicide methods with season of birth, gender, age, urban-rural residence, marital status, year of suicide, and season of suicide. For the 20 years 1961- 1980 (693cases), psychiatric in-patient and out-patient records were also examined for any history of psychiatric contacts and psychiatric diagnoses. In two mutually independent samples, we investigated the DIB in relation to the season of birth. Results: In the Stockholm sample, those born during February to April had significantly lower CSF levels of 5-HIAA, and those born during October to January had significantly higher CSF levels of HVA, HVA/5-HIAA, and HVA/MHPG, as well as (non-significantly) higher levels of 5-HIAA. Those with an intermediate score of section II (impulse action patterns) of the DIB had significantly higher CSF levels of 5-HIAA and HVA, and they were significantly more likely to have been born during October to January. In the Västerbotten register, those born during February to April were significantly more likely to have preferred hanging rather than poisoning or petrol gases, and conversely for those born during October to January. These associations with suicide methods were found for the total sample and for those without any history of psychiatric contacts, but not for those with psychiatric contacts. Conclusions: Suicidal behaviour shows statistically significant variation according to the season of birth, most probably mediated by a variation in an independent trait of vulnerability to suicide based on neurodevelopmental parameters, particularly the serotonergic system. The suicidal process differs between those who seek psychiatric care compared to those who do not, reflecting differences in the diagnostic spectra and in the extent of mental illness. / <p>Härtill 6 delarbeten.</p>

Previous studies on the secondary metabolites of Formosan octocoral Isis hippuris were collected only at Green Island. In the course of our studies on secondary metabolites from marine organisms, the acetone-solubles of the Formosan octocoral Isis hippuris collected at Orchid Island has led to the isolation of eleven polyoxygenated steroids (1¡V11), along with two known compounds (12 and 13). The structures of these compounds were determined on the basis of their spectroscopic and physical data, including NMR, IR, MS, etc. The cytotoxicity against of A-549 (human lung epithelial carcinoma), HT-29 (human colon adenocarcinoma), and P-388 (mouse lymphocytic leukemia) cells, and anti-HCMV (human cytomegalovirus) activity of metabolites 1¡V13 were evaluated. Compounds 12 and 13 displayed cytotoxicity against P-388 cell line with ED50 values of 3.2 and 3.6 £gg/mL, respectively. Compound 12 exhibited cytotoxicity against A-549 cell line with an ED50 value of 3.8 £gg/mL. Compound 8 exhibit inhibitory activity against HCMV, with EC50 values of 2.0 £gg/mL.

Numerous bioactive secondary metabolites including sesquiterpenoids,diterpenoids, meroditerpenoids, and steroids have been isolated from the soft corals of the genus Nephthea. In order to search for novel bioactive substances from marine organisms, we have investigated the secondary metabolites of the organic extract of the soft coral Nephthea chabrolii collected at San-Hsian-Tai. Chromatographic fractionation of the acetone-soluble has led to the isolation of four 19-oxygenated steroids 1¡V4 and two 19-norergosterols 5, 6, along with twelve known compounds 7¡V18. The structures of these compounds were determined on the basis of their spectroscopic analysis data (1H NMR, 13C NMR, 1H¡V1H COSY, HSQC, HMBC, NOESY, IR, and HRESIMS), physical data and compared with the literature data. The cytotoxicity against of A-549 (human lung epithelial carcinoma), HT-29 (human colon adenocarcinoma), and P-388 (mouse lymphocytic leukemia) cells, and anti-HCMV (human cytomegalovirus) activity of compounds 1¡V6 were evaluated. Metabolites 1¡V6 displayed cytotoxicity against P-388 cell line with ED50 values of 0.93, 1.05, 1.20, 1.74, 1.19, 1.19 £gg/mL. However, none of them exhibited inhibitory activity against HCMV (human cytomegalovirus).