This thesis describes the development of new methods for thepreparation of enantiomerically pure methyl branched alkylcompounds and their use as building blocks in the synthesis ofstereoisomerically pure pheromones of pine sawflies. The high regioselectivity, enantioselectivity and activityof lipases in organic solvent in conjunction withenvironmentally compatible reaction conditions have madelipase-catalysed synthesis an attractive alternative toconventional synthetic methods in organic chemistry. The lipasefromPseudomonas cepacia(PCL) was used in kineticresolutions of primary 2- methylalcohols by acylation of thealcohols with vinyl acetate/vinyl butyrate. For alcoholsstudied, PCL showed moderate enantioselectivity(E= 10-20) towards 3-alkyl- or 3- cycloalkylsubstitutedprimary 2-methylpropanols, whereas 3-aryl-2-methyl-1-propanolswere accepted with high E-values(E&gt;100). Esterification of substituted methylcarboxylic acids withprimary alcohols catalysed byCandida rugosalipase (CRL) was found to be anenantioselective reaction. In general, CRL showed highselectivity towards(S)-2-methylcarboxylic acids(E= 15-70) and also towards(R)-3-methylcarboxylic acids(E= 15-40). For substrates having a double bond located5-6 bonds from the carbonyl moiety a two-fold enhancement ofenantioselectivity value(E-value) was obtained compared to their saturatedanalogues,E≈ 15-25. Furthermore, the enantioselectivity of CRL towards a seriesof 3- to 8-methyldecanoic acids were studied. CRL surprisinglyshowed enantiorecognition for all of these acids within theE-value range of 2.3-68. Interestingly, whereas the lipaseshowed S-preference when the methyl group was situated ateven-numbered carbons, R-preference was observed for thesubstrates with the methyl group at odd-numbered carbons. In order to establish the stereoisomeric composition of thenatural sex pheromone of the pine sawflyMicrodiprion pallipes, all sixteen individual isomers of3,7,11-trimethyl-2-tridecanol (and their propionate esters)found in this species were synthesised in highdiastereoisomeric purities, 95.3-97.6%. The syntheses werebased on six enantiomerically pure building blocks, the fourstereoisomers of 1-lithio-2,6-dimethyloctane and the twoenantiomers ofcis-3,4- dimethyl-g-butyrolactone. <b>Keywords:</b>lipase,Pseudomonas cepacia,Candida rugosa, enantioselective, enantiomeric ratio,primary 2-methylalcohol, substituted-methylcarboxylic acid,pine sawfly,Microdiprion pallipes, sex pheromone,3,7,11-trimethyl-2-tridecanol, stereoisomer.

<p>This thesis describes the development of new methods for thepreparation of enantiomerically pure methyl branched alkylcompounds and their use as building blocks in the synthesis ofstereoisomerically pure pheromones of pine sawflies.</p><p>The high regioselectivity, enantioselectivity and activityof lipases in organic solvent in conjunction withenvironmentally compatible reaction conditions have madelipase-catalysed synthesis an attractive alternative toconventional synthetic methods in organic chemistry. The lipasefrom<i>Pseudomonas cepacia</i>(PCL) was used in kineticresolutions of primary 2- methylalcohols by acylation of thealcohols with vinyl acetate/vinyl butyrate. For alcoholsstudied, PCL showed moderate enantioselectivity<i>(E</i>= 10-20) towards 3-alkyl- or 3- cycloalkylsubstitutedprimary 2-methylpropanols, whereas 3-aryl-2-methyl-1-propanolswere accepted with high E-values<i>(E</i>>100).</p><p>Esterification of substituted methylcarboxylic acids withprimary alcohols catalysed by<i>Candida rugosa</i>lipase (CRL) was found to be anenantioselective reaction. In general, CRL showed highselectivity towards<i>(</i>S)-2-methylcarboxylic acids<i>(E</i>= 15-70) and also towards<i>(</i>R)-3-methylcarboxylic acids<i>(E</i>= 15-40). For substrates having a double bond located5-6 bonds from the carbonyl moiety a two-fold enhancement ofenantioselectivity value<i>(</i>E-value) was obtained compared to their saturatedanalogues,<i>E</i>≈ 15-25.</p><p>Furthermore, the enantioselectivity of CRL towards a seriesof 3- to 8-methyldecanoic acids were studied. CRL surprisinglyshowed enantiorecognition for all of these acids within theE-value range of 2.3-68. Interestingly, whereas the lipaseshowed S-preference when the methyl group was situated ateven-numbered carbons, R-preference was observed for thesubstrates with the methyl group at odd-numbered carbons.</p><p>In order to establish the stereoisomeric composition of thenatural sex pheromone of the pine sawfly<i>Microdiprion pallipe</i>s, all sixteen individual isomers of3,7,11-trimethyl-2-tridecanol (and their propionate esters)found in this species were synthesised in highdiastereoisomeric purities, 95.3-97.6%. The syntheses werebased on six enantiomerically pure building blocks, the fourstereoisomers of 1-lithio-2,6-dimethyloctane and the twoenantiomers of<i>ci</i>s-3,4- dimethyl-g-butyrolactone.</p><p><b>Keywords:</b>lipase,<i>Pseudomonas cepaci</i>a,<i>Candida rugos</i>a, enantioselective, enantiomeric ratio,primary 2-methylalcohol, substituted-methylcarboxylic acid,pine sawfly,<i>Microdiprion pallipe</i>s, sex pheromone,3,7,11-trimethyl-2-tridecanol, stereoisomer.</p>

Cystic fibrosis (CF) is a single gene Mendelian disorder characterized by pulmonary disease and pancreatic insufficiency. Pulmonary disease is the major cause of death in CF patients. Although some cystic fibrosis transmembrane conductance regulator (CFTR) genotypes are associated with less severe disease, patients possessing the same genotype show great variation in pulmonary disease severity and progression. Genes involved in modulating the inflammatory response and genes increasing susceptibility to infection are proposed as modifiers of pulmonary disease severity. Polymorphisms selected for based on evidence that they affect the function of the gene and prevalence of the putative risk allele: 1) antiprotease gene alpha-1-antitrypsin (alpha-1-AT), 2) innate immunity genes: mannose binding lectin (MBL2) (promoter [G→C] at -221 and codon 52 (Arg52Cys, D allele), 54 (Gly54Asp, B allele), and 57 (Gly57Glu, C allele), and pulmonary surfactant genes SPA-1 (Arg219Trp), SPA-2 (Thr9Asn, Lys223Gln) and SPD (Thr11Met), 3) antioxidant genes GSTM1 and T1 (gene deletion polymorphisms), GSTP1 (Ile105Val) and GCLC repeats, 4) mucin genes (MUC2 and MUC5B). Pulmonary disease progression and survival in patients with chronic Burkholderia cepacia complex (BCC) infection were also investigated controlling for genomovar and RAPD type of the organism. BCC infection was associated with more severe pulmonary disease progression and worse survival. Alpha-1-AT genotype was not a major contributor to variability of pulmonary disease severity, but the results suggest that alpha-1-AT plasma levels during pulmonary infections may be affected by poor nutritional status. We showed similar pulmonary disease progression and MBL2 genotype. Contrary to the previous literature, wild-type MBL2 genotype was associated with steeper decline in pulmonary disease over time following chronic infection with BCC, but genotype was not associated with increased susceptibility to BCC infection. We showed inconsistant results for the pulmonary surfactant gene polymorphisms, GSTM1, T1 and GSTP1 polymorphisms, and number of repeats for GCLC and MUC5B depending on the phenotype investigated. We conclude that some of the variability in pulmonary disease severity and progression in CF is explained by polymorphisms in secondary genes.

Cystic fibrosis (CF) is a single gene Mendelian disorder characterized by pulmonary disease and pancreatic insufficiency. Pulmonary disease is the major cause of death in CF patients. Although some cystic fibrosis transmembrane conductance regulator (CFTR) genotypes are associated with less severe disease, patients possessing the same genotype show great variation in pulmonary disease severity and progression. Genes involved in modulating the inflammatory response and genes increasing susceptibility to infection are proposed as modifiers of pulmonary disease severity. Polymorphisms selected for based on evidence that they affect the function of the gene and prevalence of the putative risk allele: 1) antiprotease gene alpha-1-antitrypsin (alpha-1-AT), 2) innate immunity genes: mannose binding lectin (MBL2) (promoter [G→C] at -221 and codon 52 (Arg52Cys, D allele), 54 (Gly54Asp, B allele), and 57 (Gly57Glu, C allele), and pulmonary surfactant genes SPA-1 (Arg219Trp), SPA-2 (Thr9Asn, Lys223Gln) and SPD (Thr11Met), 3) antioxidant genes GSTM1 and T1 (gene deletion polymorphisms), GSTP1 (Ile105Val) and GCLC repeats, 4) mucin genes (MUC2 and MUC5B). Pulmonary disease progression and survival in patients with chronic Burkholderia cepacia complex (BCC) infection were also investigated controlling for genomovar and RAPD type of the organism. BCC infection was associated with more severe pulmonary disease progression and worse survival. Alpha-1-AT genotype was not a major contributor to variability of pulmonary disease severity, but the results suggest that alpha-1-AT plasma levels during pulmonary infections may be affected by poor nutritional status. We showed similar pulmonary disease progression and MBL2 genotype. Contrary to the previous literature, wild-type MBL2 genotype was associated with steeper decline in pulmonary disease over time following chronic infection with BCC, but genotype was not associated with increased susceptibility to BCC infection. We showed inconsistant results for the pulmonary surfactant gene polymorphisms, GSTM1, T1 and GSTP1 polymorphisms, and number of repeats for GCLC and MUC5B depending on the phenotype investigated. We conclude that some of the variability in pulmonary disease severity and progression in CF is explained by polymorphisms in secondary genes.

Cystic fibrosis (CF) is a single gene Mendelian disorder characterized by pulmonary disease and pancreatic insufficiency. Pulmonary disease is the major cause of death in CF patients. Although some cystic fibrosis transmembrane conductance regulator (CFTR) genotypes are associated with less severe disease, patients possessing the same genotype show great variation in pulmonary disease severity and progression. Genes involved in modulating the inflammatory response and genes increasing susceptibility to infection are proposed as modifiers of pulmonary disease severity. Polymorphisms selected for based on evidence that they affect the function of the gene and prevalence of the putative risk allele: 1) antiprotease gene alpha-1-antitrypsin (alpha-1-AT), 2) innate immunity genes: mannose binding lectin (MBL2) (promoter [G→C] at -221 and codon 52 (Arg52Cys, D allele), 54 (Gly54Asp, B allele), and 57 (Gly57Glu, C allele), and pulmonary surfactant genes SPA-1 (Arg219Trp), SPA-2 (Thr9Asn, Lys223Gln) and SPD (Thr11Met), 3) antioxidant genes GSTM1 and T1 (gene deletion polymorphisms), GSTP1 (Ile105Val) and GCLC repeats, 4) mucin genes (MUC2 and MUC5B). Pulmonary disease progression and survival in patients with chronic Burkholderia cepacia complex (BCC) infection were also investigated controlling for genomovar and RAPD type of the organism. BCC infection was associated with more severe pulmonary disease progression and worse survival. Alpha-1-AT genotype was not a major contributor to variability of pulmonary disease severity, but the results suggest that alpha-1-AT plasma levels during pulmonary infections may be affected by poor nutritional status. We showed similar pulmonary disease progression and MBL2 genotype. Contrary to the previous literature, wild-type MBL2 genotype was associated with steeper decline in pulmonary disease over time following chronic infection with BCC, but genotype was not associated with increased susceptibility to BCC infection. We showed inconsistant results for the pulmonary surfactant gene polymorphisms, GSTM1, T1 and GSTP1 polymorphisms, and number of repeats for GCLC and MUC5B depending on the phenotype investigated. We conclude that some of the variability in pulmonary disease severity and progression in CF is explained by polymorphisms in secondary genes. / Medicine, Faculty of / Medicine, Department of / Experimental Medicine, Division of / Graduate

This diploma thesis deals with the study of the utilization of Raman spectroscopy and Raman tweezers for analysis and isolation of polyhydroxyalkanoates (PHA) producing bacteria. Using gas chromatography with FID detection, we determined the polyhydroxybutyrate (P(3HB)) content of the PHA biomass of bacterial strains Burkholderia cepacia, Halomonas halophila, Cupriavidus necator H16 and its mutant strain Cupriavidus necator H16/PHB-4 and Lactobacillus delbrueckii, which is not a producer of polyhydroxyalkanoates but this bactrea was selected as representative of Gram-positive bacteria. Subsequently, thanks to Raman microspectroscopy, Raman tweezers and FT-IR spectrometer in combination with Raman FT-module, we were able to confirm or disprove the presence of P(3HB) in bacteria. Furthermore, the thesis describes Cupriavidus necator H16, which is a model organism for the production of P(3HB), and his mutant strain Cupriavidus necator H16/PHB-4. The bacterial strain Cupriavidus necator H16 was cultivated in a production mineral medium of various nitrogen contents, while cultivation was also carried out in liquid Nutrient Broth. By this cultivation we were able to reach various P(3HB) content in bacterial biomass, the spectra were subsequently compared with the spectrum of the bacterial strain Cupriavidus necator H16/PHB-4. Raman spectroscopy is well used to characterize the composition of individual bacterial cells, is a fast, versatile, and virtually non-invasive tool for studying cells.

This master thesis deals with polyhydroxyalkanoates (PHA) and their role in bacterial biofilms. In the theoretical part the polyhydroxyalkanoates, bacterial biofilm and the relationship between them were reviewed. The experimental part focused on differences in PHA production by planktonic and biofilm cells. In order to study selected topic, bacterial strains of Burkholderia cepacia and Burkholderia sacchari were cultivated using a CDC biofilm reactor. The attention was paid to quantity and especially to the form in which PHA occurs in planktonic and biofilm cells. Results of Raman spectroscopy have shown that PHA exists exclusively in native amorphous form in planktonic bacterial cells. On the other hand, in biofilm PHA occurs also in a partially crystalline form. In addition, the resistance of planktonic and biofilm cells against various stress factors and the effect of osmotic stress on PHA production was tested too. According to the results of the experiment, when the bacteria were exposed to different stress factors (high temperature, low temperature, presence of detergent and so forth) biofilm cells showed a higher stress resistance than planktonic cells. Apart from slowing cell growth and reproduction, increased osmotic pressure in the culture medium also caused decrease of PHA production. In addition, planktonic cells responded to external stimuli more sensitively than biofilm ones.

This diploma thesis is focused on study of chemical and enzymatical hydrolysis of raw wood material. The aim of this work was to find the suitable method for pretreatment of selected lignocellulose materials. The theoretical part deals with characterization of lignocellulosic material and its components. There are also subscribed various pretreatment methods and their effect on hydrolysis of sawdust. In experimental part of the work the most appropriate approach of pretreatment and hydrolysis of sawdust was studied. Criteria for the selection of suitable method was concentration of saccharides as desired product of hydrolysis and also concentration of the most important microbial inhibitors - polyphenols. Application of 96% ethanol or 5% H2O2 were identified as the most promising pretreatment methods which enhanced yields of fermentable sugars about 30 %. Further, we also performed cultivation of bacteria Burkholderia cepacia and bacteria Burkholderia sacchari using solution obtained by hydrolysis of lignocellulose material.

The aim of this work was study of the detoxification of lignocellulose material hydrolysates and to investigate sawdust suitability as a substrate for microbial production of PHA by bacteria Burkholderia cepacia and Burkholderia sacchari. In the experimental part of the work the most suitable way of detoxification of model and real hydrolysate was studied. After that, detoxification methods used were evaluated. Criteria for evaluation were concentration of polyphenols as the most important microbial inhibitors and reduction saccharides as the main carbon substrate. Furthermore, fermentability of the hydrolysates was also tested by cultivation of two bacteria capable of PHA accumulation. Burkholderia sacchari demonstrated higher ability to accumulate PHA then Burkholderia cepacia. Then in the summary – most effective way for detoxification was ‚overliming‘. Major increase of PHB in biomass was obtained when Burkholderia sacchari was cultivated on media gained by application of overliming of real lignocellulose hydrolysate. However, total gains of PHB were more likely low and then sawdust can not be considered as a substrate for PHB production at industrial scale.

No description available.