published_or_final_version / Microbiology / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / Microbiology / Doctoral / Doctor of Philosophy

published_or_final_version / Microbiology / Master / Master of Philosophy

No description available.

Results of previous research on whole-cell cultures of Penicillium chrysogenum have suggested that acetyl CoA, without being converted to malonyl CoA, supplies the two carbon units for the elongation of palmitic acid. The purpose of this study was to determine the mode of elongation of 1-t4C palmityl CoA by a 20,000 x g mitochondrial pellet from P. chrysogenum.Acetyl CoA or malonyl CoA was incubated with radioactivelylabeled palmityl CoA for 20 minutes. Avidin was added to some experimental reaction mixtures. The resulting fatty acids were saponified, extracted with hexane, methylated with diazomethane, and purified by thin layer chromatography. The methyl esters were separated and identified by gas-liquid chromatography. The radioactivity of each methyl ester was determined by liquid scintillation spectrometry.Elongation of palmityl CoA was observed in the presence of acetyl CoA, but not in the presence of malonyl CoA. The addition avidin produced a greater proportion of short-chained fatty acidsthe expense of palmitic acid, but did not decrease the percentage of long-chained fatty acids produced.A high proportion of label was recovered in the C18:3 fatty acid, linolenic acid. This suggested that two pathways of linolenic acid synthesis may be operating in this organism.Methods for detection and control of cancer encompass a large area of today's research. Recent use of granulomas as a model for such detection and control may be a promising field, especially for monitoring tumor antigens and immune responses. These granuloma systems are increasingly becoming vehicles in the study of tumor immunology. Although granulomas may be induced naturally by means of foreign bodies i.e. viral, fungal, or bacterial agents, new methods are being established to produce artificial granuloma systems. These systems include chemical or foreign body implantations followed by tumor vaccine challenges. The research presented here involved the use of a golf ball-induced granuloma for the purpose of establishment of a detection system for immune responses. The use of a golf ball-induced granuloma provided a closed system for monitoring cell-mediated and humoral responses to tumor antigens. Immune responses were monitored by means of hematocrits (packed blood cell counts), white blood cell differential counts, and electrophoretic results.Hematocrit results indicated no great immune response to the closed vaccine injected granulomasystems. Observations made on differential white blood cell counts indicated decreasing neutrophil/lymphocyte ratios for cellular immune responses. Electrophoretic results for granuloma fluids indicated decreases in albumin levels concurrent with increases in peak two,and complete loss of peak three following vaccination. Responses to tumor specific antigens in the form of cell-mediated immune responses are indicated by the results presented in this research. Utilization of the golf ball-induced granuloma system provided a means of separating the cell-mediated and humoral immune responses.Tumor specific antigens elicited various immune responses and provide hope for future identification of tumors by this method. Future development and utilization of the golf ball-induced granuloma system may be potential means of monitoring cell-mediated immune responses to tumor malignancies.

Reproduction de : Thèse de doctorat : cMicrobiologie et biocatalyse industrielles : Toulouse, INPT : 2007. / Titre provenant de l'écran-titre. Bibliogr. 236 réf.

Reproduction de : Thèse de doctorat : Pharmacie. Mycologie et mycochimie : Université de Nantes : 2007. / Bibliogr.

published_or_final_version / Microbiology / Doctoral / Doctor of Philosophy

Penicillium chrysogenum was investigated to determine whether there are lipoproteins specifically associated with fatty acid synthesis and in what fraction of P. chrysogenum they are located. Radio-activity labeled free fatty acids and CoA thioester substrates were added to cell-free extracts and incubated. Samples of these incubation mixtures were analyzed for lipoprotein content with electrophoresis and for the presence of radioactivity with a radiochromatogram scanner and scintillation spectrometer.Label from fatty acyl thioester substrates migrated with protein fractions having alpha-2 or beta mobility showing the existence of lipoproteins associated with fatty acid metabolism in cell-free extracts of P. chryogenum. The percent distribution of radioactivity from the thioester incubations supported previous work in this laboratory on the desaturase system and suggested the presence of a long chain fatty acyl-CoA-ACP synthetase enzyme in Penicillium chrysogeum.

Aspergillus niger and Penicillium candidum were grown and harvested on days 6 and 8, which corresponded to their maximal biomasses and lipoxygenase (LOX) activities. The extracts were enriched with ammonium sulfate precipitation at 30 to 70% and 20 to 60% of saturation, respectively. The LOX activity was assayed with linoleic, linolenic and arachidonic acids as substrates. Both enriched microbial LOXs demonstrated preferential substrate specificities towards free fatty acids, over acyl esters of linoleic acid. The LOXs had the highest catalytic efficiency values (ratio of V max to Km) for linolenic acid biocatalysis. Major and minor pH optima at 5.0 and 10.5 were observed for A. niger, whereas for P. candidum they were at 6.0 and 8.5. Normal phase high-performance liquid chromatography (NP-HPLC) and gas-liquid chromatography/mass spectrometry (GC/MS) characterization of end products revealed that both LOXs produced the 10-hydroperoxide of linoleic acid (10-HPOD) at 15 to 16% of total isomers detected, respectively. Chiral studies of the P. candidum LOX catalyzed hydroperoxides revealed an excess in the production of (S) stereo-isomers resultant from linoleic, linolenic and arachidonic acids bioconversion. Penicillium camemberti was grown and harvested at its maximal biomass and LOX activity. The microbial extract was ultrafiltered (30 kDa NMWCO) and KCI (7.5 ppm) was added prior to lyophilization for the stabilization of enzyme activity. The LOX and hydroperoxide lyase (HPL) activities were assayed using linoleic acid and the 10-HPOD as substrates, respectively. The post-lyophilization residual activities were 93% and 223% for LOX and HPL, respectively. The long-term storage stability (-80°C) of the extract (KCI 7.5 ppm) was ~100% after 8 and 4 weeks for LOX and HPL, respectively. The investigated stabilizing chemical additives included glycine, mannitol, glycerol, sucrose and polyethylene glycol. The lowest Kinactivation values were observed with glycine with 0.136 and 0.0296 for LOX and HPL, respectively. Thermostability studies indicated that 5 and 10% (w/v) mannitol and glycine effectively stabilized LOX and HPL, respectively. Immobilization of an enzymatic extract from P. camemberti containing LOX and HPL activities was performed on EupergitRTMC and EupergitRTMC250L-iminodiacetate (IDA), respectively. The free and immobilized extracts both possessed LOX activity with a pH optimum of 6.0, whereas pH 6.0 and 4.0 were the optima of the HPL activity for free and immobilized extract, respectively. Optimal LOX reaction temperatures were 30 and 55°C for the free and immobilized extract, respectively, whereas 45 and 30°C were determined for the HPL activity of the free and immobilized extract, respectively. Long-term stability (-80°C) of the immobilized extract containing LOX and HPL activity showed residual activities of 82.6 and 93.8% after 4 and 8 weeks, respectively.