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The present study is the first molecular epidemiological study in Lithuania that covers the evaluation of resistance of both Acinetobacter spp. and E. coli to various antibiotic groups, as well as virulence gene identification, phylogenetic isolation, application, and analysis of different genotyping methods. This study describes 194 Acinetobacter spp. and 256 multidrug-resistant E. coli isolates from hospitalised patients diagnosed with sepsis. Antibiotic susceptibility genes, which have been minimally studied in Lithuania, have been characterised in this study. These include the OXA group β-lactamases, metallo-β-lactamases, and carbapenemases. Resistance to aminoglycoside-, quinolone-, and polymyxin-encoding genes have not been extensively studied in Europe and have never been identified in Lithuania prior to this study. Similarly, regulatory genes for efflux pumps, which are increasingly being detected worldwide, were identified in Lithuania for the first time. Genes encoding virulence factors responsible for the invasion and pathogenesis of ExPEC strains in the human body, have also been identified for the first time in Lithuania. Phylogenetic groups of E. coli isolates from different Lithuanian hospitals were also identified in this study, which further supplemented the results of previous studies in Lithuania. Moreover, it was determined that 45.9% of all Acinetobacter spp. isolates exhibited an β-lactams resistance gene combination of blaOXA subgroup-3-blaOXA subgroup-1-blaOXA51-blaOXA sugroup-2-blaOXA-subgroup-4-blaVIM-1-blaTEM-92. Moreover, the most common resistance gene combination in E. coli isolates was tetA-strB-sul2-blaTEM-blaNDM-strA-fosA-blaAIM-sul3-aadA-blaCTX-M-9, which caused resistance to β-lactams, aminoglycosides, sulphonamides, fosfomycin, and tetracyclines. The most common virulence gene combination was fuyA-fimH-iroN, where fuyA and iroN encode siderophores and fimH is responsible for bacterial adhesion to host cells. Phylogenetic group determination was performed for all E. coli isolates. The isolates belonged to four phylogenetic groups: A, B1, B2, and F. Group A isolates were detected at a significantly higher frequency (79.3% of all isolates) than isolates of groups B1 (0.8%), B2 (15.6%), and F (4.3%). Acinetobacter spp. and E. coli isolates from different Lithuanian hospitals were described in detail by different genotyping methods, with a strong focus on the analysis of the genotypic profiles and the investigation of possible associations between the isolation year and the particular hospital. BOX-PCR genotyping analysis was performed on all 194 Acinetobacter spp. isolates, a total of 191 BOX-PCR profiles were identified, which were separated into six clusters. In total, 235 BOX-PCR profiles of E. coli isolates were obtained, where all profiles were separated into 14 genotypic clusters. Moreover, it was observed determination of resistance and virulence genes to genotyping profiles and results showed potential changes in these genes. Also, at this work MLVA genotyping was used to describe the VNTR profiles of studied bacteria and results could be used for further analysis of future distribution Acinetobacter spp. and E coli multidrug resistant strains and their potential treatment strategies in different healthcare institutions.

Human pathogenic bacteria are increasingly gaining antimicrobial resistance. For this reason, new natural bioactive substances are being sought. Caves, because of their oligotrophic conditions, are considered to be an excellent source of antimicrobials. Studies over the past decades have shown that the microorganisms detectable in caves are able to synthesize secondary metabolites which might inhibit the growth of pathogens. During this work, the search of genes for the synthesis of antimicrobials was carried out in strains characterized by phenotypic antimicrobial activity in the previous scientific group. Qualitative and quantitative expression analysis of the genes for the synthesis of the bioactive substances in selected strains was also performed. After gene search, it was decided to work with 3 strains - VR1, VR5 and VR26. The phylogenetic analysis of these strains showed that the VR1 strain belongs to the Rhodococcus genus and is closest to the species Rhodococcus erythreus and Nocardia coellaca. The VR5 strain belongs to the genus Arthrobacter and is the closest to the species Arthrobacter sulfureus, and the VR26 strain belongs to the genus Pseudomonas and is closest to the species Pseudomonas frederiksbergensis. The qualitative analysis of the expression revealed that the expression of the genes for the synthesis of antimicrobials took place in all 3 strains, at all stages of growth, independently to the richness of the nutrient medium. For quantitative expression analysis VR26 strain was selected. It was found that the weakest expression of the gene was carried out during exponential growth, most effectively during stationary growth.

Analysis of bioactive compound synthesis gene transcription in Krubera – Voronja Cave bacterial Because of increasing emergence of antibiotic resistant bacteria, scientists are constantly trying to discover for new bioactive compounds. A promising environment for such a search - caves. A limited resource of available energy in such habitats, forces organisms to compete through production of antimicrobial agents. Many bacteria that show biological activities have already been isolated, although the bioactive compound production genes remain unexplored. Four bacterial strains isolated from Kruber-Voronja cave samples were analyzed during the work. In the genomes of these microorganisms, genes for the production of bioactive substances have been identified: Streptomyces sp. - Type I and Type II PKS genes and NRPS gene; Pseudarthrobacter sp. - one NRPS gene; Pseudomonas sp. - two different NRPS genes; Planomicrobium sp. - one gene coding for Type I PKS. Growth curves were constructed for all four strains by growing microorganisms nutrient rich and nutrient poor enviroments. Total RNA were extracted from the cells in the exponential growth phase, in the transition to stationary growth and in the stationary growth phase. In case of Pseudarthrobacter sp. strain NRPS expression was observed only in nutrient poor medium, during all growth phases. For Streptomyces sp. Type I PKS expression was observed only during exponential growth in rich media, and Type II PKS at all growth stages, both in rich and poor media. Streptomyces sp. expression of the NRPS gene, similar to Pseudarthrobacter sp. strain, observed only in nutrient poor media, during all growth stages.For Pseudomonas sp. the expression of one NRPS gene was not observed and the other NRPS gene expression was observed under all conditions tested. The quantitative expression analysis using specific quantitative PCR primers showed that in case of Streptomyces sp. strain PKS genes D_18_PKS_1 and D_18_PKS_2 expression levels were completely different depending on the composition of the nutrient amountin growth media, and the expression of the gene L_5_PKS_1 was higher in nutrient poor media and independent of the growth phase. In case of Pseudomonas sp. strain expression of L_3_NRPS_3 gene increased over all growth phases but was not dependent on the composition of the culture medium. The expression of the gene I_13_NRPS_1 was significantly higher during exponential growth in nutrient poor media, and the expression of the gene A_16_NRPS_ depended on both the composition of growth media and growth phase.

Chitin is the second most abundant natural biopolymer that is found in exoskeletons of crustaceans, incuticles of insects, in cell walls of fungi and yeasts, even in protozoa. This polymer helps to sustain the shape of arthropods and fungi, also it protects them from environmental impact, besides it is the source of nutrients for various bacteria species. Since chitin has a complex structure it is difficult to decompose it, however chitinolytic systems (comprised from chitinolytic enzymes) in microorganisms are able to decompose this polymer effectively. Chitin polymer degrading chitinolytic enzymes are found in every kingdom of living organisms. A vast variety of useful products, and chitinolytic enzymes amongst them, are being produced by bacteria, especially Actinobacteria phylum bacteria, that are found in soil. Streptomyces genus bacteria from Actinobacteria phylum often produces more than one chitinolytic enzyme at a time. Bacterial strain, named BiG9, was isolated from nature and showed chitinolytic activity. Phylogenetic analysis showed that this chitinolytic strain belongs to Streptomyces genus with 99 % of identity to Streptomyces albolongus and Streptomyces cavourensis. Partialy purified chitinolytic enzyme showed maximum activity (determined by zymography method) in 37 degrees Celsius and pH 5. The measured molecular weight of chitinolytic enzyme was about 27 kDa. Chitinolytic enzyme retains activity over a wide temperature range (6 – 50 degrees Celsius), and wide pH range (4 – 9 pH). Thermostability of this chitinolytic enzyme is improved by calcium ions and activity is stimulated by ferrous ions, inhibited by manganous and magnesium ions.

Screening for Mesophilic Chitin-Degrading Microorganisms and Characterization of Their Activity Chitin is one of the most common polysaccharides in nature, which is usually found in fungi, cuticula of insects or outer skeleton of crustaceans. The high abundancy of this polysaccharide and difficulties in its degradation are promoting scientists to search for new bacterial strains which would have a chitinolytic activity. These new strains could help to improve degradation of chitinous compounds and have an industrial usage. The aim of this study was to search for new bacterial strains which would have a chitinolytic activity. After picking one with the best activity, the enzymes from the strain were extracted and characterized. Based on phylogenetic analysis it was showed that selected strain belongs to Streptomyces genus and is closely related to Streptomyces sampsonii species. It was demonstrated that selected strain secretes four chitinolytic enzymes which are 120 kDa, 27 kDa, 25 kDa and 17 kDa in sizes, also it was studied that intracellular enzyme filtrate contained additionally 230 kDa, 100 kDa, 35 kDa, 32 kDa and 20 kDa chitinolytic enzymes. It was demonstrated that this group of enzymes has their optimal activity when temperature ir 30 ºC – 37 ºC also pH 3,0 and pH 5,0. Also, it was studied that this group of enzymes is activated by Mn2+, Na+, or Zn2+ ions and inhibited by Fe2+ and Fe3+ ions.

Increased demand of nowadays consumers for less processed, with no chemical preservatives but beneficial to health food products forces the research on compatibility studies of functional food components – probiotic bacteria and prebiotics, aiming to create effective synbiotics. Probiotic bacteria produce various antibacterial substances and bacteriocins among them. These proteinaceous molecules attracted enormous attention due to the widespread pathogenic bacteria resistance to conventional antibiotics, while bacteriocins are considered being the best alternative. In this work lactic acid bacteria type strains, probiotic yogurt isolates and thermophilic spore-forming 118 strain, producing antibacterial substances, were investigated. Different ability to assimilate prebiotic oligosaccharides and their influence on antibacterial activity of investigated strains was shown during this research. Antibacterial activity between thermophilic and lactic acid bacteria and their activity spectra against pathogenic bacteria were also evaluated. New bacteriocin, secreted by 118 strain, belonging to Aeribacillus sp., was characterized with properties especially beneficial for its application in food industry. Probiotic potential of investigated thermophilic and lactic acid bacteria strains was also evaluated. According to the obtained results, 4 combinations of investigated bacteria and prebiotic oligosaccharides were proposed as the most perspective for creation of new synbiotics.

Increased demand of nowadays consumers for less processed, with no chemical preservatives but beneficial to health food products forces the research on compatibility studies of functional food components – probiotic bacteria and prebiotics, aiming to create effective synbiotics. Probiotic bacteria produce various antibacterial substances and bacteriocins among them. These proteinaceous molecules attracted enormous attention due to the widespread pathogenic bacteria resistance to conventional antibiotics, while bacteriocins are considered being the best alternative. In this work lactic acid bacteria type strains, probiotic yogurt isolates and thermophilic spore-forming 118 strain, producing antibacterial substances, were investigated. Different ability to assimilate prebiotic oligosaccharides and their influence on antibacterial activity of investigated strains was shown during this research. Antibacterial activity between thermophilic and lactic acid bacteria and their activity spectra against pathogenic bacteria were also evaluated. New bacteriocin, secreted by 118 strain, belonging to Aeribacillus sp., was characterized with properties especially beneficial for its application in food industry. Probiotic potential of investigated thermophilic and lactic acid bacteria strains was also evaluated. According to the obtained results, 4 combinations of investigated bacteria and prebiotic oligosaccharides were proposed as the most perspective for creation of new synbiotics.

Lipases and esterases produced by Geobacillus bacteria is a promising and important area in basic research and industrial applications. In this work the significance of Asp371, Phe375 and Tyr376 from C- terminal region for the efficient functionality of Geobacillus sp. 95 lipase (GD-95) was shown for the first time and new carboxylesterase (GDEst-95) produced by Geobacillus sp. 95 strain with a molecular size of 55 kDa was identified. In further experiments GDEst-95 esterase together with GD-95 lipase were used for the construction of the first fused lipolytic chimeric biocatalyst GDEst-lip. Because of their physicochemical and kinetic properties GDEst-95 esterase and fused GDEst-lip enzyme have a high potential for application in various industrial areas. This work also demonstrated that identical domain fusion strategy (GDLip-lip and GDEst-est) is useful for creating of new biocatalysts. It was shown that usage of several fused domains can modulate the activity and physicochemical characteristics of target enzymes for industrial applications. In this study three new Geobacillus lipases were also identified. These proteins expanded the existing information about physicochemical properties of Geobacillus lipases. Furthermore, genes of Geobacillus lipases were subjected to DNA shuffling and epPCR experiments to create more thermostable and more thermoactive lipolytic enzymes.

Lipases and esterases produced by Geobacillus bacteria is a promising and important area in basic research and industrial applications. In this work the significance of Asp371, Phe375 and Tyr376 from C- terminal region for the efficient functionality of Geobacillus sp. 95 lipase (GD-95) was showen for the first time and new carboxylesterase (GDEst-95) produced by Geobacillus sp. 95 strain with a molecular size of 55 kDa was identified. In further experiments GDEst-95 esterase together with GD-95 lipase were used for the construction of the first fused lipolytic chimeric biocatalyst GDEst-lip. Because of their physicochemical and kinetic properties GDEst-95 esterase and fused GDEst-lip enzyme have a high potential for application in various industrial areas. This work also demonstrated that identical domain fusion strategy (GDLip-lip and GDEst-est) is useful for creating of new biocatalysts. It was shown that usage of several fused domains can modulate the activity and physicochemical characteristics of target enzymes for industrial applications. In this study three new Geobacillus lipases were also identified. These proteins expanded the existing information about physicochemical properties of Geobacillus lipases. Furthermore, genes of Geobacillus lipases were subjected to DNA shuffling and epPCR experiments to create more thermostable and more thermoactive lipolytic enzymes.

Solid keratin-rich waste management is one of essential research area in nowadays. Conventional chemical and high thermal keratin waste decomposition methods are fully explored and not enough effective for future biotechnolgy perspectives. However, traditional keratin-rich waste decomposition methods could be replaced by environmentally-friendly and economical microbial keratin waste biodegradation methods without energy wastage and essential amino acids and nutrition elements loss. Therefore, microbial bio-decomposition are attractive approach to keratin or keratin-like waste manage without any nutrition loss in eco-friendly process environment. Increased attention has been diverted to these keratinolytic peptidases because of their important potential uses in biomedicine, pharmaceutics, cosmetics and keratin waste bioconversion industries associated to the hydrolysis of keratin. Keratinolytic proteinases are next generation molecular tools for keratin hydrolysis and production of small value-added bio-active peptides. For the first time, we identified, purified and characterized native keratinolytic peptidase (BtKER) from Bacillus thuringiensis AD-12. For the first time, we created chimeric (SynKer-TT and SynKer-TM) keratinolytic peptidases with improved physicochemical properties. Eventually, we demonstrated that metabolically active secretomes from keratinolytic microorganisms can be used for eco-friendly enzymatic keratin waste management in lab-scale system.

Solid keratin-rich waste management is one of essential research area in nowadays. Conventional chemical and high thermal keratin waste decomposition methods are fully explored and not enough effective for future biotechnolgy perspectives. However, traditional keratin-rich waste decomposition methods could be replaced by environmentally-friendly and economical microbial keratin waste biodegradation methods without energy wastage and essential amino acids and nutrition elements loss. Therefore, microbial bio-decomposition are attractive approach to keratin or keratin-like waste manage without any nutrition loss in eco-friendly process environment. Increased attention has been diverted to these keratinolytic peptidases because of their important potential uses in biomedicine, pharmaceutics, cosmetics and keratin waste bioconversion industries associated to the hydrolysis of keratin. Keratinolytic proteinases are next generation molecular tools for keratin hydrolysis and production of small value-added bio-active peptides. For the first time, we identified, purified and characterized native keratinolytic peptidase (BtKER) from Bacillus thuringiensis AD-12. For the first time, we created chimeric (SynKer-TT and SynKer-TM) keratinolytic peptidases with improved physicochemical properties. Eventually, we demonstrated that metabolically active secretomes from keratinolytic microorganisms can be used for eco-friendly enzymatic keratin waste management in lab-scale system.

The full understanding of laws of nature cannot be accomplished without comprehensive characterisation of collagenolysis as collagen is a major structural protein in organisms. The diversity of collagenolytic peptidases from non-pathogenic bacteria remains only fragmentary characterized, whereas the characterization of the diversity of collagenolytic peptidases from eukaryotes and pathogenic bacteria are far more comprehensive. The determined constitutive production of collagenases by moderate thermophile Geobacillus thermoleovorans DSM 15325 confirms the ability of thermophiles to be ever ready for collagen uptake. The identification of constitutive collagenolytic peptidases variety produced by geobacilli lead to substantial development of collagen degradation model and confirms importance of collagenolytic potential for adaptive plasticity of bacteria. Determined M3 family M3B subfamily oligopeptidases, secreted by geobacilli, characteristics indicate oligopeptidases nutritional importance for bacteria during the adaption for environmental changes. Combination of oligopeptidases characteristics also outlines biotechnological applicability of this thermostable hydrolase. Determined U32 family peptidase from geobacilli characteristics ensure objective understanding of U32 family peptidases characteristics and functional importance. U32 family peptidases ability to specifically in vitro interact with dsRNA was never determined for collagenolytic peptidases previously.

The aim of the study was to investigate the diversity and ecological properties of Dothideomycetes and Sordariomycetes in the alluvial black alder forests and the pine forest affected by cormorants. For the first time in Lithuania, detailed investigations were carried out on the diversity and distribution of Dothideomycetes and Sordariomycetes in the above-mentioned forests. The checklist of the study fungi was complied. 72 Dothideomycetes and Sordariomycetes species new to Lithuania were identified and original descriptions of their morphology were provided. The influence of abiotic factors on the diversity of woody and herbaceous plants fungi was evaluated for the first time in Lithuania. The results of the study enhanced the knowledge about the distribution of Dothideomycetes and Sordariomycetes in the alluvial forests protected in Europe, which may be used to protect the biologic diversity of these habitats. The properties of the diversity and distribution of studied fungi in the pine forest affected by cormorants allow assessing the impact of the hypertrophication, caused by these birds on the pine forest mycobiota. Darbo tikslas – ištirti dotidėjomicetų (Dothideomycetes) ir sordarijomicetų (Sordariomycetes) įvairovę bei ekologines ypatybes aliuviniuose juodalksnynuose ir kormoranų pažeistame pušyne. Darbe pirmą kartą Lietuvoje detaliai ištirta aliuviniuose juodalksnynuose ir kormoranų pažeistame pušyne dotidėjomicetų ir sordarijomicetų rūšių įvairovė ir paplitimas. Sudarytas šiuose miškuose aptiktų grybų rūšių sąvadas. Išaiškintos 72 naujos Lietuvai tirtų aukšliagrybūnų rūšys ir pateikti originalūs jų morfologijos aprašymai. Pirmą kartą Lietuvoje įvertinta aplinkos veiksnių įtaka sumedėjusių bei žolinių augalų dotidėjomicetų ir sordarijomicetų įvairovei. Darbo rezultatai pagilino žinias apie šių grybų paplitimo dėsningumus Europoje saugomuose aliuviniuose miškuose, kas gali būti panaudota šių buveinių biologinės įvairovės išsaugojimui. Kormoranų pažeistame pušyne nustatyti tirtų grybų rūšinės sudėties ir paplitimo ypatumai leidžia įvertinti šių paukščių sukeliamos hipertrofikacijos poveikį pušyno mikobiotai.

Melatoninas – tamsiuoju paros metu kankorėžinėje liaukoje gaminamas hormonas. Šviesa nakties metu slopina melatonino produkciją. Melatoninas yra svarbus organizmo biologinis reguliatorius. Jis reguliuoja paros ir sezono bioritmus, gliukozės metabolizmą, lytinių liaukų aktyvumą, širdies ir kraujagyslių sistemos veiklą, virškinimo trakto veiklą, kitų endokrininių liaukų aktyvumą, imuninės sistemos veiklą. Literatūroje paskelbta nemažai duomenų apie imunoreguliacines melatonino savybes. Melatoninas panaikina su amžiumi susijusias užkrūčio liaukos ir blužnies involiucijas, skatina imuninės sistemos ląstelių proliferaciją, pagerina imuninį atsaką. Tyrinėjant imunoreguliacines melatonino savybes daugeliu atvejų taikomi tyrimo metodai, kurių metu atliekamos melatonino injekcijos arba pašalinama pagrindinė melatonino gamybos vieta - kankorėžinė liauka. Tačiau ankstesnių eksperimentų metu melatonino gamyba retai buvo natūraliai slopinama, laikant gyvūnus pastovaus apšvietimo sąlygomis. Disertacijos darbuose buvo taikytas pastovaus apšvietimo metodas modelinėje pelių sistemoje. Taip buvo siekta atkurti pamaininio darbo sąlygas ir ištirti galimą melatonino trūkumo poveikį žmonių, dirbančių pamaininį darbą, imuninei sistemai. Gauti rezultatai rodo, kad, laikant peles pastovaus apšvietimo sąlygomis, sutrinka jų imuninės sistemos homeostazė: antikūnų gamyba, leukocitų, granuliocitų migravimas. Dirbant pamaininį darbą reikėtų atkreipti dėmesį į jo poveikį imuninei sistemai ir įvertinti... [toliau žr. visą tekstą] Melatonin – is a hormone produced by the pineal gland during the dark time. Light during the night suppresses melatonin production. Melatonin is an important biological body regulator: it controls daily and seasonal biorhythms, glucose metabolism, gonadal activity, cardiovascular system, gastrointestinal tract and the activity of the immune system There is a lot of scientific information about the immunoregulative properties of melatonin. Melatonin modulates the development of some organs of the immune system, cell differentiation, immune response and cytokine production. The imunomodulatory activity of melatonin is usually determined by the following experimental models: surgical pinealectomy, in vivo treatment with melatonin or in vitro treatment of the immune cells with melatonin. However, during the experiments while keeping animals under constant light conditions, melatonin production was rarely naturally inhibited. For the experiments of this study, the method of constant lighting in the mouse model was applied. Our aim was to artificially evoke shift work conditions in order to find out how the deficiency of melatonin production influences the immune system of the shift workers. The results obtained showed that keeping mouse under constant lighting conditions the homeostasis of the immune system (antibody production, leukocyte and granulocyte migration) is disrupted. Therefore, shift work and its influence on the immune system could be considered as a factor for... [to full text]

Darbo tikslas – ištirti dotidėjomicetų (Dothideomycetes) ir sordarijomicetų (Sordariomycetes) įvairovę bei ekologines ypatybes aliuviniuose juodalksnynuose ir kormoranų pažeistame pušyne. Darbe pirmą kartą Lietuvoje detaliai ištirta aliuviniuose juodalksnynuose ir kormoranų pažeistame pušyne dotidėjomicetų ir sordarijomicetų rūšių įvairovė ir paplitimas. Sudarytas šiuose miškuose aptiktų grybų rūšių sąvadas. Išaiškintos 72 naujos Lietuvai tirtų aukšliagrybūnų rūšys ir pateikti originalūs jų morfologijos aprašymai. Pirmą kartą Lietuvoje įvertinta aplinkos veiksnių įtaka sumedėjusių bei žolinių augalų dotidėjomicetų ir sordarijomicetų įvairovei. Darbo rezultatai pagilino žinias apie šių grybų paplitimo dėsningumus Europoje saugomuose aliuviniuose miškuose, kas gali būti panaudota šių buveinių biologinės įvairovės išsaugojimui. Kormoranų pažeistame pušyne nustatyti tirtų grybų rūšinės sudėties ir paplitimo ypatumai leidžia įvertinti šių paukščių sukeliamos hipertrofikacijos poveikį pušyno mikobiotai. The aim of the study was to investigate the diversity and ecological properties of Dothideomycetes and Sordariomycetes in the alluvial black alder forests and the pine forest affected by cormorants. For the first time in Lithuania, detailed investigations were carried out on the diversity and distribution of Dothideomycetes and Sordariomycetes in the above-mentioned forests. The checklist of the study fungi was complied. 72 Dothideomycetes and Sordariomycetes species new to Lithuania were identified and original descriptions of their morphology were provided. The influence of abiotic factors on the diversity of woody and herbaceous plants fungi was evaluated for the first time in Lithuania. The results of the study enhanced the knowledge about the distribution of Dothideomycetes and Sordariomycetes in the alluvial forests protected in Europe, which may be used to protect the biologic diversity of these habitats. The properties of the diversity and distribution of studied fungi in the pine forest affected by cormorants allow assessing the impact of the hypertrophication, caused by these birds on the pine forest mycobiota.

Melatonin – is a hormone produced by the pineal gland during the dark time. Light during the night suppresses melatonin production. Melatonin is an important biological body regulator: it controls daily and seasonal biorhythms, glucose metabolism, gonadal activity, cardiovascular system, gastrointestinal tract and the activity of the immune system There is a lot of scientific information about the immunoregulative properties of melatonin. Melatonin modulates the development of some organs of the immune system, cell differentiation, immune response and cytokine production. The imunomodulatory activity of melatonin is usually determined by the following experimental models: surgical pinealectomy, in vivo treatment with melatonin or in vitro treatment of the immune cells with melatonin. However, during the experiments while keeping animals under constant light conditions, melatonin production was rarely naturally inhibited. For the experiments of this study, the method of constant lighting in the mouse model was applied. Our aim was to artificially evoke shift work conditions in order to find out how the deficiency of melatonin production influences the immune system of the shift workers. The results obtained showed that keeping mouse under constant lighting conditions the homeostasis of the immune system (antibody production, leukocyte and granulocyte migration) is disrupted. Therefore, shift work and its influence on the immune system could be considered as a factor for... [to full text] Melatoninas – tamsiuoju paros metu kankorėžinėje liaukoje gaminamas hormonas. Šviesa nakties metu slopina melatonino produkciją. Melatoninas yra svarbus organizmo biologinis reguliatorius. Jis reguliuoja paros ir sezono bioritmus, gliukozės metabolizmą, lytinių liaukų aktyvumą, širdies ir kraujagyslių sistemos veiklą, virškinimo trakto veiklą, kitų endokrininių liaukų aktyvumą, imuninės sistemos veiklą. Literatūroje paskelbta nemažai duomenų apie imunoreguliacines melatonino savybes. Melatoninas panaikina su amžiumi susijusias užkrūčio liaukos ir blužnies involiucijas, skatina imuninės sistemos ląstelių proliferaciją, pagerina imuninį atsaką. Tyrinėjant imunoreguliacines melatonino savybes daugeliu atvejų taikomi tyrimo metodai, kurių metu atliekamos melatonino injekcijos arba pašalinama pagrindinė melatonino gamybos vieta - kankorėžinė liauka. Tačiau ankstesnių eksperimentų metu melatonino gamyba retai buvo natūraliai slopinama, laikant gyvūnus pastovaus apšvietimo sąlygomis. Disertacijos darbuose buvo taikytas pastovaus apšvietimo metodas modelinėje pelių sistemoje. Taip buvo siekta atkurti pamaininio darbo sąlygas ir ištirti galimą melatonino trūkumo poveikį žmonių, dirbančių pamaininį darbą, imuninei sistemai. Gauti rezultatai rodo, kad, laikant peles pastovaus apšvietimo sąlygomis, sutrinka jų imuninės sistemos homeostazė: antikūnų gamyba, leukocitų, granuliocitų migravimas. Dirbant pamaininį darbą reikėtų atkreipti dėmesį į jo poveikį imuninei sistemai ir įvertinti... [toliau žr. visą tekstą]

Pektinas sudaro didžiausią makromolekulių masę, kuri randama pirminės augalų ląstelių sienelės bei tarpinės plokštelės, atsakingos už tarpląstelinę adheziją, sudėtyje. Pektininė matrica sukuria pagrindą celiuliozės-glikanų tinklui, lemia jo tąsumą, taip pat atsako už porų susidarymą bei yra pagrindinė adhezinė medžiaga tarp ląstelių (Willats, McCartney et al. 2001). Šio polisacharido pagrindą sudaro α-(1-4)-glikozidiniais ryšiais sujungtos D-galakturono rūgšties liekanos (GalpA) ir mažesnė dalis kitų sacharidų, tokių kaip L-ramnozė, arabinozė, galaktozė ir ksilozė. GalpA karboksigrupės yra iš dalies esterintos metoksigrupių ir iš dalies arba visiškai neutralizuotos natrio, kalio ar amonio jonų. Pektiną degraduojantys fermentai, trumpiau vadinami pektinazėmis, sintetinami pačių augalų, taip pat fitopatogeninių bakterijų ir grybų (http://pec.biodbs.info/listofMicroorganisms. .html). Šie fermentai yra klasifikuojami pagal tris kriterijus: a) specifiškumą tam tikram polisacharidui (substratui) arba keliems; b) substrato grandinės skėlimo reakcijos mechanizmą; c) skėlimo vietą substrato grandinėje. Netrukus po šių fermentų atradimo, susidomėta jų pramoniniu pritaikomumu, kuris pasirodė besąs ganėtinai platus: sulčių, kavos, arbatos, augalinio aliejaus, popieriaus ir gyvūnų ėdalo gamyboje, tekstilės pramonėje ir net nuotekų valyme (Kashyap, Vohra et al. 2001). Šiuo metu dar didesnis dėmesys skiriamas termostabiliems pektiną degraduojantiems fermentams, kurie pramonėje gali... [toliau žr. visą tekstą] Plants, bacteria and fungi are known to synthesize pectic polysaccharides degrading enzymes called pectinases. These enzymes are important for plant growth and development, fruit ripening, phytopathogenesis etc. The aim of this study was to select biotechnologically important strains of endospore- forming bacteria with the highest pectinolytic activity as well as characterize pectinolytic enzymes from the crude protein extract. 8 out of 80 tested strains were selected for the experiments of growth curve measurement. PCR and 16S rRNA gene sequence analysis ascertained that two pectinolytic strains with the highest activity are closely related to Geobacillus thermodenitrificans NG80-2. Accordingly, these two strains were named Geobacillus sp. strain No. 3 and No. 4. Further analysis was performed with Geobacillus sp. strain Nr. 3 which was grown in the liquid medium with pectin. The crude protein extract was prepared using supernatant of the culture and then analysed spectrofotometrically for lyases and hydrolases activity, pectic substrates specificity, and thermostability. For further examinations electrophoretic and zymografic analyses were used. It was demonstrated that Geobacillus sp. strain No. 3 secretes at least two enzymes: pectin and polygalacturonic acid lyase and pectin hydrolase. For lyase, molecular mass (60-70 kDa), temperature optimum (60 °C) and pH optimum (7.0) were determined.

Geobacillus thermodenitrificans JK1 kamieno termostabilios ksilanazės geno klonavimas, ekspresija ir fermento savybių analizė Ksilanas - pagrindinis hemiceliuliozės komponentas ir antras pagal paplitimą polisacharidas gamtoje. Šis biologinis polimeras yra chemiškai heterogeniška bei kompleksiška medžiaga, kurios efektyviam degradavimui reikalingos multifermentinės sistemos. Tokios sistemos yra plačiai paplitusios tarp įvairių prokariotinių ir eukariotinių mikroorganizmų. Pagrindinį vaidmenį ksilano degradacijoje atlieka endo-1,4-β-ksilanazės. Ksilanazės pasižyminčios termostabilumu yra plačiai taikomas popieriaus ir maisto pramonėje, pašarų gamyboje bei biokonversijoje. Darbo metu buvo atrinktas JK1 kamienas, gebantis sekretuoti ksilanolizinius fermentus į ląstelės išorę. Remiantis 16S rRNR ir spo0A genų sekų palyginimais bei filogenetine analize nustatyta, kad tiriamas kamienas buvo artimiausias G. thermodenitrificans NG80-2 kamienui, kuriame taip pat nustatytas ksilanazės genas. Pagal šią geno seką sukurti pradmenys amplifikuojantys JK1 kamieno ksilanazės geną (1224 bp). Ksilanazės genas klonuotas ir perkeltas į ekspresijos vektorių (pET-28c(+)), sukonstruojant rekombinantinę ksilanazę su His inkarine uodega (50,979 kDa), kas leido ekspresuoti baltymą E. coli ir atlikti gryninimą nikelio afininės chromatografijos metodu. Šiame darbe pirmą kartą charakterizuota G. thermodenitrificans JK1 kamieno ksilanazė. Rekombinantinio fermento optimalios veikimo sąlygos - 70 °C... [toliau žr. visą tekstą] Cloning, expression and biochemical characterization of the thermostable xylanase from Geobacillus thermodenitrificans strain JK1 Xylan is a major structural polysaccharide in plant cells, and is the second most abundant polysaccharide in nature. Due to its heterogeneity and complexity, the complete hydrolysis of xylan requires a variety of cooperatively acting enzymes. These xylanolytic enzyme systems are quite widespread among prokariotic and eukaryotic microorganisms. Endo-1,4-β-xylanase is considered to play the most important role in xylan degradation. Thermostable xylanases have potential applications in a wide range of industrial processes, including pulp and paper industry, food and animal feeds industry and bioconvertion. In this study a thermophilic JK1 strain producing xylanolytic enzymes was isolated from compost sample. The 16S rRNR and spo0A genes sequence analysis indicated that JK1 strain had the highest similarity with G. thermodenitrificans NG80-2 strain. Based on the xylanase gene sequence from G. thermodenitrificans NG80-2 strain, degenerated primers were designed for amplification of xylanase-coding gene (1224 bp) from JK1 strain. Subsequently, the amplified gene was cloned into expression vector (pET-28c(+)) and the recombinant xylanase with a polyhistidine-tag was designed (50,979 kDa). Biochemical properties of the purified by nickel affinity chromatography recombinant xylanase from G. thermodenitrificans JK1 strain were further characterized. The... [to full text]

Bendrąja prasme kolagenolizė - dalinė arba visiška konkretaus kolageno tipo molekulės proteolizė, vykstanti in vivo (fiziologinių ar patologinių procesų metu) ar in vitro. Vandens molekulė, kaip įprasta hidrolizės reakcijoms, būtinas antrasis kolagenolizės proceso substratas. Kolagenolizę katalizuoja kolagenoliziniu specifiškumu pasižyminčios peptidazės. Kolagenai -vieni svarbiausių baltymų tiek atliekamų funkcijų, tiek biotechnologinio panaudojimo atžvilgiais. Jų panaudojimas neįmanomas be kolagenolizės proceso, taigi -kolagenolizinių peptidazių. Tik nedaugeliui peptidazių būdingas kolagenolizinis specifiškumas. Eukariotų ir prokariotų skirtumai kolageno kaip kolagenolizės substrato produkavimo galimybės atžvilgiu, taip pat netapačios šio fibrilinio baltymo biologinės funkcijos šiuose gyvybės domenuose lėmė skirtumus tarp prokariotų ir eukariotų kolagenolizinių peptidazių. Nors prokariotinės kolagenolizinės peptidazės tiriamos vis intensyviau, daugiau dėmesio skiriama patogeninių kamienų (Clostridium, Porphyromonas genčių) produkuojamoms kolagenazėms. Tuo tarpu nepatogeninių prokariotų kolagenazių tyrimai vykdomi rečiau. Ilgainiui tai gali lemti nevisavertį prokariotų kolagenazių biotechnologinio potencialo išnaudojimą bei iš dalies riboti pačio kolageno taikymą, sprendžiant konkrečias problemas. Biotechnologinio panaudojimo poreikiams tenkinti būtina klonuoti kolagenolizinių peptidazių genus, pritaikant efektyvias ekspresijos sistemas. Darbo tikslas: Atlikti termofilinių... [toliau žr. visą tekstą] Collagens are one of the most important proteins including their functions and biotechnological application. The biotechnological application of collagens wouldn‘t be possible without the process of collagenolysis which is catalysed by collagenolytic peptidases. The aim of this work was to perform transcriptional analysis of U32.002 (Helicobacter-type) collagenolytic peptidase gene and its cloning as well as basic analysis. The analysis of U32.002 peptidase gene transcription was performed after the extraction of total RNA from G. lituanicus DSM 15325T cells that were in two different stages of growth. Reverse transcription assays were performed after total RNA extraction. After the process of reverse transcription samples of cDNA were used for the diagnostic PGR that was carried out by using five different primers. This gene was cloned with and without putative signal/pro sequence in order to produce the preparation of U32.002 peptidase. Full sequence of U32.002 peptidase gene was cloned into pTZ57R/T and then into the expression vector pET28c(+). U32.002 gene without putative signal/pro sequence was cloned into pJET1.2, and then into pET28c(+). SDS-PAGE and MALDI-TOF analyses were performed in order to determine the fact of expression in E. coli BL21 (DE3). Full scale expression optimisation was performed, as well. The influence of calcium and zinc ions to the structural stability of U32.002 peptidase with putative signal/pro sequence was analysed. The collagenolytic... [to full text]

Collagens are one of the most important proteins including their functions and biotechnological application. The biotechnological application of collagens wouldn‘t be possible without the process of collagenolysis which is catalysed by collagenolytic peptidases. The aim of this work was to perform transcriptional analysis of U32.002 (Helicobacter-type) collagenolytic peptidase gene and its cloning as well as basic analysis. The analysis of U32.002 peptidase gene transcription was performed after the extraction of total RNA from G. lituanicus DSM 15325T cells that were in two different stages of growth. Reverse transcription assays were performed after total RNA extraction. After the process of reverse transcription samples of cDNA were used for the diagnostic PGR that was carried out by using five different primers. This gene was cloned with and without putative signal/pro sequence in order to produce the preparation of U32.002 peptidase. Full sequence of U32.002 peptidase gene was cloned into pTZ57R/T and then into the expression vector pET28c(+). U32.002 gene without putative signal/pro sequence was cloned into pJET1.2, and then into pET28c(+). SDS-PAGE and MALDI-TOF analyses were performed in order to determine the fact of expression in E. coli BL21 (DE3). Full scale expression optimisation was performed, as well. The influence of calcium and zinc ions to the structural stability of U32.002 peptidase with putative signal/pro sequence was analysed. The collagenolytic activity of U32.002 peptidase was analysed by using zymography of native collagen, type I. The U32.002 peptidase of G. lituanicus DSM 15325T is transcribed constitutively during the exponential phase of growth, whereas only fragments of U32.002 gene transcript are observed in the later growth phases. It is evident that zinc ions increase structural thermostability of U32.002. The homodimer of U32.002 peptidase with putative signal/pro sequence was able to digest native type I collagen at 50 °C; pH 7,4.

Cloning, expression and biochemical characterization of the thermostable xylanase from Geobacillus thermodenitrificans strain JK1 Xylan is a major structural polysaccharide in plant cells, and is the second most abundant polysaccharide in nature. Due to its heterogeneity and complexity, the complete hydrolysis of xylan requires a variety of cooperatively acting enzymes. These xylanolytic enzyme systems are quite widespread among prokariotic and eukaryotic microorganisms. Endo-1,4-β-xylanase is considered to play the most important role in xylan degradation. Thermostable xylanases have potential applications in a wide range of industrial processes, including pulp and paper industry, food and animal feeds industry and bioconvertion. In this study a thermophilic JK1 strain producing xylanolytic enzymes was isolated from compost sample. The 16S rRNR and spo0A genes sequence analysis indicated that JK1 strain had the highest similarity with G. thermodenitrificans NG80-2 strain. Based on the xylanase gene sequence from G. thermodenitrificans NG80-2 strain, degenerated primers were designed for amplification of xylanase-coding gene (1224 bp) from JK1 strain. Subsequently, the amplified gene was cloned into expression vector (pET-28c(+)) and the recombinant xylanase with a polyhistidine-tag was designed (50,979 kDa). Biochemical properties of the purified by nickel affinity chromatography recombinant xylanase from G. thermodenitrificans JK1 strain were further characterized. The optimal activity of recombinant enzyme was obtained at 70 °C and pH 6,0. It was shown, that this enzyme is thermostable and cellulase-free.