Funkcionalizované hybridní polymerní struktury pro biomedicínské aplikace

Tato dizertační práce se věnuje syntéze a charakterizaci nových hybridních struktur využitelných v biomedicíně. Tyto systémy jsou zde rozděleny do dvou hlavních skupin: modifikované přírodní polymery a syntetické amfifilní blokové kopolymery, přičemž obě tyto skupiny byly studovány jako potenciální theranostická činidla pro medicinální aplikace. Co se týče první skupiny, přírodní polysacharidy glykogen a manan byly vybrány jako výchozí materiály pro přípravu nových nanokonjugátů, určených pro multimodální detekci in vivo. Protože roubování polysacharidů pomocí syntetických polymerů obecně zpomaluje biodegradační procesy, oba polysacharidy byly modifikovány dvěma různými způsoby tak, aby vytvořily konečné "nanosondy", přičemž jeden způsob využíval roubování pomocí poly(2-methyl-2-oxazolinu). Připravené nanokonjugáty byly funkcionalizovány pomocí N- hydroxysukcimidového esteru, díky čemuž výsledné materiály nesly fluorescenční značku a značku pro magnetickou rezonanci. Finální materiály byly testovány in vitro a in vivo, přičemž bylo zjištěno, že jsou plně biokompatibilní a biodegradovatelné a zároveň vykazují užitečné vlastnosti pro praktické využití v biomedicíně. Glykogen byl modifikován tak, aby nesl allylové a propargylové skupiny, a poté byl lyofilizován z vodného roztoku, aby mohl vytvořit...This doctoral thesis is dedicated to the synthesis and characterization of novel functionalized hybrid structures for biomedical purposes. Systems reported in this work can be subdivided into the two main groups: natural-based materials and synthetic amphiphilic block copolymers. Both groups were studied as perspective theranostic agents for medical applications. In the first group, natural polysaccharides glycogen and mannan were selected as starting materials for preparation of novel nanoconjugates that possess ability for multimodal detection in vivo. Because grafting of natural macromolecules with synthetic polymers generally slows down the biodegradation rate, both polysaccharides were modified in two different ways to form nanoprobes with or without poly(2-methyl-2-oxazoline)s chains. The prepared nanoconjugates were functionalized with N-hydroxysuccinimide-activated fluorescence and magnetic resonance imaging labels. The resulting materials were tested both in vitro and in vivo and were shown to be completely biocompatible, biodegradable and exhibit some extra benefits in terms of their practical usage in biomedicine. Glycogen was functionalized with allyl and propargyl groups with following freeze-drying from aqueous solutions to form nano- and microfibrous materials. The presence of both...Katedra fyzikální a makromol. chemieDepartment of Physical and Macromolecular ChemistryPřírodovědecká fakultaFaculty of Scienc

Ecological and physiological peculiarities of bryophytes on a post-technogenic salinized territory

Taxonomic, biomorphological and ecological structures of bryophytes, their reproductive strategy and the main mechanisms of tolerance in the conditions of salinization were investigated. Bryophytes are the pioneers that have colonized the territory of a tailing storage that holds liquid waste from potassium-magnesium concentrate production of the Mining and Chemical Enterprise "Polymineral". Due to excess salts, the soil solution in the shore area of the tailing pond acquires high osmotic pressure. Three experimental plots which differed significantly in the level of the substrate salinity were laid at the distance of 3, 6 and 9 m from the reservoir for experimental studies. Water extracts of the substrates from the test sites showed the highest concentrations for sulfates – 10.4–64.6 mg Eq/100 g of soil and chlorides – 7.6–43.3 mg Eq/100 g of soil. It was established that the investigated areas of the tailing storage territory differed in the biochemical activity of the substrate, which was evaluated by its redox potential. On the areas of the uncovered substrate it was the lowest – 230 mV, which indicates anaerobiosis in conditions of very high salinization and moisture. Higher ROP values were determined at the sites of bryophyte cover distribution – 295–330 mV. The aim of the study was to determine the features of taxonomic, biomorphological and ecological structures of bryophytes, their reproductive strategy and to establish the main mechanisms of adaptation to the conditions of salinization on the tailing storage territory. 24 species and 3 varieties of bryophytes, belonging to 12 families and 16 genera were found on the shore of the tailing storage pond. The results of biomorphological and ecological analysis of bryophytes indicate the uneven conditions of the habitats and their considerable ecological plasticity. Among the bryophytes, mesophytes, xeromesophytes and meso-eutrophs, eutrophs with a life-form of low dense and loose turf dominated. In salinization conditions, dioicous acrocarpous mosses prevailed, the fertile turf of which, depending on the influence of abiotic factors, differed significantly in the number of sexual shoots, their ratio and productivity. Bulbils were found only on the tips of Bryum argenteum shoots. Along with Salicornia europaea L., a euhalophyte, the leading role in the initial stage of overgrowth of the tailing storage area most often belonged to Didymodon rigidulus, Bryum argenteum, Funaria hygrometrica and Barbula unguiculata. The process of formation of bryophyte cover occurred along a gradient of decrease in salt concentration at the experimental sites. Adaptation of bryophytes to substrate salinity is due to a change in metabolic processes, which is manifested in an increase of the total content of carbohydrates and an increase of the cation exchange capacity of moss cell walls, which is the primary barrier that reduces the toxic effect of ions under salt stress

Role of the bryophytes in substrate revitalization on a post-technogenic salinized territory

This work aims to investigate the role of the bryophyte cover in substrate revitalization on a post-technogenic salinized territory. The influence of the moss cover on the organic carbon content, actual acidity, redox potential, and the content of the main ecological and trophic groups of microorganisms in the substrate of the tailings storage of the Stebnyk Mining and Chemical Enterprise "Polymineral" was investigated. Bryophytes significantly affect the tailings storage saline substrates. They colonize areas with a very strong and strong degree of salinity, which are unsuitable for other plants. It was indicated that pioneer moss species promote the accumulation of organic matter in saline substrates of the tailings storage. Under moss turfs, the amount of organic carbon increased 2.2–5.0 times, compared with its content in the uncovered substrate. The high variability of the organic matter content is determined by the species characteristics of mosses, primarily their life form. The dense-turf species Didymodon rigidulus and Pthychostomum pseudotriquetrum var. bimum accumulated the most organic matter. The thickness of the litter under the moss turf of these species was much greater than in Barbula unguiculata and Funaria hygrometrica, which form loose turf. We assessed the specificity of the accumulation of organic carbon in the turfs of the studied mosses. It founded that most organic matter accumulated in the dead parts of the moss turf. In the green parts of the shoots of these moss species the amount of organic carbon was 3–4 times less, which indicates a relationship between litter capacity and content of carbon in the substrate under moss turfs. We investigated the influence of mosses on the actual acidity of the tailings storage substrate. Moss turfs promote the increase of acidity of the aqueous solution of the tailings substrate by 0.2–0.5 units. The tailings storage substrates are characterized by a reduction regime. The redox potential of the substrate under moss cover significantly depended on the species characteristics of mosses. Under the moss cover, the redox potential increased by 1.2–1.4 times, compared with the index for the substrate without moss cover. We studied the influence of moss cover on microbial biomass and the quantity of some ecological-trophic groups of microorganisms in the substrates of the tailings storage. The amount of microbial biomass under moss turfs increased depending on the degree of the substrate salinization and the species characteristics of the mosses. In areas with a very high degree of salinization under the moss turfs of Didymodon rigidulus and Funaria hygrometrica, the microbial biomass index increased almost two times, compared with the uncovered substrate. We found a significant increase in the quantity of the main ecological and trophic groups of microorganisms (saprophytes, cellulose-destroying bacteria, oligonitrophils and nitrogen fixers) in the substrate under the moss cover. Thus, pioneer moss species have a complex effect on the saline substrate of tailings storage. They accumulate organic matter, increase the acidity of the upper layer, improve the redox regime of the substrate and promote the development of soil microbiota

SimReg1 is a master switch for biosynthesis and export of simocyclinone D8 and its precursors

Analysis of the simocyclinone biosynthesis (sim) gene cluster of Streptomyces antibioticus Tü6040 led to the identification of a putative pathway specific regulatory gene simReg1. In silico analysis places the SimReg1 protein in the OmpR-PhoB subfamily of response regulators. Gene replacement of simReg1 from the S. antibioticus chromosome completely abolishes simocyclinone production indicating that SimReg1 is a key regulator of simocyclinone biosynthesis. Results of the DNA-shift assays and reporter gene expression analysis are consistent with the idea that SimReg1 activates transcription of simocyclinone biosynthesis, transporter genes, regulatory gene simReg3 and his own transcription. The presence of extracts (simocyclinone) from S. antibioticus Tü6040 × pSSimR1-1 could dissociate SimReg1 from promoter regions. A preliminary model for regulation of simocyclinone biosynthesis and export is discussed

Bryophytes on the devastated territories of sulphur deposits and their role in restoration of dump substrate

Bryophytes possess a wide ecological diapason allowing them to populate substrates of technogenic origins which are scarcely suitable or completely unsuitable for viability of vascular plants. 49 bryophyte species, which belong to 2 divisions, 3 classes, 8 orders, 17 families, and 33 genera have been found on the dump territory of sulphur extraction of the mining-chemical enterprise “Sirka” (Yavoriv district, Lviv region). Seven transects, three on the north slope (base, slope, top), three on the south slope and one on the plateau were laid for sample selections. 20 investigated 0.5 × 0.5 m plots located 2 m apart were analyzed within each 10 × 10 m transect. Specific composition, life forms, projective cover, biomass of bryophytes, numbers of male, female and sterile plants, moisture content in the turfs, pH and physiological investigation of mosses were determined on each plot. The quantitative analysis of the biomorphological structure allowed us to establish the dependence of the spread of life forms on exposition and slope height; essential variability of the projective cover and moss biomass. Bryophyte cover plays an essential part in optimization of the moisture regime and surface layer temperature of technogenic substrates, improving the conditions of growth localities. We established that on the dump the dominant moss species are dioecious with a high level of reproductive effort (sexual and sexless), with short ontogenesis and age of first reproduction, which provides the chance to produce the maximum number of progeny in the minimum period and to form a complete moss cover. The analysis of seasonal moss photosynthesis dynamics has demonstrated the adaptability of moss photosynthetic apparatus to contrasting climatic conditions and the ability to support the intensity of photosynthetic processes on a rather stable level during the vegetative period. Our research showed that bryophytes play an important role in productivity of plant cover on the post-technogenic territories of sulphur extraction. It was found that bryophytes play a role in accumulation of organic carbon and biogenic elements in the substrate of the sulphur extraction dump . Carrying out research of specific composition dynamics and species activity is the precondition for revealing the essence of the dynamic processes taking place in the structure of the bryophyte communities on devastated territories and the influence of these processes on the formation of vegetation on dump complexes

Regulation of antibiotic production in Actinobacteria: new perspectives from the post-genomic era

The antimicrobial activity of many of their natural products has brought prominence to the Streptomycetaceae, a family of Gram-positive bacteria that inhabit both soil and aquatic sediments. In the natural environment, antimicrobial compounds are likely to limit the growth of competitors, thereby offering a selective advantage to the producer, in particular when nutrients become limited and the developmental programme leading to spores commences. The study of the control of this secondary metabolism continues to offer insights into its integration with a complex lifecycle that takes multiple cues from the environment and primary metabolism. Such information can then be harnessed to devise laboratory screening conditions to discover compounds with new or improved clinical value. Here we provide an update of the review we published in NPR in 2011. Besides providing the essential background, we focus on recent developments in our understanding of the underlying regulatory networks, ecological triggers of natural product biosynthesis, contributions from comparative genomics and approaches to awaken the biosynthesis of otherwise silent or cryptic natural products. In addition, we highlight recent discoveries on the control of antibiotic production in other Actinobacteria, which have gained considerable attention since the start of the genomics revolution. New technologies that have the potential to produce a step change in our understanding of the regulation of secondary metabolism are also described

Functionalized hybrid polymer structures for biomedical applications

This doctoral thesis is dedicated to the synthesis and characterization of novel functionalized hybrid structures for biomedical purposes. Systems reported in this work can be subdivided into the two main groups: natural-based materials and synthetic amphiphilic block copolymers. Both groups were studied as perspective theranostic agents for medical applications. In the first group, natural polysaccharides glycogen and mannan were selected as starting materials for preparation of novel nanoconjugates that possess ability for multimodal detection in vivo. Because grafting of natural macromolecules with synthetic polymers generally slows down the biodegradation rate, both polysaccharides were modified in two different ways to form nanoprobes with or without poly(2-methyl-2-oxazoline)s chains. The prepared nanoconjugates were functionalized with N-hydroxysuccinimide-activated fluorescence and magnetic resonance imaging labels. The resulting materials were tested both in vitro and in vivo and were shown to be completely biocompatible, biodegradable and exhibit some extra benefits in terms of their practical usage in biomedicine. Glycogen was functionalized with allyl and propargyl groups with following freeze-drying from aqueous solutions to form nano- and microfibrous materials. The presence of both..

The Use of Combinatorial Methods for Sound Scanning of Objects

In article considers application of combinatorial methods for research of sound images scan by the medium of integer sequences – ideal ring bundles as comfortable mathematical models for synthesis and optimization of systems with non-uniform structure. By using combinatorial approaches quality of methods based on the passage of sound signals through the tissues can be significantly improved. Studies show that the use of integer sequences based on the ideal ring bundles in information conversion tasks ensures simplicity of hardware application

Mannan-Based Nanodiagnostic Agents for Targeting Sentinel Lymph Nodes and Tumors

Early detection of metastasis is crucial for successful cancer treatment. Sentinel lymph node (SLN) biopsies are used to detect possible pathways of metastasis spread. We present a unique non-invasive diagnostic alternative to biopsy along with an intraoperative imaging tool for surgery proven on an in vivo animal tumor model. Our approach is based on mannan-based copolymers synergistically targeting: (1) SLNs and macrophage-infiltrated solid tumor areas via the high-affinity DC-SIGN (dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin) receptors and (2) tumors via the enhanced permeability and retention (EPR) effect. The polymer conjugates were modified with the imaging probes for visualization with magnetic resonance (MR) and fluorescence imaging, respectively, and with poly(2-methyl-2-oxazoline) (POX) to lower unwanted accumulation in internal organs and to slow down the biodegradation rate. We demonstrated that these polymer conjugates were successfully accumulated in tumors, SLNs and other lymph nodes. Modification with POX resulted in lower accumulation not only in internal organs, but also in lymph nodes and tumors. Importantly, we have shown that mannan-based polymer carriers are non-toxic and, when applied to an in vivo murine cancer model, and offer promising potential as the versatile imaging agents

Genomic insights into evolution of AdpA family master regulators of morphological differentiation and secondary metabolism in streptomyces

Erworben im Rahmen der Schweizer Nationallizenzen (http://www.nationallizenzen.ch)The AdpA protein from a streptomycin producer Streptomyces griseus is a founding member of the AdpA family of pleiotropic regulators, known to be ubiquitously present in streptomycetes. Functional genomic approaches revealed a huge number of AdpA targets, leading to the claim that the AdpA regulon is the largest one in bacteria. The expression of adpA is limited at the level of translation of the rare leucyl UUA codon. All known properties of AdpA regulators were discovered on a few streptomycete strains. There are open questions about the true abundance and diversity of AdpA across actinobacterial taxa (and beyond) and about the possible evolutionary forces that shape the AdpA orthologous group in Streptomyces. Here we show that, with respect to the TTA codon, streptomycete adpA is more diverse than has been previously thought, as the genes differ in presence/position of this codon. Reciprocal best hits to AdpA can be found in many actinobacterial orders, with a domain organization resembling that of the prototypical AdpA, but other configurations also exist. Diversifying positive selection was detected within the DNA-binding (AraC) domain in adpA of Streptomyces origin, most likely affecting residues enabling AdpA to recognize a degenerate operator. Sequence coding for putative glutamine amidotransferase (GATase-1) domain also shows signs of positive selection. The two-domain organization of AdpA most likely arose from a fusion of genes encoding separate GATase-1 and AraC domains. Indeed, we show that the AraC domain retains a biological function in the absence of the GATase-1 part. We suggest that acquisition of the regulatory role by TTA codon is a relatively recent event in the evolution of AdpA, which coincided with the rise of the Streptomycetales clade and, at present, is under relaxed selective constraints. Further experimental scrutiny of our findings is invited, which should provide new insights into the evolution and prospects for engineering of an AdpA-centered regulatory network