Introduction: Immunochemical methods have significant importance in biomedical research. The benefit of immunochemical methods is their high sensitivity and accuracy. The aim of the work was to use immunochemical methods in investigation of cancer biomarkers and degenerative diseases. A total of four studies were performed. Two studies were focused on the search for new biomarkers of prostate cancer, the third study was focused on evaluation of FGF23 in patients with hyperparathyroidism, and the fourth study looked at the assessment of hyaluronic acid in synovial fluid of patients with knee gonarthrosis. Methods and patients: Study 1: Concentrations of Chromogranin A, Endoglin, TIMP-1 and thymidine kinase 1 (TK1) in serum and Endoglin, SPINK-1, Annexin, TIMP-1 in urine were measured in 58 patients with the (mean age 68 years, range 45 - 82 years) with clinically diagnosed prostate cancer and in 30 healthy individuals with the (mean age 64 years, range: 55 - 78 years) without clinically relevant urological history and PSA values in the reference range. Subsequently, serum TK1 levels were measured in 169 patients (mean age 62 years, range 45- 82 years) with prostate cancer and in a control group of 39 healthy men (mean age 61 years, range 54-78 years). The TK1 assay was also used to determine the...

This master thesis is a part of the project TAČR TREND, which aim is the development of new ingredient for treatment of dry eye syndrome. Developed ingredient is derivative of hyaluronic acid, which is in this thesis compared with native hyaluronic acids and commercial eye drops. Aim of this thesis is to describe rheological and tribological properties of measured samples in terms of viscosity, mucoadhesivity and coefficient of friction. Coefficient of friction was measured on created tribological model of the eye in pin-on-plate configuration which represents physiologically relevant sliding speeds of blinking. Comparison with native hyaluronic acids and commercial products of eye drops showed, that derivative reaches higher values of viscosity, has mucoahesive properties and has low values of coefficient of friction. Those key parameters show that modification of hyaluronic acid is promising way for drug development for dry eye disease.

Hydratace patří mezi nejdůležitější faktory ovlivňující sekundární strukturu a tím i funkci molekul v živých systémech. Díky vysoké afinitě tvoří molekuly vody specifické struktury jejichž složení a fyzikální vlastnosti jsou ovlivněny přítomností studované látky. Hyaluronan patří mezi biomolekuly s obrovskou schopností vázat a zadržovat vodu. Cílem této práce bylo prozkoumat hydratační vlastnosti hyaluronanu o různé molekulové hmotnosti a vyčíslit množství molekul vody v jednotlivých hydratačních vrstvách. V první části práce byla využita metoda diferenční kompenzační kalorimetrie. V druhé části diplomové práce, na základě rozdílné kompresibility, byla vázaná voda studována metodou vysoko rozlišovací ultrazvukové spektroskopie.

Předmětem této práce bylo popsat agregační vlastnosti kyseliny hyaluronové (HA) a jejích hydrofobně modifikovaných derivátů. Za tímto účelem byly připraveny vodné roztoky kyseliny a dvou derivátů, lišící se stupněm substituce a druhem anorganické soli jako prostředím. Koncentrační řady čisté kyseliny hyaluronové a jejích derivátů byly studovány dvěma metodami, tensiometricky a spektrometricky. Povrchové vlastnosti roztoků byly měřeny s použitím kroužku Du Noüy. Zkoumáno bylo také solubilizační chování ve vodě a v chloridu sodném pomocí dvou sond: Coomassie brilantní modři a sudanu III. Amfifilní deriváty hyaluronanu mohou skutečně tvořit hydrofobní agregáty v roztoku a adsorbovat se na rozhraní vzduch–voda. Tato vlastnost je důležitá v souvislosti s transportními systémy léčiv, protože právě hydrofobní domény agregátů hyaluronanu mohou být použity jako nosiče hydrofobních léčiv. Byly zjištěny kritické agregační koncentrace roztoků derivátů hyaluronanu (tensiometricky a spektrometricky), které odpovídají právě těm koncentracím, při kterých vznikají hydrofobní domény.

This diploma thesis is focused on the study of physical and chemical properties of hyaluronan and cationic surfactant. As the cationic surfactant Septonex was used. The influence of the environment on the system, the effect of molecular weight of hyaluronan, and its concentration was studied. Then, the study of the influence and the effects of concentration of Septonex on the interaction of hyaluronan-surfactant followed. Different methods of measurement were chosen to characterize these ionokomplexes. The critical micelle concentration of the surfactant itself was measured, and then also with the addition of hyaluronan by spectrofluorimetry with fluorescent probe pyren. It was found, that hyaluronan forms gel with Septonex. On this basis, gels were prepared for three different molecular weights of hyaluronan – 300 kDa, 806 kDa and 1697 kDa. Gels were prepared in a ratio of hyaluronan – surfactant 1:1. In gels prepared in this way, the influence of environmental water and 0.15 M NaCl was studied and it was found that at 0.15 M NaCl clear gels are formed. Selected samples of the gels were then measured with oscillatory testing and the rheological behavior of gels of Septonex was studied. As the last method the turbidimetric measurement was chosen, which characterized the turbidity point in the gradual addition of Septonex to sodium hyaluronate solution. Again, the effect of the molecular weight of hyaluronan and its concentration in two environments - water and 0.15 M NaCl was evaluated. It was found that 0,15 M NaCl suppresses formation of turbidity and formation of precipitates.

The electrospinning process of polymer solutions is affected by many different parameters that can be divided into solution parameters, process parameters, and ambient parameters. This study is focused on characteristics of a polymer and its solution, which are ranging into the solution parameters. The aim of this study is to compare poly(ethylene oxide) characteristics in the relation to electrospinning of hyaluronic acid, to find the difference between them using available analytic, thermic and spectral methods, and to define the influence of properties on polymeric solutions behavior during electrospinning. It was find that poly(ethylene oxides) obtained from two different sources behave differently in an electrostatic field, although their molar mass is declared as the same. It was also confirmed that the electrospinning process of polymeric solutions is affected mainly by their viscosity and conductivity. This two properties are determined especially by the molar mass of polymer and also by the content of impurities or content of some another polymer during electrospinning of mixed solution.

This diploma thesis deals with preparation and subsequent characterization of albumin (BSA) and mixed hyaluronan-albumin (HA+BSA) hydrogels. The experimental characterization was based on the rheological measurements of the influence of ionic strength, pH and molar mass of HA on the gelation process and the properties of formed gels, as well as the time stability of these properties. Following object of the study was swelling of hydrogels in water and buffers with different pH value. Characterization was extended by thermal analysis, morphology description using scanning electron microscopy (SEM) and study of transport properties of hydrogels for selected model substance. Due to increasing ionic strength, the gel point was shifted towards higher temperature values and the resulting gels exhibited higher values of complex moduli. In case when pH was lower than isoelectric point of BSA, attractive electrostatic interactions between albumin and hyaluronan took place and the faster gelation occurred, compared to the case when pH was higher than isoelectric point of BSA. On the contrary, values of complex moduli increased with increasing pH. The gel samples prepared in the presence of low molecular weight HA reached higher values of complex moduli, compared to samples with the high molecular weight HA. Rheological properties of BSA gels were stable over time, whereas the mixed HA+BSA were characterized by significant decrease of complex moduli during the first three days of storage. Due to heating up to 100 °C, more than 80 % of the weight of studied samples was lost, as a result of water evaporation. Further heating of hydrogels, up to 250 °C, did not cause other changes associated with weight loss, in contrast with crystalline powder BSA, in which another weight loss at 220 °C was observed, as a result of the beginning degradation. Swelling studies, as well as SEM images, indicated higher crosslink density of BSA gels, compared to HA+BSA samples, which exhibited higher values of swelling ratio. The lowest values of equilibrium swelling ratio were observed at pH 4, which is close to the isoelectric point of albumin. Calculated diffusion coefficients relative to diffusivity of methylene blue in water ranged between the order of 10E-2 for diffusion into physiological solution and the order of 10E-3 for the diffusion into water. Ongoing transport of model dye from gel to solution was significantly slower, compared to its diffusion in water, as a result of gel porosity and electrostatic immobilization of positively charged molecules of methylene blue on the negatively charged BSA and HA chains.

The aim of this diploma thesis was to study the structure and to evaluate the properties of the hydrogel prepared by the interaction of the aminolclay with the biopolymer. Representatives of the biopolymers were selected from low to medium molecular weight sodium hyaluronate and sodium polystyrenesulfonate. On the basis of the experiments carried out, it was found that phase separation takes place only when the aminolclay interacts with medium molecular weight hyaluronan (MMW). In the experimental part, analyzes of this sample were carried out in order to determine the formation of phase-separated hydrogel by influencing the solution by ionic strength, investigation of hydrogel extinction in various organic solvents, stability of hydrogel under extreme temperature conditions, the effect of storage on its degradation, etc. Viscoelastic properties were experimentally proven by rheology and thermal analysis detected binding water. Inhibition of microorganisms was confirmed by antibacterial diffusion assays. All experiments were carried out for the use of the Aj-HyA hydrogel (MMW) in the field of medical applications, specifically for the modern method of wet wound healing of the skin.

6 1. Abstrakt Hyaluronidázy jsou významnou skupinou enzymů odpovědných za štěpení kyseliny hyaluronové, která je jednou z hlavních složek pojivové tkáně obratlovců. U většiny krevsajících členovců je tento enzym přítomen ve slinách a usnadňuje sání tím, že se podílí na průniku kůží a zvětšení potravní léze v místě bodnutí. Vzniklé fragmenty extracelulární matrix mohou navíc modulovat lokální imunitní odpověď hostitele a zvyšovat pravděpodobnost přenosu patogenů. V naší práci jsme se zaměřili na průkaz hyaluronidázové aktivity u klíštěte Ixodes ricinus (Acari: Ixodidae) a zástupců čtyř rodů ovádů (Diptera: Tabanidae). V slinných žlázách klíšťat se hyaluronidázovou aktivitu detekovat nepodařilo. Naopak, ve slinných žlázách ovádů jsme prokázali velmi silnou aktivitu u všech studovaných druhů a dále ji charakterizovali biochemickými a elektroforetickými metodami. Hyaluronidázy ovádů štěpí hyaluronan i chondroitin sulfát; enzymy jednotlivých druhů se mírně liší molekulovou hmotností, pH optimem a citlivostí k redukujícím podmínkám. Abstract (in English) Hyaluronidases are an important group of enzymes responsible for cleaving hyaluronic acid, which is a major component of the extracellular matrix of vertebrates. In bloodsucking arthropods these enzymes are frequently present in saliva. Salivary hyaluronidases...

Cell-derived (CDM) matrix scaffolds provide a 3-dimensional (3D) matrix material that recapitulates a native, human extracellular matrix (ECM) microenvironment. CDMs are a heterogeneous source of ECM proteins with a composition dependent on the cell source and its phenotype. CDMs have several applications, such as for development of cell culture substrates to study stromal cell propagation and differentiation, as well as cell or drug delivery vehicles, or for regenerative biomaterial applications. Although CDMs are versatile and exhibit advantageous structure and activity, their use has been hindered due to the prolonged culture time required for ECM deposition and maturation in vitro. Macromolecular crowding (MMC) has been shown to increase ECM deposition and organization by limiting the diffusion of ECM precursor proteins and allowing the accumulation of matrix at the cell layer. A commonly used crowder that has been shown to increase ECM deposition in vitro is Ficoll, and was used in this study as a positive control to assess matrix deposition. Hyaluronic acid (HA), a natural crowding macromolecule expressed at high levels during fetal development, has been shown to play a role in ECM production, organization, and assembly in vivo. HA has not been investigated as a crowding molecule for matrix deposition or development of CDMs in vitro. This dissertation focused on 2 aims supporting the development of a functional, human dermal fibroblast-derived ECM material for the delivery deliver an antimicrobial peptide, cCBD-LL37, and for potentially promoting a pro-angiogenic environment. The goal of this thesis was to evaluate the effects of high molecular weight (HMW) HA as a macromolecular crowding agent on in vitro deposition of ECM proteins important for tissue regeneration and angiogenesis. A pilot proteomics study supported the use of HA as a crowder, as it preliminarily showed increases in ECM proteins and increased retention of ECM precursor proteins at the cell layer; thus supporting the use of HA as a crowder molecule. In the presence of HA, human dermal fibroblasts demonstrated an increase in ECM deposition comparable to the effects of Ficoll 70/400 at day 3 using Raman microspectroscopy. It was hypothesized that HA promotes matrix deposition through changes on ECM gene expression. However, qRT-PCR results indicate that HA and Ficoll 70/400 did not have a direct effect on collagen gene expression, but differences in matrix crosslinking and proteinase genes were observed. Decellularized CDMs were then used to assess CDM stiffness and endothelial sprouting, which indicated differences in structural organization of collagen, and preliminarily suggests that there are differences in endothelial cell migration depending on the crowder agent used in culture. Finally, the collagen retained in the decellularized CDM matrix prepared under MMC supported the binding of cCBD-LL37 with retention of antimicrobial activity when tested against E.coli. Overall, the differences in matrix deposition profiles in HA versus Ficoll crowded cultures may be attributed to crowder molecule-mediated differences in matrix crosslinking, turnover, and organization as indicated by differences in collagen deposition, matrix metalloproteinase expression, and matrix stiffness. MMC is a valuable tool for increasing matrix deposition, and can be combined with other techniques, such as low oxygen and bioreactor cultures, to promote development of a biomanufactured CDM-ECM biomaterial. Successful development of scalable CDM materials that stimulate angiogenesis and support antimicrobial peptide delivery would fill an important unmet need in the treatment of non-healing, chronic, infected wounds.