The reproductive biotechnologies currently represent a major scientific discipline based on a sufficient quantity of wholesome oocytes matured in vitro conditions to acquire permits for their effectiveness. The meiotic maturation is a complex process where a wide range of factors is involved in the regulation. One of these factors are small gaseous molecules, so-called gasotransmitters. These gases demonstrate number of physiological functions in the organism and the latest discovered gasotransmitter is hydrogen sulfide (H2S). The aim of this thesis was to test the hypothesis according to which there are enzymes present in porcine oocytes that produce H2S (CBS, CTH, 3-MPST), their distribution is in interaction and their joint actions influence the process of the meiotic maturation. The resulting data were obtained on the basis of immunocytochemical staining and colocalization analysis. The results proofed the presence of H2S-releasing enzymes in porcine oocytes. It was also confirmed the function of these enzymes in relation to the regulation of the meiotic maturation when oocytes cultured with triple inhibitors of H2S-releasing enzymes matured more slowly. The results also showed medium correlation dependence of enzymes CBS, CTH and 3-MPST. The combination of CBS and 3-MPST resulted in high correlation dependence which confirmed their interaction both in immature oocytes and in vitro matured oocytes. It is evident that so far studied effects of H2S are only a fraction of skills which this signal molecule possesses. There exists a need for future experiments to help us describe and explain the acting mechanisms of H2S.

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Forest rings are large features common in Ontario’s boreal forests that comprise circular topographic depressions in carbonate mineral soil that are filled with peat. This thesis documents differences in peat and soil chemistry along transects across the “Bean” and “Thorn North” rings, which are centered on accumulations of CH4 and H2S, respectively. Within the mineral soil, ring edges are characterized by strong negative anomalies in pH, ORP and carbonate, as well as positive anomalies of Al, Fe and Mn in the results of aqua regia and hydroxylamine-hydrochloride digestions. Within the peat, positive carbonate and pH anomalies are recorded. This antithetic relationship suggests vertical migration of carbonate species from clay to peat. An inverse relationship exists between ORP, versus redox inferred from aqua regia. Strong ORP lows occur where oxidized products show highest concentrations. This is interpreted to reflect the proliferation of autotrophic organisms occupying the strong redox gradient at the ring edge.

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Elastin like peptides (ELPs)—derived from the protein elastin—are widely used as thermoresponsive components in biomaterials due to their LCST (lower critical solution temperature) behavior at a characteristic transition temperature (Tt). While linear ELPs have been well investigated, few reports focused on branched ELPs. Using lysine (Lys, with an additional side-chain amine) as branching units, ELP dendrimers were synthesized by solid-phase peptide synthesis (SPPS) with up to 155 amino acid residues. A secondary structure change with decreasing ratio of random coil and increasing ratio of β-turn upon heating, which is typical of linear ELPs, was confirmed by circular dichroism spectroscopy for all peptides. Conformational change did not show evident dependence on topology, while a higher Tt was observed for dendritic peptides than for their linear control peptides with the same number of GLPGL repeats. Variable-temperature small-angle X-ray scattering (SAXS) measurements showed a size increase and fractal dimension upon heating, even below the Tt. These results were further confirmed by cryogenic transmission electron microscopy (cryo-TEM), and micro differential scanning calorimetry (micro-DSC), revealing the presence of aggregates below the Tt. These results indicated the presence of a pre-coacervation step in the LCST phase transition of the ELP dendrimers. We further prepared hydrogels by crosslinking hyaluronic acid (HA) with ELP dendrimers. We invesigated their physical properties with scanning electron microscopy (SEM), swelling tests, SAXS, and model drug loading/release experiments. Most of the HA_denELP hydrogels retained transparent upon gelation, but after lyophilization and reswelling remained opaque for days. This reswelling process was carefully investigated with time-course SAXS studies, and was attributed to forming pre-coacervates in the gelation step, which slowly reswelled during rehydration. We then prepared hydrogels with H2S-releasing aroylthiooxime (SATO) groups and showed human neutrophil elastase-responsive H2S-releasing properties with potential applications in treating chronic diseases with recurring inflammation. Furthermore, we prepared a series of wedge-shaped triblock polyethylene glycol (PEG)-ELP dendrimer-C16 (palmitic acid) conjugate amphiphiles with adjustable Tts. Various techniques were used to investigate their hierarchical structures. The triblock PEG-peptide-C16 conjugate amphiphiles were thermoresponsive and showed a morphology change from small micelles to large aggregates. However, the hydrophilic shell and strong tendency for micelle formation limited the thermoresponsive assembly, leading to slow turbidity change in the LCST transition. The secondary structure was twisted from conventional β-sheet, and the thermoresponsive trend observed in typical ELP systems was not observed, either. Variable temperature NMR showed evidence for coherent dehydration of the PEG and ELP segments, probably due to the relatively short blocks. Utilizing the micelles with hydrophobic cavity, we were able to encapsulate hydrophobic drugs, with promising applications for localized drug release in hyperthermia. / Doctor of Philosophy / Elastin like peptides (ELPs) are similar to the protein elastin in terms of amino acid sequence. They are used widely as thermoresponsive (change in properties at different temperatures) components in biomaterials due to their abnormally lower solubility at higher temperatures. While linear ELPs have been thoroughly investigated, few investigations in ELP dendrimers have been studied. Dendrimers are molecules that branch in a controlled way to achieve sphere-like structures with rich surface functionalities. We synthesized the ELP dendrimers by using lysine amino acids as branching units. A protein secondary structure change, typical of ELPs, was observed for all peptide dendrimers. Secondary structure transitions showed no dependence on the molecular branching/linear structures, but a higher transition temperature (T_t) was observed for dendritic peptides than for their linear control peptides with the same number of amino acids. Various techniques confirmed the existence of aggregates below the T_ts, which was never reported before. We further fabricated hydrogels that mimic the native extracellular matrix, by connecting hyaluronic acid (HA) with ELP dendrimers. Interestingly, most of the hydrogels studied retained transparent upon gelation, but after freeze-drying and addition of water remained opaque for days. This phenomenon was attributed to forming of small aggregates in the gelation step, which resulted in slow reswelling. We then prepared hydrogels with H₂S-releasing groups, which showed human neutrophil elastase-responsive H₂S-releasing properties with potential applications in treating chronic diseases with recurring inflammation. We then prepared a series of wedge-shaped triblock poly (ethylene glycol) (PEG)- ELP dendrimer-alkyl chain molecules. The triblock molecules were thermoresponsive and showed a change from small spheres to large aggregates. However, the hydrophilic shell limited the thermoresponsive assembly, leading to slow turbidity change in the LCST transition. We found evidence of coherent assembly of the PEG and ELP parts, probably due to the relatively short polymer chains. Utilizing the micelles with hydrophobic cavity, we were able to encapsulate hydrophobic drugs, with promising applications for localized drug release for cancer treatment.

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Hydrogen sulfide (H2S) is an endogenously produced signaling gas involved in numerous cellular functions. At the appropriate concentration, exogenous administration of this gasotransmitter regulates vasodilation, promotes angiogenesis of endothelial cells, and generally exhibits beneficial effects as an anti-inflammatory and antioperoxidative agent. H2S is also capable of acting as a gaseous chemotherapeutic agent. Therefore, the therapeutic potential of exogenous delivery of H2S is vast. The delivery of H2S is complicated by its gaseous nature. Under physiologically relevant conditions, H2S is rapidly depleted from solution by oxidation and/or degassing. Therefore, direct exogenous delivery is difficult. To date, most studies have employed Na2S as a convenient H2S source. However, the rapid surge in H2S concentration upon Na2S dissolution followed by its rapid decline poorly mimics the sustained production of low concentrations of H2S that occurs in biological systems. We synthesized a library of S-aroylthiooximes (SATOs)—H2S-releasing compounds that more aptly mimic in vivo H2S concentrations. SATOs are synthesized via reaction of a S-aroylthiohydroxylamine and an aldehyde or ketone. SATOs release H2S in response to a thiol functionality. H2S release from SATOs could be controlled, with H2S release half-lives on the order of minutes to hours. SATO chemistry was utilized to prepare H2S-releasing polymers. Copolymers prepared using RAFT polymerization could be functionalized with SATOs with conversions > 99%, and these polymers released H2S on a similar timescale to our small molecule donors, confirming the viability of SATO formation as a post-polymerization modification strategy. SATO-functionalized polymer amphiphiles were prepared that self-assembled into micelles or vesicles based on their composition. H2S was released from these polymer assemblies more slowly than from the small molecules and statistical polymers. These H2S-releasing micelles were employed in in vitro cytotoxicity studies. H2S released from the micelles was found to be selectively toxic to human colon cancer cells compared with healthy fibroblasts. These polymeric micelle donors outperformed existing H2S donors in terms of their toxicity towards cancer cells. The observed enhanced toxicity was suspected to arise from the slow and sustained release of H2S from the micelles. / Ph. D. / Hydrogen sulfide (H2S) is biologically relevant gas involved in numerous cellular functions. At the appropriate concentration, administration of this gasotransmitter exhibits potentially beneficial effects in multiple biological systems. H2S is also capable of acting as a gaseous chemotherapeutic agent. Therefore, the therapeutic potential of H2S is vast. The delivery of H2S is complicated by its gaseous nature. Under physiologically relevant conditions, H2S is rapidly depleted from solution by oxidation and/or degassing. Therefore, direct external delivery is difficult. To date, most studies have employed used sulfide salds as a convenient H2S source. However, these poorly mimic the production of low concentrations of H2S that occurs in biological systems. We synthesized a library of S-aroylthiooximes (SATOs)—H2S-releasing molecules that more aptly mimic H2S concentrations in the body. SATOs can be triggered to release H2S by biologically relevant compounds. H2S release from SATOs could be controlled over minutes to hours. SATO chemistry was utilized to prepare H2S-releasing polymers. Copolymers were prepared using and functionalized with SATOs, and these polymers released H2S on a similar timescale to our small molecule donors. SATO-functionalized nanoparticles were also prepared. H2S was released from these nanoparticles assemblies more slowly than from the small molecules and polymers. H2S released from the micelles was found to be selectively toxic to human colon cancer cells compared with healthy cells. These nanoparticle donors outperformed existing H2S donors in terms of their toxicity towards cancer cells. The observed enhanced toxicity was suspected to arise from the slow and sustained release of H2S from the nanoparticles.

Made available in DSpace on 2014-12-17T15:42:01Z (GMT). No. of bitstreams: 1 LuizCDB_DISSERT.pdf: 2635764 bytes, checksum: a9af996a27546dc8a59f234cdabe7aad (MD5) Previous issue date: 2012-12-14 / In this work were synthesized matrix-based commercial white clay in its composition having large amounts of kaolinite and quartz, with a certain percentage of iron oxide for use as an adsorbent for hydrogen sulfide (H2S). To characterize the effect of initial matrix techniques were used to characterize XRD, FTIR, XRF and TG. The initial clay mineral matrix was placed in contact with 0.1 molar solutions of the salts of Co2+, Ni2+, Cr3+ and a solution 0.1 g / 100ml rhodamine B. During the synthesis process, the solutions were placed in contact with the initial matrix for a period of 48 hours in order to have ion exchange with the clay mineral. To check the amount of exchanged metals, we used the technique of X-ray Fluorescence (XRF). After synthesis was initiated the process of adsorption of H2S, where the arrays were placed in the reactor, then by passing a stream of hydrogen sulfide. The matrix along with the reactor was weighed before and after to measure the amount of gas adsorbed. Based on the gravimetric data the matrix which had the highest performance of the adsorption matrix was exchanged with Ni2+ ions, obtaining a result of 11.13 mg H2S / g matrix, then the matrix coated with rhodamine B which was reached 10.13 mg H2S / g matrix / Neste trabalho foram sintetizadas matrizes a base de argila branca comercial que possui em sua composi??o grande quantidade de caulinita e quartzo, com certo percentual de ?xido de ferro, para uso como adsorvente de sulfeto de hidrog?nio (H2S). Para efeito de caracteriza??o da matriz inicial foram utilizadas as t?cnicas de caracteriza??o DRX, FTIR, FRX e TG. A matriz argilomineral inicial foi colocada em contato com solu??es 0,1 molar dos sais de Co2+, Ni2+, Cr3+ e com uma solu??o 0,1 g/ 100ml de rodamina B. Durante o processo de s?ntese, as solu??es foram colocadas em contato com a matriz inicial por um per?odo de 48 horas a fim de haver troca i?nica com o argilomineral. Para verificar a quantidade de metais trocados, foi utilizada a t?cnica de Fluoresc?ncia de Raios X (FRX). Ap?s a s?ntese foi iniciado o processo de adsor??o de H2S, onde as matrizes foram colocadas no reator, passando em seguida por um fluxo cont?nuo de sulfeto de hidrog?nio. A matriz juntamente com o reator foi pesada antes e depois para aferir a quantidade de g?s adsorvida. Com base nos dados gravim?tricos a matriz que obteve o maior desempenho de adsor??o foi a matriz trocada com ?ons Ni2+, obtendo um resultado de 11,13 mg de H2S/ g de matriz, seguida pela matriz recoberta com rodamina B a qual chegou-se a 10,13 mg de H2S/ g de matriz