Formaldehyde cross-linking has been used to study protein-protein interactions in cells. Its short spacer arm, ability to permeate through cell membrane and the reversibility of the cross-linking reaction makes this a desirable cross-linker for in vivo studies. Although it has been widely used as a cross-linking reagent, the detailed chemistry behind protein cross-linking is not well understood. In vitro studies conducted under extended incubation periods (2 days) have shown that a multitude of amino acids are reactive to formaldehyde and that residue accessibility appears to play a role in reactivity. How applicable these findings are to formaldehyde cross-linking studies done under in vivo conditions (10-20 min incubations) is unclear. The chemistry of formaldehyde cross-linking was therefore investigated in model peptides under conditions similar to those used in in vivo studies. It was observed that only a subset of amino acids (amino termini and side chains of lysine and tryptophan) that were found reactive under extended incubation times was reactive in the much shorter incubation period. No cross-linking was detected between peptides, and elevating the peptide and formaldehyde concentrations resulted in only a minimal amount of cross-linked peptides. The relationship between residue accessibility and formaldehyde reactivity was assessed in model proteins that contain a more complex tertiary structure. It was shown that the extent of formaldehyde reactivity was dependent on the state of protein unfolding, i.e., solvent accessibility of reactive residues, and that an unfolded protein showed a significantly higher number of formaldehyde-induced modifications than a folded form, with lysine being the predominant reactive site. Formaldehyde treatment of proteins in their native form resulted in a low number of modifications even under an increased incubation time, suggesting that the protein remains folded during the course of the reaction. This is important for in vivo cross-linking studies where specificity and stability of protein-protein interactions is dictated by protein tertiary structure. / Science, Faculty of / Chemistry, Department of / Graduate

Předložená diplomová práce v teoretické části shrnuje základní fakta o kolagenu, bílkovině, která je nejrozšířenější v lidském organismu a oxidované celulóze používané v medicíně po několik desetiletí. Hlavním tématem této časti je síťování kolagenu, které je důležitým faktorem pro stabilizaci kolagenu podporující odolnost proti jeho degradaci. Jako síťující činidlo lze použít právě oxidovanou celulózu, která má kromě hemostatického účinku i funkční karboxylové skupiny vhodné k síťování proteinů. Praktická část práce byla zaměřena na sledování vzájemného chování směsí roztoků oxidované celulózy a kolagenu. Filmy či pěnové lyofilizáty připravené z těchto polymerních směsí by mohly sloužit jako účinná hemostatika nebo jako antibakteriální krytí ran podporující hojení. Byla zvolena řada hmotnostních poměrů mezi kolagenem a oxidovanou celulózou (9:1, 3:1, 5:3, 1:1, 1:2, 1:3, 1:9) se zachováním konstantního množství kolagenu, ale se vzrůstajícím množstvím celulózy. Jejich schopnost chemicky se vázat a umožnit tak vznik amidových vazeb mezi volnými aminovými skupinami kolagenu a karboxylovými skupinami oxidované celulózy byla sledována pomocí dvou UV-VIS spektroskopických metod, které využívají barevné reakce chemického činidla (ninhydrinu či 2,4,6 trinitrobenzensulfonové kyseliny) se zbylými volnými aminovými skupinami kolagenu. Karboxylové skupiny oxidované celulózy byly navíc aktivovány jak v polymerním roztoku tak i ve formě filmu směsí činidel 1-ethyl-3-(3-dimethylaminopropyl)karbodiimidu a N hydroxysukcinimidu (EDC/NHS). Pomocí infračervené spektroskopie s Fourierovou transformací (FT-IR) byly vyšetřeny změny vzorků na úrovni sekundární struktury kolagenu. Stabilita připravených směsí byla sledována ve formě filmu pomoci hydrolytické degradace při 37 °C. Morfologické změny na dvou typech lyofilizovaných vzorků, vymražených rychle při 196 °C nebo pomaleji při -30 °C, byly sledovány pomocí rastrovací elektronové mikroskopie (SEM). Při přípravě polymerních směsí se obě složky (kolagen i celulóza) se vzrůstajícím obsahem celulózy srážely až do poměru 1:1. UV-VIS analýzy potvrdily pokles volných –NH2 skupin poukazující na síťování kolagenu s celulózou shodně s nárůstem odolnosti vůči hydrolytické degradaci získané z měření úbytků hmotností připravených filmů. Od poměru 1:2 se složky již nesrážely, polymerní roztok byl homogenní, ale z důvodu nárůstu počtu volných aminových skupin od tohoto poměru výše lze usoudit, že celulóza fungovala v malém obsahu pouze jako fyzikální síťovalo a po dosažení rovnovážného stavu s kolagenem funguje spíše jako rozpouštědlo. Tímto způsobem zřejmě mohlo dojít ke změnám kolagenu až na úrovni sekundární struktury zaznamenané pomocí FT-IR. Aktivace karboxylových skupin celulózy činidly EDC/NHS nebyla prokázána. Poměry složek ovlivnily i porozitu a velikosti pórů připravených lyofilizátů určených pomocí SEM. Do poměru 1:1 byla porozita skafoldů vymražených kapalným dusíkem mezi 46 – 60 %, po dalším přidání celulózy stoupla až na 81 % (u poměru 1:9). Průměrná velikost pórů samotného kolagenu byla velice malá (14 ± 5 m), oproti oxidované celulóze (79 ± 24 m), proto přídavek celulózy vždy zvýšil velikost pórů na cca 55 m s výjimkou poměru 1:9, mající vysokou průměrnou velikost pórů (186 ± 76 m) a velmi pravidelnou strukturu připomínající včelí plást, kterou má i samotná celulóza.

No description available.

Die Arbeit umreißt das Krankheitsbild der Myopie (Kurzsichtigkeit) und deren unterschiedliche Ausprägungen, im Speziellen der progressiven und pathologischen Myopie. Hierbei wird ein Einblick in die Symptomatik, die anatomischen Ursachen und die heutigen medizinischen Interventionen gegeben. Hierdurch wird die Problematik einer zu „weichen“ Sklera (Lederhaut des Auges) und des damit einhergehenden fortschreitenden Augenwachstums deutlich. Im Zentrum der Arbeit steht ein neuer therapeutischer Ansatz zur vorbeugenden Behandlung der pathologischen Myopie; das Riboflavin/Blaulicht Cross-Linking der Sklera des Kaninchenauges. Dessen Wirkungsweise ermöglicht die biomechanische Versteifung von kollagenem Gewebe. Aus diesem Sachverhalt ergibt sich die Fragestellung der Arbeit: Ist das sklerale Riboflavin/Blaulicht Cross Linking geeignet das Augenwachstum im Tiermodell (junge Kaninchen) verträglich zu hemmen? Operationsbeeinflussende Parameter wie die Riboflavin-Durchdringungsdauer der Sklera und die sklerale Lichtdurchlässigkeit werden untersucht und für die Optimierung der Operationsmethode herangezogen und diskutiert. Zur Einschätzung des Versuchsansatzes werden die im Methodikteil dargelegten Anwendungen an adulten und jungen Kaninchen/Kaninchenaugen durchgeführt. In Tierversuchen wird die Schadensschwelle in Abhängigkeit der Blaulichtintensität, des Alters und der Pigmentierung untersucht, wobei histologische, immunhistochemische und elektronenmikroskopische Verfahren angewendet werden. Der inhibitorische Einfluss des Riboflavin/Blaulicht Cross-Linkings auf das Augenwachstum kann im Jungtiermodell durch verschiedene metrische Verfahren und MRT-Untersuchungen belegt werden.

Le doctorant n'a pas fourni de résumé en français. / The research described in this thesis is directed to study an externally stimulated DDS that incorporates a hydrogel as the matrix for the therapeutic agent. The research does not investigate a particular site for the delivery of the therapeutic agent. However, the aim of this research program is to develop various hydrogel formulations with desirable characteristics and structures from which the drug release can be controlled with applied external energy in the form of low-frequency ultrasound. To accomplish this, two types of natural hydrogels from agarose and chitosan and one type of synthetic hydrogel from PVA were fabricated. Parameters that affect the structure were varied for each type of hydrogel in order to study the effect of structural changes on drug loading and release capacity of hydrogels. Next, the obtained hydrogels were assessed for the delivery of Theophylline as the model drug.Among the three types of hydrogels, chitosan was found to have the fastest swelling rates and the higher water uptakes while the least swelling was found with PVA hydrogels and then agarose hydrogels crosslinked at pH 12. Regarding the mechanical stability of hydrogels, the ranking of the elastic modulus was PVA hydrogels (highest), then agarose hydrogels and chitosan copolymers (lowest). It seemed that the more mechanically stable structure of the PVA hydrogels correlated with a reduced mobility of water, in comparison to the greater mobility of water in the mechanically weaker chitosan copolymers.The stimulated and passive release of Theophylline from those hydrogel carriers showed how ultrasound, as an external energy, stimulates and controls the release of the drug. The measurements confirmed that it is only the energy imparted by the longitudinal ultrasonic waves that act on the polymeric network. The mechanism by which the ultrasound affects the release is considered as a form of a ratchet motor. The polymer chains play the role of the “ratchet” steps and the ultrasonic waves accelerate the particle movement in the release media. Hence, once the ultrasound is applied, the particles descend chain-to-chain (i.e. step-by-step) driven down their concentration gradient by the applied energy until they reach the surface of the hydrogel and hence are released into the surrounding media.Increasing the ultrasound intensity vastly accelerates the drug release. Indeed a higher intensity equals a higher energy transferred from the ultrasonic waves to the drug particles, resulting in faster and less controlled release. This also depends on the type of drug carrier structure. If the hydrogel carrier is mechanically stable, such as the PVA samples or the agarose hydrogels crosslinked at pH 12, the effect of high ultrasound intensity is much less compared to a less mechanically stable carrier such as the chitosan blends. Ultrasound applied for a longer period of time increases the amount of drug released, with the consequent effect of increasing the amount of heat generated in the hydrogel. Generally, a longer duration of the applied energy results in a greater amount of energy absorption, and an increase in friction and heat generation. These effects are important considerations in relation to the heat sensitivity of the drug to be delivered and the thermal characteristics of the polymeric carrier.This PhD research has demonstrated that both natural and synthetic hydrogels coupled to an ultrasonic energy source provides a controllable DDS, which provide some novel outcomes and contributions to the body of knowledge in the field of controlled drug delivery.

A major step in the biosynthesis of many membrane proteins is their insertion into the membrane of the endoplasmic reticulum (ER). The insertion of a multi-spanning membrane protein is a complex process since several transmembrane (TM) domains have to be correctly integrated in order to enable its correct assembly. At present it is unclear how the integration of multiple TM domains is co-ordinated by the ER translocon. The aim of this study was to analyse the molecular environment of the TM domains of a model seven TM domain protein, opsin, so as to better understand the mechanism by which integration occurs. For this purpose, stable 'integration intermediates' of defined lengths representing distinct stages of opsin biosynthesis were generated by in vitro translation of truncated mRNA in the presence of semi-permeabilised cells. Cysteine-mediated, site-specific cross-linking and immunoprecipitation were employed to examine the environment of these integration intermediates. In addition, cysteine-specific modification reagents with different physical properties were used to investigate the environment of opsin TM3 during its insertion at the ER membrane. Opsin TM domains exhibit unique patterns of adduct formation with the ER translocon components, Sec61α and Sec61β. TM1 associates with the Sec61 complex at two distinct stages during nascent chain extension, and this behaviour is dependent on the presence of subsequent TM domains. The re-association of TM1 with the transloconmay well facilitate the co-ordinated integration of TMs 1-3 into the lipid bilayer. Opsin TM4 exits the Sec61 complex as soon as the subsequent TM domain is synthesised, while TM5, TM6 and TM7 remain associated with the ER translocon throughout protein synthesis, suggesting their concerted release upon chain termination. Evidence is provided that opsin is integrated via a single Sec61 heterotrimer, despite the fact that the ER translocon appears to consist of multiple copies of the Sec61 complex. On the basis of this work, a model is presented describing the complete integration of opsin at the ER membrane.

Cross-linking in soy proteins is hypothesised to have an impact on the texture of tofu. In vitro incubation showed soy proteins and its two fractions, glycinin and β-conglycinin, were cross-linked using glutaraldehyde, formaldehyde, glyceraldehyde and transglutaminase (TGA). Increasing concentration of these carbonyl compounds and TGA, and temperature of the carbonyl compounds treatment, increased the reactivity of cross-linking. Glutaraldehyde was the most reactive in forming aggregated proteins, followed by formaldehyde and glyceraldehyde. Both carbonyl moieties of glutaraldehyde are believed to be essential for the rapid cross-linking reaction. In the unfractionated soy proteins, β-conglycinin had a higher reactivity than glycinin. In in vitro incubation using TGA, soy proteins served as good substrates for TGA, in which β-conglycinin was more susceptible to TGA than glycinin in the unfractionated soy proteins. The addition of TGA, and 1 and 2 mM glutaraldehyde prior to soymilk boiling in situ resulted in a small number of cross-linked proteins, which correspond to an increase in fracture force. The addition of glutaraldehyde after soymilk boiling resulted in a slight decrease in fracture force compared to the control. At higher concentrations of glutaraldehyde (15 and 30 mM), soy proteins were mostly cross-linked, regardless of addition before or after soymilk boiling. Highly cross-linked proteins resulted in a significant decrease in the fracture force. For TGA treatment, the fracture force was increased with increasing TGA concentration from 1000 to 5000 ppm, added either before or after soymilk boiling. However, the TGA treatment showed only a small quantity of cross-linking. It is hypothesised that TGA hydrolysed glutamine of proteins to glutamate and changed the functional properties of proteins. Upon examination of the microstructure, it was found that the TGA treatment resulted in a fine-stranded network, compact structure and less porosity. These characteristics resulted in a higher fracture force. In contrast, in the glutaraldehyde treatment, the network consisted of a higher porosity, loose network and diffuse structure, which gave lower fracture force. Thus, it appears that substrate modification to the structure of the soy proteins may have a greater impact than the number of cross-links. These findings are likely to have implications for production of soy products with a wide range of textures by manipulating the soy protein properties.

No description available.

This research was focused on the study of the polymer dormant radical systems, species containing free radical structures that have longer lifetimes and greater stability than radicals in general. In order to understand the nature and reactivity of the dormant radicals, polymeric systems capable of producing dormant free radicals were synthesised. In addition, the use of these novel polymeric materials in a range of applications were studied. Those applications exploited the nature of the dormant radical groups and included controlled modifications in the polymeric structure, heterogeneous catalysis and chromatographic separations.

Master of Science / Department of Biochemistry / Michael R. Kanost / Resilin is a rubber-like cuticular protein found in many insect species. Resilin is important for jumping and flying of those insects due to the properties of high elasticity and efficient energy storage. Some recombinant proteins or peptides derived from resilin sequences have been synthesized to produce biomaterials that mimic the remarkable properties of resilin. This research focused on resilin in the red flour beetle, Tribolium castaneum. A cDNA for T. castaneum resilin was inserted into plasmid vectors for expression of resilin in Escherichia coli or Bacillus subtilus. Resilin produced in E. coli was used as antigen to produce a rabbit antiserum. Resilin synthesized by B. subtilis as a secreted protein was purified and used for biochemical studies. Resilin is highly expressed in the late pupal stage, and in hind wings, but not found in elytra of pharate adults, indicated by RT-PCR and immunoblot analysis. Recombinant resilin could be cross-linked in the presence of horseradish peroxidase and hydrogen peroxide, detected by appearance of a high molecular weight band on SDS-PAGE, which had blue fluorescence under ultraviolet light, presumably due to dityrosine linkages. RNA interference was used to knock down resilin expression in T. castaneum. Immunoblot and RT-PCR analyses indicated that resilin expression was successfully decreased by RNAi. However, the knockdown adults exhibited no apparent differences in morphology, behavior or life span from control beetles. Blue fluorescence under ultraviolet illumination has frequently been used as an indication of the presence of resilin containing dityrosine cross-links in insect tissues such as wings, wing tendons and leg joints. A similar blue fluorescence was observed in hind wings of T. castaneum. However, this fluorescence was not decreased in hind wings of beetles in which resilin expression was knocked down by RNA interference. There was a blue fluorescence in the hind wings of knockdown beetles, which was similar in distribution to that in wings of control insects. This result suggests that the observed blue fluorescence in T. castaneum hind wings is derived not only from cross-linked resilin but also from components other than resilin, perhaps other cuticular proteins that contain dityrosine cross-links.