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.

The objective of this study was to improve canola protein gelation properties with the use of enzymes. Both cross-linking and limited proteolysis were explored. Enzyme treatments were performed prior to heat induced gelation. A texture analyzer, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and scanning electron microscopy were used to characterize the resulting networks. Enzymatic cross-linking with transglutaminase was shown to improve the gelation of canola protein isolate (CPI). To the contrary, proteolysis with trypsin, ficin and bromelin, did not enhance the gelation properties of CPI.

Maintaining an awareness of collaborators' actions is critical during collaborative work, including during collaborative visualization activities. Particularly when collaborators are located at a distance, it is important to know what everyone is working on in order to avoid duplication of effort, share relevant results in a timely manner and build upon each other's results. Can a person's brushing actions provide an indication of their queries and interests in a data set? Can these actions be revealed to a collaborator without substantially disrupting their own independent work? I designed a study to answer these questions in the context of distributed collaborative visualization of tabular data. Participants in my study worked independently to answer questions about a tabular data set, while simultaneously viewing brushing actions of a fictitious collaborator, shown directly within a shared workspace. I compared three methods of presenting the collaborator's actions: brushing & linking (i.e. highlighting exactly what the collaborator would see), selection (i.e. showing only a selected item), and persistent selection (i.e. showing only selected items but having them persist for some time). My results demonstrated that persistent selection enabled some awareness of the collaborator's activities while causing minimal interference with independent work. Other techniques were less effective at providing awareness, and brushing & linking caused substantial interference. These findings suggest promise for the idea of exploiting natural brushing actions to provide awareness in collaborative work. / Graduate / 0984 / amirhos.hajiz@gmail.com

The objective of this study was to improve canola protein gelation properties with the use of enzymes. Both cross-linking and limited proteolysis were explored. Enzyme treatments were performed prior to heat induced gelation. A texture analyzer, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and scanning electron microscopy were used to characterize the resulting networks. Enzymatic cross-linking with transglutaminase was shown to improve the gelation of canola protein isolate (CPI). To the contrary, proteolysis with trypsin, ficin and bromelin, did not enhance the gelation properties of CPI.

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.

abstract: In the 1970s James Watson recognized the inability of conventional DNA replication machinery to replicate the extreme termini of chromosomes known as telomeres. This inability is due to the requirement of a building block primer and was termed the end replication problem. Telomerase is nature's answer to the end replication problem. Telomerase is a ribonucleoprotein which extends telomeres through reverse transcriptase activity by reiteratively copying a short intrinsic RNA sequence to generate 3' telomeric extensions. Telomeres protect chromosomes from erosion of coding genes during replication, as well as differentiate native chromosome ends from double stranded breaks. However, controlled erosion of telomeres functions as a naturally occurring molecular clock limiting the replicative capacity of cells. Telomerase is over activated in many cancers, while inactivation leads to multiple lifespan limiting human diseases. In order to further study the interaction between telomerase RNA (TR) and telomerase reverse transcriptase protein (TERT), vertebrate TERT fragments were screened for solubility and purity following bacterial expression. Soluble fragments of medaka TERT including the RNA binding domain (TRBD) were identified. Recombinant medaka TRBD binds specifically to telomerase RNA CR4/CR5 region. Ribonucleotide and amino acid pairs in close proximity within the medaka telomerase RNA-protein complex were identified using photo-activated cross-linking in conjunction with mass spectrometry. The identified cross-linking amino acids were mapped on known crystal structures of TERTs to reveal the RNA interaction interface of TRBD. The identification of this RNA TERT interaction interface furthers the understanding of the telomerase complex at a molecular level and could be used for the targeted interruption of the telomerase complex as a potential cancer treatment. / Dissertation/Thesis / Ph.D. Chemistry 2011

SOUZA, Geraldo Herbert Beltrão de. Integral Linking para o espaço hiperbólico. 2016. 35 f. Dissertação (Mestrado em Matemática)- Centro de Ciências, Universidade Federal do Ceará, Fortaleza, 2016. / Submitted by Rocilda Sales (rocilda@ufc.br) on 2016-09-19T15:13:15Z No. of bitstreams: 1 2016_dis_ghbsouza.pdf: 464546 bytes, checksum: a010067c092935c226c9771b9f96d399 (MD5) / Approved for entry into archive by Rocilda Sales (rocilda@ufc.br) on 2016-09-19T15:13:53Z (GMT) No. of bitstreams: 1 2016_dis_ghbsouza.pdf: 464546 bytes, checksum: a010067c092935c226c9771b9f96d399 (MD5) / Made available in DSpace on 2016-09-19T15:13:53Z (GMT). No. of bitstreams: 1 2016_dis_ghbsouza.pdf: 464546 bytes, checksum: a010067c092935c226c9771b9f96d399 (MD5) Previous issue date: 2016 / This research aimed to find a comprehensive formula that calculates the linking number between two submanifolds of a visible hypersurface of hyperbolic space, which will be defined in the text. The motivation for this was the article "HIGHER-DIMENSIONAL LINKING INTEGRALS " whose authors are Clayton Shonkwiler and David Shea Candle-Vick. Which article Shonkwiler and Vela-Vick derive an integral formula for two submanifolds of a visible hypersurfaces of Euclidean space. Trying to adapt the idea of them, we were behind a full formula for the hyperbolic case, following the same script, but using the geometric structure of the hyperbolic space. Moreover, it is noteworthy that the Shonkwiler article and Vela-Vick is quite succinct, leaving several arguments and unexplained passages, which also led us to go back to explain in more detail all the arguments of them and thus a " concept new " and very important had to be made, such a concept we call "conical variety," which is not a deferenciável variety of apparel and so we had to develop a little degree theory for such sets. Finally, we gave work to express " application of hyperbolic Gauss ", in order that it desempenhasse the same role that the application of Euclidean Gauss played in article Shonkwiler and Vela-Vick. / Esta dissertação teve como objetivo encontrar uma fórmula integral que calcula o linking number entre duas subvariedades de uma hipersuperfície visível do espaço hiperbólico, que será definida no texto. A motivação para isso foi o artigo "HIGHER-DIMENSIONAL LINKING INTEGRALS", cujos autores são Clayton Shonkwiler e David Shea Vela-Vick. Em tal artigo Shonkwiler e Vela-Vick derivam uma fórmula integral para duas subvariedades de uma hipersuperfície visível do espaço euclidiano. Tentando adaptar a ideia deles, fomos atrás de uma fírmula integral para o caso hiperbólico, seguindo o mesmo roteiro, porém utilizando a estrutura geométrica do espaço hiperbolico. Além disso, vale ressaltar que o artigo de Shonkwiler e Vela-Vick é bastante suscinto, deixando vários argumentos e passagens inexplicados, o que também nos levou a ir atrás de explicar com maiores detalhes toda a argumentação deles e assim, um conceito \novo"e bastante importante teve que ser apresentado, tal conceito denominamos "variedade cônica", que não é uma variedade deferenciável de fato e por isso tivemos de desenvolver um pouco a teoria do grau para tais conjuntos. Por fim, nos demos a trabalho de expressar a "aplicação de Gauss hiperbólica", com a finalidade de que ela desempenhasse o mesmo papel que a aplicação de Gauss euclidiana desempenhou no artigo de Shonkwiler e Vela-Vick.

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

Abstract Academic support in IT Governance focuses static viewpoints on IT Governance. IT Governance in a global context has to cater for intensive competition, cultural diversity, and various fluctuating economic conditions. A static model of IT Governance and organisation cannot adequately address these issues. A major goal of this Dissertation is to develop a Framework for component-based IT Governance using dynamic Linkage Mechanisms to allow the determination of an optimum (resource-) flow. Linkage Mechanisms are dynamic arrangements and IT Governance Service Value Channels between IT Governance Components in the IT Governance Domain and allow communication between them. The (resource-) flow is based on transactions. These dynamic networks of relationships allow for greater flexibility and agility in acquiring, combining and deploying required IT Resources and IT assets. In this new research approach methods of the Object-oriented Modelling and Operations Research are applied to IT Governance disciplines. The result of this Dissertation is a generic, model-based, reusable and expandable 'building block' for the component-based design of an IT Governance Domain. This building block is composed of process oriented models, structured processes, strategies for optimisation and application use cases. It is shown how the architecture for the performance of a concrete IT Governance Domain is instrumented and in this way a component-based IT Governance is performed. Possible addressees of the results of this Dissertation are following: - ITG architects and (software-) developers who would like to implement the components, services or a platform for component-based ITG; - ITG managers or Chief Information Officers (CIO) who would like to construct a better understanding for requirements and relationships in the context ITG; - Auditors who would like to control and audit the conformity of an ITG Domain against a generic requirements catalogue; - Authors of Standards and Best Practice-Frameworks in the ITG area as-well-as scientists who are working with related research questions in the context ITG.

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.