As Hsp70 são proteínas centrais no sistema de homeostasia proteica da célula. Pelo fato delas estarem envolvidas em uma grande variedade de processos relacionados ao enovelamento correto das proteínas, elas estão envolvidas em processos como envelhecimento, doenças degenerativas, como Alzheimer, e alguns tipos de câncer. Uma das etapas essenciais no seu ciclo funcional é a troca de ADP por ATP, um processo que é acelerado pelos fatores de troca de nucleotídeos (NEFs) que, em bactérias e mitocôndrias, correspondem à proteína GrpE. Por razões ainda não bem compreendidas, duas isoformas estão presentes nas mitocôndrias de humanos, a GRPEL1 e a GRPEL2. Pouco se sabe da dinâmica da interação destas com a Hsp70 mitocondrial de humanos (mortalina) porque havia uma dificuldade em se obter esta proteína na sua forma solúvel e funcional (atualmente superada). Dessa forma, o presente trabalho de pesquisa busca caracterizar os aspectos bioquímicos e biofísicos dessas proteínas junto à mortalina, visando compreender a dinâmica da interação entre elas, contribuir para a elucidação da rede de interações das Hsp70 e compreender o porquê de 2 isoformas estarem presentes em mamíferos. Para isto, ensaios in vitro das proteínas mortalina, GRPEL1 e GRPEL2 recombinantes foram realizados. Elas foram expressas e purificadas por cromatografia de afinidade ao Ni2+ e gel filtração. As GrpEs tiveram seus graus de pureza e enovelamento correto avaliadas por SDS-PAGE e dicroísmo circular. Suas estruturas terciárias e quaternárias foram avaliadas através da cromatografia de exclusão molecular analítica e do crosslinking químico. Com as proteínas tendo sido purificadas, ensaios de interação molecular foram realizados através do pulldown, do ITC e, adicionalmente, foram feitos ensaios de agregação para investigar um possível papel das GrpEs no processo de agregação térmica da mortalina. Todas as proteínas puderam ser obtidas solúveis e com alto grau de pureza. Os ensaios de pulldown validaram a interação entre a mortalina e as GrpEs, mas essas interações não foram detectadas no ITC. Por fim, não houveram evidências de que as GrpEs atuem no sentido de prevenir a agregação térmica da mortalina. / Hsp70 are proteins that play a central role in cellular protein homeostasis. Because they are involved in a variety of processes related to protein folding, they are also involved in processes such as aging, degenerative diseases like Alzheimer and certain types of cancer. One of the essential steps in the Hsp70 functional cycle is the exchange of ADP for ATP, a process accelerated by the nucleotide exchange factors (NEF´s) which, in bacteria and mitochondria, corresponds to GrpE protein. For reasons not well understood yet, two isoforms are present on human mitochondria, GRPEL1 and GRPEL2. Little is known about the dynamics of their interaction with human mitochondrial Hsp70 (mortalin) because it was difficult to produce this protein in its soluble and functional form (now overcome by co-expression strategies with one co-chaperone). That being said, the current research work seeks to characterize the biochemical and biophysical aspects of those proteins together with mortalin in order to comprehend the dynamics of their interaction, to contribute on the elucidation of the Hsp70 interaction network and to comprehend why two isoforms are present. For this, in vitro assays of the recombinant proteins mortalin, GRPEL1 and GRPEL2 were carried out. They were expressed and purified by Ni2+ affinity chromatography and gel filtration. Both GrpEs had their degree of purity and correct folding assessed by SDS-PAGE and circular dichroism. Their tertiary and quaternary structures were evaluated by analytical size exclusion chromatography and chemical crosslinking. Having the proteins being purified, molecular interactions assays were done with pulldown, ITC and, additionally, aggregation assays were carried out to investigate a possible role played by GrpEs in the thermal aggregation process of mortalin. All the proteins could be obtained soluble and with a high degree of purity. Pull-down assays validated the interaction between mortalin and GrpEs, but this interaction could not be detected by ITC. Lastly, there was no evidence that GrpEs acted out preventing the thermal aggregation process of mortalin.

The overall objective of the work described in this thesis was to characterise the three genes Hv14.3.3c, HvMAPKK1 and HvFKBP41, in terms of a role in defence and stress response signalling. These genes had previously been found to be differentially expressed in compatible versus incompatible interactions of barley with the fungus Rhynchosporium secalis, suggesting a possible role in the plant defence response, while current literature suggests these genes may also play a role in signal transduction, possibly under a broad range of stresses, including abiotic as well as biotic. Two main approaches were undertaken to characterise gene function: expression analysis and the identification of protein-protein interactions. To facilitate expression analysis, full length cDNA fragments of each gene were first obtained using bioinformatics, RACE and genomic walking techniques. Expression was then investigated using quantitative real-time RT-PCR. The results of the expression analysis confirmed that the candidate genes were in fact differentially expressed during infection, suggesting a role in the defence response of barley against R. secalis. Analysing their expression in the context of other stresses and treatments, namely frost, drought and ABA, indicated their role may not be limited only to biotic stress, but include abiotic stress as well. To investigate the possibility that these genes are involved in signalling during the defence response, protein-protein interaction techniques such as yeast two-hybrid and affinity pulldowns were used to identify interacting proteins in an attempt to place the genes within a known signalling network and build and extend on these networks. Y2H screening was used successfully to identify two putative interactors of Hv14.3.3c; an EPSP (5-enolpyruvylshikimate-3-phosphate) synthase and a putative wound-induced protein, and two interactors of HvFKBP41; a Rab-type GTPase and the same wound-induced protein. From what is known about the function of these genes in the literature, they fit well with a role in stress response signalling and the potential to be involved in signalling networks with the candidate gene products and also with each other. Through the trial of many different affinity pulldown techniques, a method for identifying interacting proteins from plant extracts was successfully established, however, issues with protein identification meant that interacting proteins were not identified using this technique. Steps were then made towards confirming the interactions identified using the Y2H system. Full length cDNA sequences of the identified interactors were obtained and expression analysis performed, in the aim of investigating co-expression patterns between the genes encoding the interacting proteins and the three candidate genes, to support a potential interaction. To confirm the Hv14.3.3c-HvEPSP interaction, co-immunoprecipitation and BRET were then used, however confirmation was unsuccessful due to issues with non-specific binding in co-immunoprecipitation and technical issues trying to establish the BRET analysis system in barley. In summary, the results of this study place the candidate genes Hv14.3.3c, HvMAPKK1 and HvFKBP41 as players in signal transduction during the plant defence/stress response. With the identification of previously uncharacterised protein interactions, some progress has also been made towards placing these genes within known signalling networks and identifying potential downstream genes that could possibly play a more specific role in defence response signalling and therefore be potential targets for the generation of resistant or stress tolerant plants. / Thesis (Ph.D.) -- University of Adelaide, School of Agriculture, Food and Wine, 2009

The focus of this thesis is to characterise the interactions between GLUT4-related SNARE proteins – syntaxin4, SNAP23 and VAMP2 – and a regulatory protein, Munc18c. GLUT4 is the primary insulin-regulated glucose transporter and is presentin fat and muscle cells. GLUT4 is held in intracellular pools of vesicles until it is transported to the cell surface upon insulin stimulation. Insulin initiates a cellular signalling cascade via the insulin receptor on the cell membrane, which in turn stimulates GLUT4 vesicles to move to the cell surface where they fuse to the plasmamembrane via SNARE proteins. SNAREs are membrane-anchored proteins present on both vesicle and target membranes that form a tight complex which brings themembranes together for fusion. Fusion of vesicles to the target membrane releases the vesicular cargo.SNARE-mediated membrane fusion is a conserved mechanism that controls many other vesicle fusion processes such as neurotransmitter release and yeast vesicular trafficking. However, the regulation of the SNARE mechanism is not fully understood. SNAREs can interact with many other proteins that could act as regulatory factors,and studies have focused primarily on a group of effector proteins called Sec1p/Munc18 (SM) proteins. SM proteins were discovered and characterised because they bind to one type of SNARE protein, syntaxin. The SM protein that interacts with the GLUT4-related SNARE, syntaxin4, is Munc18c.The aim of this thesis was to investigate Munc18c interactions with SNARE proteins, principally syntaxin4, using biochemical techniques with purified recombinant proteins. This work was carried out in several stages including: 1) development of methods to produce and purify GLUT4-related SNARE proteins, SNARE complexes and Munc18c, 2) development of an assay to quantify Munc18c interactions with binding partners using surface plasmon resonance, 3) investigation into interactions between Munc18c and SNARE ternary complex, 4) characterising Munc18c interactions with syntaxin4, and 5) developing a method to produce selenomethionine-containing Munc18c in a baculovirus system to be used in structural studies. The methods and outcomes of these experiments are described inthis thesis. There were two major outcomes from this work. Firstly, Munc18c interacts with SNARE ternary complex, and secondly, Munc18c requires only the N-terminal 29residues of syntaxin4 for an interaction to occur. These results were determined using pulldown assays with purified proteins, as well as other chromatographic methods to show that protein complexes were formed. The steps taken to develop these binding assays are also discussed. Initial crystallisation conditions forMunc18c-HIS and a peptide consisting of syntaxin4 residues 1-20 have been identified using crystallisation screens. The interactions determined for Munc18c binding to Sx4 are in direct contrast to those of neuronal SM protein, Munc18a, and its interaction with neuronal SNARE proteins - Munc18a does not bind to its ternary complex and binds to the entire cytoplasmic domain of Sx1a. Rather, the Munc18c:Sx4 interactions are similar to that for the yeast SM protein, Sly1p, which can interact with both its SNARE ternary complex and with its syntaxin via the Nterminal residues. Another interesting outcome of this research was that syntaxin4 binds to metals (cobalt and nickel). This finding represents the first reported for a syntaxin interacting with metals. Preliminary results indicate that un-tagged syntaxin4 can bind to cobalt resin, and to nickel immobilised on a chip. This interesting and novel property of syntaxin4 binding was serendipitously discovered while investigating conditions for the Munc18c assay. Overall, I have shown that Munc18c, the SM protein involved in GLUT4 trafficking, interacts with SNARE proteins in a different manner to its mammalian counterpart inneurons, Munc18a, and is more like Sly1p, a yeast ER-Golgi SM protein. Munc18c interacts with SNARE complexes and only the N-terminal residues of syntaxin4.These interactions demonstrate that the regulatory mechanism for SNARE-mediated fusion is conserved between yeast and mammals. This finding has several implications for the role of Munc18c in the exocytosis of GLUT4-containing vesicles. Munc18c could act at several stages in the fusion process via syntaxin4 binding.These interactions could involve binding to other proteins (such as synip or tomosyn), conformational switching of syntaxin4 or interaction with metal ions to induce conformational changes in the proteins. Finally, these studies of GLUT4 exocytosis contribute to our understanding of glucose transport disorders such as Type 2 diabetes and could one day pave the way for the design of therapeutic agents.

Protein-Arrays sind das Mittel der Wahl, um eine Vielzahl von Proteinen parallel zu untersuchen. Ziele dieser Untersuchungen sind meistens Proteininteraktionsnetzwerke zu entdecken oder besser verstehen zu können. Bisher wurden die benötigten Proteine fast ausschließlich mit biologischen Methoden gewonnen. Diese bieten allerdings keinen generellen Zugang zu posttranslational-modi-fizierten (PTM)-Proteinen. Somit war es bisher nicht möglich den Einfluss von PTMs auf Protein-Protein-Interaktionen (PPIs) im Arrayformat zu untersuchen. Die chemische Synthese kann dagegen Proteine mit ortsspezifischen PTMs liefern. Daher ist es verwunderlich, dass bislang noch keine Berichte über chemisch hergestellte PTM-Protein-Arrays existieren, besonders da PTMs meist entscheidend für proteomische Interaktionsnetzwerke sind. In der vorliegenden Arbeit wird eine Methodik beschrieben, die es ermöglicht PTM-modifizierte Protein-Domänen-Arrays auf der Oberfläche zu synthetisieren und zu analysieren. Mit der Methodik wurden 20 SH3-Domänen synthetisiert und 64 PPIs gemessen. Neben vier Hefe-SH3-Domänen wurden je acht humane (Phospho)SH3-Domänen der Abl- und Arg(Abl2)-Tyrosinkinase synthetisiert und funktionell untersucht. Es wurde gefunden, dass die Ligandenspezifität von Abl-SH3-Domänen durch Phosphorylierung feinreguliert wird. Je nach Phosphorylierungsmustern wurde die Affinität für spezifische Liganden erhöht oder erniedrigt. Der Ursprung dieser Phosphoregulierung wurde für die Abl-SH3-Domäne mit Hilfe der NMR-Spektroskopie und durch Zellexperimente versucht zu entschlüsseln und weiter validiert. / Protein-arrays are the method of choice to investigate a variety of proteins in a parallel fashion. Objectives of these studies are mostly to discover or to investigate protein interaction networks. So far, the necessary proteins were almost exclusively gained by biological methods. Unfortunately, generic access to proteins bearing post-translational modifications (PTM) is not provided by these techniques. Therefore, it was not possible to investigate the impact of PTMs on protein-protein-interactions (PPIs) on arrays so far. Chemical synthesis in contrast offers proteins with site-specific PTM incorporation. In this context, it is surprising, that chemical methods of PTM-protein array synthesis remained virtually unexplored, especially since these modifications are usually crucial for proteomic interaction networks. In this thesis, a methodology is described, that allows to synthesize and functional analyse post-translationally modified protein domain arrays on the surface. By using this methodology, 20 SH3 domains were synthesized and 64 protein-pep-tide interactions were measured. In addition to 4 yeast SH3 domains, 8 human (phospho) SH3 domains of the Abl and Arg(Abl2) tyrosine kinase were synthesized and functionally investigated. The experiments revealed that phosphorylation might serve as a means to fine tune the ligand recognition. Depending on the phosphorylation pattern the affinity to specific interaction partners were enhanced or reduced. The origin of this phosphoregulation was further investigated for the Abl SH3 domain by means of NMR spectroscopy and cellular experiments.

Background: To learn how to swim with proper technique takes fairly large amount of time and practice to learn and anelite swimmer spends 6-7 days training for improving aerobic capacity, anaerobic capacity and strength training for energy saving technique. Freestyle is the fastest swimming style and is performed in many different distances, 50m is classified as a sprint and the 400m as a middle-distance. The research is inconclusive if there is a correlation in the lower body and the time in 50m and 400m and mostly in the middle distance which gives this study the importance reducing these uncertainties. Aim: The aim of this study was to determine the strength of the correlation between the upper- and lower body muscle strengthwith time in 50m and 400m freestyle and to analyze which of the variables of height, sex, upper- and lower body strength contribute to velocity in sprint and middle distance in competitive swimmers. Method:A total of 14 participants (3 men and 11 women) participated in the study. The participants were tested at three occasions. The first was to determine their three-repetition maximum (3RM) in the squat and lat-pulldown. The second occasion was the collection the time in 50m freestyle and the third was to collect the time in 400m freestyle. Relative strength (kg/kg BW; %) and absolute strength (kg) in 1RM was calculated and correlated with the time in 50m and 400m freestyle. Analysis was done to see which variables of height, sex, relative strength in the squat and lat-pulldown contribute the most to the time in freestyle. Result: The result show that there was a high correlation between the absolute strength in the squat and the time in 50m (r=-0.769) a moderate correlation in the absolute strength in lat-pulldown and the 50m freestyle sprint (r=-0.513). There was also a moderate correlation for the relative strength in the lat-pulldown and 50m freestyle (r=-0.599). The 400m correlate with the relative strength in both lat-pulldown(r=-0.563) and the squat (r=-0.555). The lat-pulldown contributed most to the time in 50m freestyle as well as the male sex. Conclusions: The absolute strength in the squat had a high correlation to the time in 50m freestyle swim. The 400m there was a moderate correlation to the relative strength in the squat and lat-pulldown showing that for the overall performance in middle-distance the relative strength has the advantage over absolute strength. The relative and absolute strength in upper body correlated to both 50m and 400m freestyle and could therefore strengthen the importance of upper body strength in sprint and middle distance as previous researchers has stated. The upper body strength is the best predictor of time in 50m.

This document elaborates on the subject of video interlacing and its removal. It describes the interlacing of video, its history and the reasons that led to its use. The document also explains why it is necessary to remove interlacing and the basic methods that are used for it. It describes the proposed deinterlacing algorithm and its implementation, including description of inpainting and block matching. Included are also test results of both quality and speed of my deinterlacing algorithm. The final chapter describes the implementation as a console application and a DLL library.

Ubiquitylation is an indispensable post-translational modification system in eukaryotic cells that leads to the covalent attachment of a small ubiquitin (Ub) protein onto a target. The traditional and best-characterized role for ubiquitylation is a fundamental regulatory mechanism whereby target proteins are tagged with a characteristic Lys48-linked Ub chain that signals for their elimination through proteasomal degradation. Challenging this conventional wisdom is the finding that some ubiquitylated proteins are modified by Ub chains linked through Lys63, providing a molecular signal that is thought to be structurally and functionally distinct from Lys48-linked Ub chains. Of further interest and significance is that the Lys63-linked Ub chains are apparently synthesized through a novel biochemical mechanism employing a unique complex formed between a true Ub conjugating enzyme (E2), Ubc13, and an E2-variant (Uev), Mms2 (or Uev1A). The goal of this thesis was to employ structural and functional approaches in order to better characterize the Ubc13-Mms2 Lys63-linked Ub conjugation complex. <p>Error-free DNA damage tolerance (DDT) in the budding yeast is dependent on Lys63-linked Ub chains synthesized by Ubc13-Mms2 and thus provided the opportunity to experimentally test the function of the human UBC13 and MMS2 genes in a simple model organism. Human UBC13 and MMS2 were each shown to function in place of their yeast counterparts and in accordance, human Ubc13 was shown to physically interact with yeast Mms2, and vice versa. Two human MMS2 homologs were also tested and it was determined that UEV1A but not UEV1B can function in place of mms2 in yeast DDT. Physical interactions were observed between Ubc13 and Uev1A, but not between Ubc13 and Uev1B, suggesting that Ubc13-Uev complex formation is required for function. <p>In collaboration with a research group at the University of Alberta, crystal structure and NMR data were used to develop a mechanistic model for the conjugation of Lys63-linked Ub chains by the Ubc13-Mms2 heterodimer, whereby the special orientation of two Ub molecules facilitates a specific Ub-Ub linkage via Lys63. In order to help support the in vitro model and to determine how the Ubc13-Mms2 structure relates to biological function, I used a structure-based approach to direct the creation of point mutations within four key regions of the Ubc13-Mms2 heterodimer; the Ubc13 active-site, the Ubc13-E3 (Ub ligating enzyme) interface, the Mms2-Ub interface, and the Ubc13-Mms2 interface. <p>Underscoring the importance of the Ub conjugation by Ubc13-Mms2, a Ubc13-C87S active-site mutation was created that could bind to Mms2 but was unable to function in DDT. Regarding the Ubc13-E3 interface, a single Ubc13-M64A point mutation had a potent effect on disrupting Ubc13 function in DDT, as well as its physical interaction with Rad5, TRAF6, and CHFR. The results suggest that different RING finger E3s use the same Ubc13 surface to sequester the Ub conjugation activity of Ubc13-Mms2. Two human Mms2 mutations at Ser32 and Ile62, which are contained within the Mms2-Ub interface, were found to reduce the ability of Mms2 to bind Ub. When the corresponding yeast mutations are combined, a synergistic loss in DDT function is observed. The relative orientation of Ser32 and Ile62 suggests that the Mms2 and Tsg101 Uev families use different Uev surfaces to physically interact with Ub. A 200 ìM dissociation constant for the wild-type Mms2-Ub interaction was also determined. The systematic mutagenesis and testing of 14 Ubc13-Mms2 interface residues led to mutants with partial or complete disruption of binding and function. Using this data, a model involving the insertion of a specific Mms2-Phe residue into a unique Ubc13 hydrophobic pocket was created to explain the specificity of Mms2 for Ubc13, and not other E2s. In addition, the dissociation constant for the wild-type Ubc13-Mms2 heterodimer was determined to be approximately 50 nM. <p>The structural and functional studies strongly support the notion that Ubc13-Mms2 complex has the unique ability to conjugate Lys63-linked Ub chains. However, several reported instances of Lys63-linked Ub chains in vivo have not yet been attributed to Ubc13 or Mms2. To address the disparity I was able to demonstrate and map a physical interaction between Mms2 and Rsp5, an E3 implicated in Lys63-linked Ub conjugation. Surprisingly, it was found that MMS2 is not responsible for the RSP5-dependent Lys63-linked Ub conjugation of a plasma membrane protein. A possible explanation for the apparent paradox is presented.

Ubiquitylation is an indispensable post-translational modification system in eukaryotic cells that leads to the covalent attachment of a small ubiquitin (Ub) protein onto a target. The traditional and best-characterized role for ubiquitylation is a fundamental regulatory mechanism whereby target proteins are tagged with a characteristic Lys48-linked Ub chain that signals for their elimination through proteasomal degradation. Challenging this conventional wisdom is the finding that some ubiquitylated proteins are modified by Ub chains linked through Lys63, providing a molecular signal that is thought to be structurally and functionally distinct from Lys48-linked Ub chains. Of further interest and significance is that the Lys63-linked Ub chains are apparently synthesized through a novel biochemical mechanism employing a unique complex formed between a true Ub conjugating enzyme (E2), Ubc13, and an E2-variant (Uev), Mms2 (or Uev1A). The goal of this thesis was to employ structural and functional approaches in order to better characterize the Ubc13-Mms2 Lys63-linked Ub conjugation complex. <p>Error-free DNA damage tolerance (DDT) in the budding yeast is dependent on Lys63-linked Ub chains synthesized by Ubc13-Mms2 and thus provided the opportunity to experimentally test the function of the human UBC13 and MMS2 genes in a simple model organism. Human UBC13 and MMS2 were each shown to function in place of their yeast counterparts and in accordance, human Ubc13 was shown to physically interact with yeast Mms2, and vice versa. Two human MMS2 homologs were also tested and it was determined that UEV1A but not UEV1B can function in place of mms2 in yeast DDT. Physical interactions were observed between Ubc13 and Uev1A, but not between Ubc13 and Uev1B, suggesting that Ubc13-Uev complex formation is required for function. <p>In collaboration with a research group at the University of Alberta, crystal structure and NMR data were used to develop a mechanistic model for the conjugation of Lys63-linked Ub chains by the Ubc13-Mms2 heterodimer, whereby the special orientation of two Ub molecules facilitates a specific Ub-Ub linkage via Lys63. In order to help support the in vitro model and to determine how the Ubc13-Mms2 structure relates to biological function, I used a structure-based approach to direct the creation of point mutations within four key regions of the Ubc13-Mms2 heterodimer; the Ubc13 active-site, the Ubc13-E3 (Ub ligating enzyme) interface, the Mms2-Ub interface, and the Ubc13-Mms2 interface. <p>Underscoring the importance of the Ub conjugation by Ubc13-Mms2, a Ubc13-C87S active-site mutation was created that could bind to Mms2 but was unable to function in DDT. Regarding the Ubc13-E3 interface, a single Ubc13-M64A point mutation had a potent effect on disrupting Ubc13 function in DDT, as well as its physical interaction with Rad5, TRAF6, and CHFR. The results suggest that different RING finger E3s use the same Ubc13 surface to sequester the Ub conjugation activity of Ubc13-Mms2. Two human Mms2 mutations at Ser32 and Ile62, which are contained within the Mms2-Ub interface, were found to reduce the ability of Mms2 to bind Ub. When the corresponding yeast mutations are combined, a synergistic loss in DDT function is observed. The relative orientation of Ser32 and Ile62 suggests that the Mms2 and Tsg101 Uev families use different Uev surfaces to physically interact with Ub. A 200 ìM dissociation constant for the wild-type Mms2-Ub interaction was also determined. The systematic mutagenesis and testing of 14 Ubc13-Mms2 interface residues led to mutants with partial or complete disruption of binding and function. Using this data, a model involving the insertion of a specific Mms2-Phe residue into a unique Ubc13 hydrophobic pocket was created to explain the specificity of Mms2 for Ubc13, and not other E2s. In addition, the dissociation constant for the wild-type Ubc13-Mms2 heterodimer was determined to be approximately 50 nM. <p>The structural and functional studies strongly support the notion that Ubc13-Mms2 complex has the unique ability to conjugate Lys63-linked Ub chains. However, several reported instances of Lys63-linked Ub chains in vivo have not yet been attributed to Ubc13 or Mms2. To address the disparity I was able to demonstrate and map a physical interaction between Mms2 and Rsp5, an E3 implicated in Lys63-linked Ub conjugation. Surprisingly, it was found that MMS2 is not responsible for the RSP5-dependent Lys63-linked Ub conjugation of a plasma membrane protein. A possible explanation for the apparent paradox is presented.

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