Indiana University-Purdue University Indianapolis (IUPUI) / Mutations in PINK1 (PTEN-induced Kinase 1) are the second most common cause of early-onset Parkinson’s Disease (PD). PINK1 is believed to maintain mitochondrial integrity by orchestrating mitophagy of dysfunctional mitochondria through phosphorylation of its substrate, Parkin. However, the effects of PD-associated mutations remain unclear. To investigate this, a PINK1 orthologue, Tribolium castaneum PINK1 (TcPINK1), was genetically engineered and purified for biochemical studies. Then, TcPINK1 was reacted against the Ubiquitin-like domain (UBL1-76) of Parkin and other proteins with a similar beta-grasp fold including Ubiquitin, ATG8, NEDD8, and SUMO using an in vitro radioisotopic filter-based kinase assay. The data revealed that TcPINK1’s preferred substrate with the highest amount of activity was UBL followed by Ubiquitin, NEDD8, and SUMO, with no activity against ATG8, which lacks a Serine residue equivalent to the phosphorylated residue in UBL. NEDD8 and SUMO were phosphorylated even though they are not substrates which suggests that PINK1 is capable of nonspecific phosphorylation of proteins with a similar fold to UBL. In addition, it is possible that the phosphorylation of Ubiquitin as reported in the literature may be nonspecific as well. TcPINK1 point mutations equivalent to the PD-associated human PINK1 mutations were genetically engineered, purified, and reacted against UBL. The P374L mutant showed a similar activity to wild type, and the A194D, G285D, and S289M mutants showed a significant decrease in activity. Since P374 resides in the C-lobe of the kinase away from the active site, the data suggest that this residue may not be involved with catalysis or with UBL binding. As A194, G285, and S289 all reside in the N-lobe near the active site, the data suggest that these point mutations may be involved with catalysis. In conclusion, the data suggest that PINK1 specificity for Parkin may involve binding outside of the UBL domain. / 2024-05-26

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal.

Darbe tiriami elektroninių užsakymų sprendimai elektroninio verslo sistemose. Siekiama nustatyti problemas, kurias turi spręsti elektroninio užsakymo sistema, ištirti egzistuojančius šių problemų sprendimo būdus bei nustatyti pastarųjų privalumus ir trūkumus. Taip pat siekiama realizuoti elektroninio užsakymo sistemą, sprendžiančią nustatytas problemas atsižvelgiant į ištirtų metodų privalumus ir trūkumus. Pagrindinis dėmesys skiriamas UBL (Universal Business Language) kalbos panaudojimui universalios elektroninio užsakymo sistemos kūrime. Darbe ištirtos švediškos sąskaitos-faktūros, daniškos sąskaitos-faktūros ir Zanzibar portalo sistemos, panaudojančios UBL. / The goal of this research is to define the main problems solved by e-order solutions, as well as to analyse the existing methods of solving these problems and research similar existing systems, and finally to create and test a model of such system. The research is focused on the implementation of UBL (Universal Busines Language) in e-business systems. It covers the methods of e-procurement systems and the methods of using UBL in realization of these systems. The research covers the existing systems, such as Swedish E-Invoice, Danish E-Invoice and Zanzibar Portal. The main problem of all existing methods and solutions is the integration of existing systems on both, buyer and seller sides. In this work, the project of an e-order system is made in attempt to create a universal solution that would ensure the inegration of any existing system.

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal

Universal Business Language (UBL) is an initiative to develop common business document schemas to provide standardization in the electronic business domain. However, businesses operate in different industry, geopolitical, and regulatory contexts and consequently they have different rules and requirements for the information they exchange. In this thesis, we provide semantic enrichment mechanisms for UBL that (i) allow automated customization of document schemas in response to contextual needs and (ii) maintain interoperability among different schema versions. For this purpose, we develop ontologies to provide machine processable representations for context domains, annotate custom components using classes from those ontologies and show that using these semantic annotations, automated discovery of components and automated customization of schemas becomes possible. We then provide a UBL Component Ontology that represents the semantics of individual components and their structural relationships and show that when an ontology reasoner interprets the expressions from this ontology, it computes equivalence and class-subclass relationships between classes representing components with similar content. Finally we describe how these computed relationships are used by a translation mechanism to establish interoperability among schema versions customized for different business context values.

Ubiquitin fold modifier 1 (Ufm1) is a member of the ubiquitin like protein (UBL) family. Like other UBLs, Ufm1 can be conjugated to protein substrates via specific E1 (Uba5), E2 (Ufc1) and E3 (Ufl1) enzymes, and removed from these substrates via the action of Ufm1-specific proteases. While Ufm1 has been implicated in endoplasmic reticulum (ER) function, its biological roles remain poorly understood. By identifying; (a) Ufm1 binding proteins, (b) protein interactors of the Ufm1 conjugation/deconjugation system, (c) Ufm1 conjugates, as well as (d) the intracellular localization of Ufm1 and its main interactors, I aimed to better characterize the biological role(s) of this poorly understood UBL.

Ubiquitin fold modifier 1 (Ufm1) is a member of the ubiquitin like protein (UBL) family. Like other UBLs, Ufm1 can be conjugated to protein substrates via specific E1 (Uba5), E2 (Ufc1) and E3 (Ufl1) enzymes, and removed from these substrates via the action of Ufm1-specific proteases. While Ufm1 has been implicated in endoplasmic reticulum (ER) function, its biological roles remain poorly understood. By identifying; (a) Ufm1 binding proteins, (b) protein interactors of the Ufm1 conjugation/deconjugation system, (c) Ufm1 conjugates, as well as (d) the intracellular localization of Ufm1 and its main interactors, I aimed to better characterize the biological role(s) of this poorly understood UBL.

In most eukaryotic organisms, the ubiquitination pathway is one of the most important and versatile signaling systems in use. It is integral to processes such as protein degradation and homeostasis, DNA repair cell cycle regulation, signaling and regulation, epigenetics, and many more. Ubiquitin (Ub) is a short polypeptide of 8.6 kDa, 76 residues that functions as a reversible post-translation modification (PTM). It furthermore contains 7 different lysine residues (K6, K11, K27, K29, K33, K48, K63), all of which can form isopeptide linkages with one another to link individual Ub moieties to form unique polyUb chains onto substrates. The type of polyUb chain a substrate gets labeled with can determine the subsequent activity of that substrate. Substrate ubiquitination is achieved through an enzymatic cascade. First, an E1-activating enzyme activates a free Ub moiety. Then Ub is transferred onto an E2-conjugating enzyme, and finally an E3 ligase interacts with both substrate and E2~Ub complex to facilitate Ub transfer onto a substrate. Within this scheme, the E2-enzyme acts as a master manipulator in that, it controls when, where and how a ubiquitin chain is transferred onto a substrate. Irregular activity of E2-conjugating enzyme has been implicated in a wide variety of diseases such as cancer, neurodegenerative diseases, muscular dystrophy, genetic azoospermia and more. While attempts have been made to inhibit other ubiquitination cascade enzymes such as E3 ligases and E1-activating enzymes, there is a strikingly small number of inhibitors specifically targeting E2 enzymes mainly due to the high degree of structural conservation that exists among members of the E2 enzyme family. In this work, we introduce 3 novel linked-domain protein inhibitors of the E2-conjugating enzyme Ube2D. We covalently attached either UHRF1 RING domain or an affinity optimized U-box domain, with UHRF1 UBL domain or UbvD1.1 (A ubiquitin variant specific for Ube2D), through a glycine-serine linker, producing 3 unique inhibitors: Ring-UBL (RU), U-box-UBL (UU), and U-box-UbvD1.1 (UUD1.1). In this way, we attempt to specifically inhibit Ube2D for two purposes : 1) While Ube2D can interact with the largest number of E3 ligases and facilitate the largest number of polyUb chains, very little is known about cellular phenotypes specifically associated with Ube2D; 2) We want to establish whether targeting the E2 enzyme in general can be utilized as a viable therapeutic treatment for cancer. We show that all three inhibitors are able to inhibit ubiquitin assays using Ube2D and using ITC we measured binding affinities of UUD1.1 (5 nM) > UUWT (300 nM) > RU WT (3 µM). Furthermore, we found that all inhibitors could prevent E1, E3 and backside binding domain interactions simultaneously, which single domain UBL could not. UU and RU showed specificity towards Ube2D when tested against APC/C and Cullin1 E3 ligases and their cognate E2 enzymes. We propose that linking domains in this way, by targeting the backside binding domains of E2 enzymes, could be a strategy that can be standardized and applied to the rest of the E2 enzyme family as well. In vivo testing must now elucidate whether these inhibitors can provide more information about the cellular role of Ube2D and whether it is a viable therapeutic target to treat cancer.

The leading effort for creating a standard semantic basis for business documents to solve the electronic business document interoperability problem came from the UN/CEFACT (United Nations Centre for Trade Facilitation and Electronic Business) Core Components Technical Specification (CCTS) through a conceptual document modeling methodology. Currently, the main challenge in using UN/CEFACT CCTS based approaches is that the document artifacts are stored in spreadsheets and this makes it very difficult to discover the previously defined components and to check their consistency. Furthermore, businesses need to customize standard documents according to their specific needs. The first XML implementation of UN/CEFACT CCTS, namely, Universal Business Language (UBL) provides detailed text-based descriptions of customization mechanisms. However, without automated tool support, it is difficult to apply the customization and to maintain the consistency of the customizations. In this thesis, these problems are addressed by providing an online e-business document design and customization environment, i.e. iSURF eDoCreator, which integrates the machine processable versions of paper-based UN/CEFACT CCTTS modeling methodology and UBL customization guidelines, accompanied with an online common UN/CEFACT CCTS based document component repository. In this way, iSURF eDoCreator environment aims to maximize re-use of available document building blocks and minimize the tedious document design and customization efforts. The environment also performs the gap analysis between different customizations of UBL to show how interoperable is the compared document models. The research leading to these results has received funding from the European Community&#039 / s FP7/2007-2013 under grant agreement n&deg / 213031, the iSURF Project.

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