This thesis presents an investigation of fluorescence resonance energy transfer (FRET) as a reporting signal for protein-protein interactions. Quantitative optical assays to measure protein binding, conjugation and deconjugation are developed and results validated by conventional biochemical techniques. The optical techniques developed provide fast, cheap, quantitative and accurate alternatives to conventional methods. Fluorescent protein fluorophores ECFP and Venus-EYFP were chosen as they are a non-interfering FRET pair and provide an inexpensive and convenient cloning-based labelling method. The small ubiquitin-like modifier SUMO and the SUMOylation pathway leading to its conjugation to target proteins is investigated as a model system. These assays are hence particularly relevant to research on post-translational modification and ubiquitin systems. In protein-protein binding assays we utilise both steady-state and time-resolved FRET detection to measure the equilibrium binding constant of the well-characterised pair SUMO1 and Ubc9. An assay in multi-well plate format is also presented, which uniquely enables repeat measurements under varying conditions and under the addition of further substances. The multi-protein binding interactions of the SUMOylation pathway including RanBP2 are analysed in binding inhibition assays. Our results clarify the role of RanBP2: a covalent SUMO1-Ubc9 link is required for the formation of a trimeric complex, although mutual binding sites are present on all three proteins. Furthermore, the binding of SUMO1 and Ubc9 is disrupted by RanBP2, which may be an essential step in transferring SUMO1 to its target protein. A FRET-based kinetic study of this conjugation process to RanGAP1 is presented. An assay to monitor the deconjugation of SUMO1 by specific proteases is established using a doubly-tagged SUMO construct. This enables a quantitative analysis of protease and substrate specificity based on real-time kinetic data, a characterisation of crude cell extracts and a high-throughput screen for protease inhibitors using FRET. A screen of the National Cancer Institute (NIC) diversity set for SenP1 inhibition reveals nine suitable compounds, which are potential anti-cancer drugs. The results of two further projects, the study of protein-protein binding by measuring small refractive index changes and the autofluorescence of normal and neoplastic cervical tissue models are also presented. In the latter, principal component analysis was used to systematically identify emission regions of significant variation between samples, enabling discrimination between healthy and pre-cancerous tissue models.

Les gènes de la famille ING : « INhibitor of Growth » (ING1-5) jouent un rôle crucial dans l'inhibition de la prolifération cellulaire, en régulant notamment le cycle cellulaire, l'apoptose et la sénescence. De plus, plusieurs études (portant majoritairement sur ING1) montrent que ces gènes sont fréquemment perdus dans de nombreux cancers. Ils pourraient donc être impliqués dans l'émergence et le développement de tumeurs. Ainsi, l'objectif de mon projet de thèse était d'étudier le gène ING2, afin d'évaluer son intérêt en cancérogénèse. Nous avons tout d'abord montré que l'expression d'ING2 (ARN et protéique) est perdue dans plus de la moitié des cancers bronchiques non à petites cellules, confortant ainsi un rôle d'ING2 comme gène suppresseur de tumeurs. Par ailleurs, nous avons montré que l'inhibition de l'expression d'ING2 conduit à des défauts de réplication et à une forte augmentation de l'instabilité génomique, mettant ainsi en évidence pour la première fois qu'ING2 est un gène suppresseur de tumeurs de type « caretaker ». Ceci permet aussi pour la première fois d'expliquer comment l'inactivation des ING, observée dans les tumeurs, pourrait contribuer à la cancérogénèse. Enfin, nous avons mis en évidence le premier mécanisme de régulation post-traductionnelle d'ING2. En effet, ING2 peut être sumoylée, et cette sumoylation est nécessaire pour son association avec le complexe de régulation Sin3A/HDAC afin de cibler ce dernier au niveau des promoteurs de gènes pour réguler leur expression. Ces travaux ont donc contribué à démontrer l'intérêt d'ING2 en cancérogénèse et à mieux comprendre ses fonctions suppressives de tumeurs. De plus, ils ont permis d'ouvrir plusieurs voies d'investigation sur les fonctions et les mécanismes de régulation des protéines ING.

The SUMO protein was named Small Ubiquitin-like MOdifier because its 3-D structure was similar to Ubiquitin. In human, three SUMO proteins were discovered, namely, SUMO-1/2/3. The recombinant ¡µ1-8, 94-95 SUMO-2 protein with 10 histidine residues at its N-terminus was expressed using E. coli. BL-21(DE3), purified at 4 oC and crystallized at room temperature. The surface properties of human SUMO-1/2/3 proteins and 3-D structure of ¡µ1-8, 94-95 SUMO-2 protein were analyzed using computer modeling and X-ray diffraction technology respectively. The two-step purification by immobilized metal ion affinity chromatography(IMAC) was developed to yield ¡µ1-8, 94-95 SUMO-2 protein that reached 60 mg/ml for crystallization. On protein expression, 120 mg protein was obtained from 6 L bacterial growth broth. Crystals of ¡µ1-8, 94-95 SUMO-2 were obtained by the hanging-drop vapor diffusion method and many different crystal forms were observed. One of single crystal with triangular plate polyhedron form diffracted to 1.6 Å resolution, the other one with rectangular polyhedron form diffracted to 1.2 Å. Analysis of the diffraction pattern suggests the crystals belong to R3 space group, the former one owned unit cell parameters a= b=75.3 Å, c=29.2 Å, £\=90¢X, £]=90¢X,£^=120¢X, and the later one owned unit cell parameters a= b=74.9 Å, c=33.2 Å and the same angles respectively. The R factor and Rfree of refinement are 0.133 and 0.190 with highly precise phase on 3-D structure of SUMO-2 protein. Comparison of crystal structure between human SUMO-2 and yeast SMT3 showed that the r.m.s. deviation of C£\ coordinate is 1.054 Å. In addition, comparison of SUMO-1 NMR structure and SMT3 crystal structure showed that the r.m.s. deviation of C£\ coordinates is 2.736 Å. Hence, the structures of SUMO-2 and SMT3 are more similar each other than those of SUMO-1and SMT3.

No description available.

The aim of this master's thesis is to design an extension of the existing simulation system for designing traffic intersections. The required extension will allow the use of the Siemens sX traffic controllers. The introduction to the topic of traffic engineering, traffic controllers configuration and the dynamic traffic control is discussed first. The next part is dedicated to describing the implementation of the existing simulation system. The following chapter describes the use of the sX traffic controllers and also describes the design of the required extension of the existing simulation system, for a purpose of enabling the use of sX traffic controllers. This is followed by a chapter describing the implementation of this extension. The last chapter is devoted to testing the whole system in terms of functionality and performance. Finally, other possibilities for the future development are outlined.

La modification post-traductionnelle par les « Small Ubiquitin-Like MOdifyers (SUMOs) » est un processus majeur de régulation qui influence plus d’une centaine de protéines. Cette modification (SUMOylation) touche plusieurs fonctions nucléaires telles que la réparation de l’ADN, la réplication et la transcription. La SUMOylation affecte une protéine le plus souvent en permettant la formation de nouvelles interactions protéine-protéines avec des facteurs de régulations qui possèdent un court segment hydrophobe dans leur séquence connu sous le nom de « SUMO interacting motif (SIM) ». Bien que les interactions SUMO-SIMs soient bien documentées, la description de leur régulation n’est pas complète. Cette thèse décrit des études fonctionnelles et structurales sur différents mécanismes de régulation des interactions SUMO-SIMs. Plus précisément, elle décrit les effets de l’acétylation et de la queue N-terminale des protéines SUMOs sur leur capacité à réguler les interactions entre SUMOs et les SIMs de trois protéines : le suppresseur de tumeur « promyelocytic leukemia (PML) », le corépresseur de transcription « Death Domain Associated Protein 6 (Daxx) » et « Protein Inhibitor of Activated STAT (PIAS) », une ligase E3 pour SUMO. La première étude décrit l’effet de l’acétylation de SUMO1 sur sa capacité à interagir avec les SIMs de PML et de Daxx. À partir d’expériences de titrage calorimétrique et d’études cristallographiques, nous avons démontré que l’acétylation précise de certains résidus conservés chez SUMO1 (K39 et K46) réduit fortement l’affinité avec les deux SIMs testés. En contraste, nous démontrons que l’acétylation du résidu K37 sur SUMO1 à un effet inhibiteur spécifique pour le SIM de Daxx. Les structures cristallographiques des complexes formés entre les variants acétylés de SUMO1 avec les SIMs concordent avec les données des titrages et suggère une plasticité dans la formation des liens sur la surface d’interaction. Partant de ce constat, nous postulons que la plasticité observée dans la structure des complexes acétylés démontre un mécanisme de régulation des interactions SUMO-SIMs par l’acétylation de résidus conservés chez SUMO1. Dans la deuxième étude, nous avons identifié un deuxième SIM à l’extrémité C-terminale de protéines de la famille (PIAS1-2-3). Nous démontrons que ce SIM est capable de lier SUMO1 et que structurellement la phosphorylation de résidus clés dans ce domaine ainsi que l’acétylation de SUMO1 peut contrôler cette interaction. Une comparaison avec le premier SIM des variants PIAS démontre que les deux SIMs sont affectés différemment par la phosphorylation et l’acétylation. En outre, nous avons déterminé que le nouveau SIM identifié joue un rôle important dans la formation d’un complexe ternaire répresseur de la transcription, formé des protéines PIAS, SUMO1 et de l’enzyme de conjugaison « UBiquitin Conjugating enzyme E2I (UBC9) ». Pris ensemble, ces résultats donnent une description atomique de l’interaction d’un nouveau SIM chez PIAS avec SUMO1 et décris comment la phosphorylation et l’acétylation peuvent sélectivement réguler la spécificité des SIM trouvés chez les variants PIAS. Finalement, dans la dernière étude, nous avons exploré le rôle de la queue N-terminale des paralogues SUMO1 et 2 sur sa capacité à moduler les interactions SUMO-SIMs. Nous avons démontré que la queue N-terminale de SUMO1, mais pas SUMO2, avait un effet auto-inhibiteur sur les interactions SUMO-SIMs et que cet effet dépendait de la présence de résidus chargés négativement présent dans le SIM. Aussi, nous avons démontré que l’effet auto-inhibiteur était spécifique à la surface d’interaction des SIMs sur SUMO1. De plus à partir d’études cristallographiques et de calorimétrie, nous avons démontré que l’effet auto-inhibiteur de la queue N-terminale de SUMO1 peut être neutralisé par la présence de zinc. La structure cristallographique du complexe entre SUMO1 et le SIM de PML démontre que le zinc stabilise la formation de liens entre des résidus chargés négativement du SIM et de la queue N-terminale de SUMO1. De plus, le zinc induit la formation d’une hélice α dans la queue N-terminale de SUMO1 qui est normalement intrinsèquement désordonnée. En résumé, cette étude donne une description atomique de l’effet de l’acétylation sur les interactions SUMO-SIMs, décris un nouveau SIM dans la famille de protéines PIAS et identifie un nouveau rôle de la queue N-terminale de SUMO1 ainsi que comment cette région peut définir la sélectivité des paralogues SUMOs. / Post-translational modification with the « Small Ubiquitin-Like MOdifyer (SUMO) » is a major regulatory process (commonly referred to as SUMOylation) that regulates hundreds of proteins associated with a diverse array of biological activities including several nuclear functions such as DNA repair, replication and transcription. SUMOylation of a protein can impact its function in many ways most often by providing an additional binding surface for forming protein-protein interactions with regulatory factors through short hydrophobic regions on their binding partners known as « SUMO interacting motif (SIM) ». Although SUMO-SIM interactions are well documented, there are nevertheless outstanding questions that still need to be addressed regarding their controlling mechanisms. This thesis reports functional and structural studies on the regulatory mechanisms that govern SUMO-SIM interactions. More precisely, we studied how acetylation and the amino-terminal tail of SUMO proteins affects the interaction of SUMO with model SIMs from three proteins: the « promyelocytic leukemia (PML) » tumor suppressor, the transcriptional corepressor « Death Domain Associated Protein 6 (Daxx) » and the SUMO E3 ligase « Protein Inhibitor of Activated STAT (PIAS) ». The first study reports the role that acetylation of SUMO1 plays on its binding to the SIMs of PML and Daxx. Isothermal Titration Calorimetry (ITC) experiments demonstrated that acetylation of SUMO1 at conserved residues (K39 and K46) dramatically reduces the binding to the SIMs of PML and Daxx. In contrast, SUMO1 acetylation at K37 dramatically reduced binding to the SIM of Daxx but only had minimal impact on binding to the SIM of PML. Crystal structures of the SUMO1 acetylated variants bound to the two SIMs support the ITC titrations and suggest that there is plasticity in SUMO-SIM interactions. The plasticity observed in the structures of these complexes would provide a robust mechanism for regulating SUMO-SIM interactions using a combination of signalling mechanisms that control post-translational modifications. In the second study, we identified and characterized a novel SIM at the C-terminal extremity of three of the four known variants of the PIAS-family proteins (PIAS1-2-3). We demonstrated that this SIM binds to SUMO1 and structurally show that phosphorylation of the SIM or acetylation at select lysine residues of SUMO1 alters this interaction. In addition, we determined that it plays an important role in the formation of ternary complex made of SUMO1, PIAS1 and the « UBiquitin Conjugating enzyme E2I (UBC9) » in human cells. Together, these results provide an atomic description of the interaction between the C-terminal SIM of PIAS proteins and SUMO1 as well as important insight into how posttranslational modifications selectively regulate the specificity of the SIMs found in PIAS1-2-3. Finally, our third study explores the intrinsically disordered N-terminal tail of SUMO paralogs and their ability to regulate SUMO-SIM interactions. We demonstrate that the N-terminal region of SUMO1, but not SUMO2, has an auto-inhibitory effect on the binding to SIMs and that this effect is dependent on the presence of acidic or phosphorylated residues that within the SIM. In addition, we also determined that this inhibition does not affect the interaction of SUMO1 with its E2 conjugating enzyme UBC9. Using titration calorimetry and crystallographic screening, we identified zinc as a negative regulator of this auto-inhibitory effect. The crystallographic structure of the complex between SUMO1 and the SIM of PML shows that zinc stabilises the formation of interactions with the negatively charged residues within the SIM and the N-terminal tail of SUMO1. Interestingly, zinc also appears to stabilize the formation of an α-helix within the N-terminal tail of SUMO1 which is normally intrinsically disordered. In summary, this thesis describes the underlying atomic regulatory mechanisms of SUMO-SIM interactions by acetylation, reveals a novel SIM within the PIAS SUMO E3 ligase family and describes an unprecedented role of the N-terminal region of SUMO1 and provides important insight on how this region can define SUMO paralog specificity.

Thesis (Master of Environmental Management)--Cape Peninsula University of Technology, 2018. / Fresh water is a renewable resource, but it is also finite, especially given environmental impacts from anthropogenic activities. Globally, there are countless signs that untreated industrial discharge into fresh watercourses is one of the main causes of ecosystem degradation. Poultry slaughterhouse wastewater (PSW) amongst the main pollutants of fresh water sources. In recent years, the world’s pre-eminent researchers have developed innovative wastewater treatment processes to treat the large quantity of wastewater generated as well as to manage the environmental health concerns arising from PSW discharged into the environment. Furthermore, increasing wastewater treatment capital costs and the implementation of increasingly rigorous government legislation to mitigate environmental pollution whilst minimizing fresh water source contamination, requires that wastewater such as PSW, be adequately treated prior to discharge. In order to assist the small-scale poultry producers in South Africa (SA), process simulation for a small-scale poultry slaughterhouse wastewater treatment plant was proposed using Sumo Wastewater treatment plant (WWTP) simulation software. Sumo is an innovative and most versatile wastewater simulation package on the market. The simulator is capable of modelling treatment plants of unlimited complexity, focusing largely on Biochemical oxygen demand (BOD), Chemical oxygen demand (COD), nitrogen and phosphorus removal; with digester, and side streams design options, being available. Considering the possible advantages in modelling and ongoing studies of implementing wastewater treatment to increase water management, anaerobic digestion of high strength wastewater such as PSW, warranted this research study. Model development from the simulation included the evaluation of numerous design options to assist small scale poultry producers, to have a variety of designs to choose from in their PSW WWTP designs. With the aid of Sumo, two models were designed in this study, namely a single-stage and a two-stage anaerobic digestion without a recycle. The PSW used as feed was obtained from a local poultry slaughterhouse (Western Cape, South Africa). Both model designs predicted the reduction of the organic matter (COD, BOD5) total suspended solids (TSS), and volatile suspended solids (VSS) in the PSW. The digester for the single stage anaerobic digestion system modelled was set to operate at steady state for 150 days under mesophilic temperature (35 ˚C) with a solid retention time (SRT) of 25 days. The COD, TSS, VSS and BOD removal efficiencies reached a maximum of 64%, 77%, 84%, and 94%, respectively, at an organic load rate (OLR) of 143.6 mg COD/L/day. A minute increase in the ammonia (NH3) and phosphate (PO3- 4) concentration was observed once the simulation was completed. As for the two-stage anaerobic digestion system, both digesters were set to perform at mesophilic temperatures (35 ˚C) and a SRT of 13 days in the first digester and 25 days in the subsequent digester. The two-stage anaerobic digestion showed better performance in comparison to the single-stage anaerobic digestion system. The COD, TSS, VSS and BOD5 removal efficiencies reached a maximum of 69%, 79%, 85%, and 96%, respectively, at an at an OLR of 143.6 mg COD/L/day. A similar trend regarding phosphate and ammonia removal was noticed in the two-stage anaerobic digestion, suggesting a tertiary treatment system to be in place for further treatment. Although, the two-stage anaerobic digestion demonstrated adequate performance, for the purpose of this study, the single-stage was the process recommended for PSW treatment, as it is less costly and will be suitable for small scale poultry producers; albeit biogas production is much higher when digesters are connected in series. The PSW treatment modelling for this study was successfully employed with the resultant effluent being compliant with the City of Cape Town (CCT) wastewater and industrial effluent by-law discharge limits. Although, both the PO3- 4 and NH3 were suggested to require further monitoring. Therefore, the poultry slaughterhouse from which the PSW was obtained will be able to safely discharge the treated wastewater proposed in this research into local water bodies, i.e. rivers in the Western Cape, SA; however, the treated PSW will not be suitable for re-use as process water.

No description available.

Traffic shockwaves are a regularly occurring phe-nomenon in traffic that are a source of irritation and delaysfor the road users. One type of shockwave is the stop-and-gowave which forces entering drivers to stop and advance slowlyuntil the wave is passed. This project aims to design a controlalgorithm through the use of models and simulations to increasethe rate at which a stop-and-go wave dissipates. To design themodel and algorithm the Simulation of Urban MObility (SUMO)simulator and the Traffic Control Interface (TraCI) were usedin conjunction with Python. The setup used for simulation wasthat of a one way, two lane highway with an artificially inducedstop-and-go wave.The designed algorithm manages to dissipate a stop-and-go wavecompletely without introducing new ones. / Trafikvågorär ett vanligt förekommandefenomen i trafiken vilketär en orsak till frustration ochförseningar. En typ av vågär startochstop vågen som tvingarförare att stanna och långsamt fortsätta genom vågen tills denpasserat. Målet med detta projektär att utveckla en kontrol-lalgoritm med hjälp av modeller och simuleringar för attökaavtagandet av en sådan våg. För att utveckla modellen ochalgoritmen används simulatorn Simulation of Urban MObility(SUMO) och Traffic Control Interface (TraCI) i kombinationmed programmeringsspråket Python. Simulering gjordes på ettnätverk bestående av en enkelriktad, tvåfilig motorväg med enkonstgjord startochstop våg.En algoritm utvecklades som kan skingra en startochstop vågutan att skapa nya. / Kandidatexjobb i elektroteknik 2020, KTH, Stockholm

No / Astrocyte reactivity is implicated in the neuronal loss underlying Alzheimer's disease. Curcumin has been shown to reduce astrocyte reactivity, though the exact pathways underlying these effects are incompletely understood. Here we investigated the role of the small ubiquitin-like modifier (SUMO) conjugation in mediating this effect of curcumin. In beta-amyloid (Aβ)-treated astrocytes, morphological changes and increased glial fibrillary acidic protein (GFAP) confirmed reactivity, which was accompanied by c-jun N-terminal kinase activation. Moreover, the levels of SUMO-1 conjugated proteins, as well as the conjugating enzyme, Ubc9, were decreased, with concomitant treatment with curcumin preventing these effects. Increasing SUMOylation in astrocytes, by over-expression of constitutively active SUMO-1, but not its inactive mutant, abrogated Aβ-induced increase in GFAP, suggesting astrocytes require SUMO-1 conjugation to remain non-reactive.