Thesis (MSc)--Stellenbosch University, 2015. / ENGLISH ABSTRACT: Since its discovery in the 1980’s, HIV has affected the lives of millions of individuals around the globe. Despite obvious need and an enormous amount of research a cure has remained elusive due to the rapid onset of mutated forms of the virus. However, there has been considerable success in reducing viral levels of infected individuals through the use of highly active antiretroviral therapy (HAART). The first-line regimen HAART mainly targets reverse transcriptase (RT) through the employment of two nucleoside RT inhibitors (NRTIs) and a nonnucleoside RT inhibitor (NNRTI). NNRTIs target an allosteric pocket situated about 10 Å from the catalytic site and cause a conformational change in the enzyme upon binding, leading to the inhibition of viral replication. There are currently 5 FDA approved NNRTIs on the market which successfully inhibit viral replication, but the use of these drugs is becoming limited due to the onset of drug resistant strains of the virus. In light of this need for the development of novel NNRTIs, we set out to explore new territory in NNRTI drug design with a goal of maintaining efficacy in the presence of both wild-type and mutated forms of HIV-1. To this end we designed three different NNRTI scaffolds along three different research thrusts. The first of these focused on the synthesis of 15 novel flexible triazole containing compounds. With these compounds we sought to achieve π-π stacking interactions with conserved amino acid residue Trp229 in the hope that we would be able to maintain efficacy in the presence of mutated forms of the virus. An additional feature included hydrogen bonding interactions to the backbone of Lys103. However, despite having thoroughly explored the triazole ring with multiple substitution arrangements, these compounds had very poor to no activity against whole cell HIV-1. Secondly we focused on the synthesis of a 4-hydroxyindole scaffold as a potential NNRTI. The focus here was to achieve interactions to Trp229 and simultaneously achieve hydrogen bonding interactions to the backbone of Lys101 at the entrance of the pocket. This was a novel concept in this class of compounds. We were able to successfully synthesize the indole core as a proofof-concept using the Knoevenagel-Hemetsberger method however; this compound had no activity against HIV-1. Lastly, in our quest to synthesize a novel NNRTI that could maintain efficacy against HIV-1 we decided to attempt to improve upon the stability of a lead indole-based compound synthesized previously within our research group. The lead compound was found to be potent with an IC50 of 1 nM but was unstable in acidic media due to the presence of a methoxy functionality situated at the 3-position on the indole. We sought to overcome this issue by introducing a substituted aryl amine functionality at this position. We were successful in synthesizing our desired compound but unfortunately it was significantly less active against whole cell HIV-1 than the lead compound. However, we were not completely deterred as there are a number of unexplored bioiososteres as possibilities to improve upon the stability of the lead compound while maintaining its excellent activity profile. / AFRIKAANSE OPSOMMING: Sedert die ontdekking van die menslike immuniteitsvirus (MIV) in die 1980’s, het die virus al die lewens van miljoene mense wêreldwyd geaffekteer. Ten spyte van die ooglopende behoefte aan ‘n geneesmiddel sowel as meer navorsing, bly ‘n keermiddel sover onbekombaar as gevolg van die verskillende mutasies wat binne die virus gebeur. Ten spyte hiervan, was daar al heelwat sukses in terme van ‘n verlaging van die virale vlakke in besmette individue deur die gebruik van hoogsaktiewe antiretrovirale terapie (HAART). As ‘n eerste behandeling, teiken HAART meestal trutranskriptase (RT) deur die inspanning van twee nukleosied trutranskriptase inhibeerders (NRTIs) en ‘n nie-nukleosied trutranskriptase inhibeerder (NNRTI). NNRTIs teiken ‘n allosteriese leemte wat ongeveer 10 Å weg van die katalitiese posisie is en veroorsaak dan ‘n konformasie verandering in die ensiem tydens die bindingsproses, wat dan lei tot die inhibisie van die virus se replikasie. Daar is tans 5 FDA goedgekeurde NNRTIs op die mark wat virale replikasie inhibeer, maar die gebruik van hierdie middels word alhoemeer belemmer as gevolg van die onwikkeling van weerstandige stamme van die virus. Met die oog op hierdie nood aan die ontwikkeling van nuwe NNRTIs, het ons gepoog om new gebiede te ondersoek in terme van die ontwerp van NNRTIs, met die doel om die effektiwiteit teen beide die wilde-tipe sowel as die gemuteerde vorme van HIV-1 te behou. Vir hierdie doeleindes het ons drie verskillende NNRTI steiers ontwerp, wat drie navorsingsdoeleindes na streef. Die eerste van hierdie doeleindes was die sintese van 15 nuwe buigsame triasool-bevattende middels. Met hierdie middels het on gepoog om π-π pakkingsinteraksies te behaal met aminosuur residu, Trp229, en sodoende die effektiwiteit van die NNRTIs in die gemuteerde vorm van die virus te behou. ‘n Additionele eienskap wat bygevoeg is, is ‘n waterstofbindingsinteraksie met die ruggraat van Lys103. Ten spyte van pogings om verskeie substitusie patrone om die triasool-ring te ondersoek, het hierdie middels baie swak tot geen aktiwiteit teen heel sel HIV-1 getoon nie. Tweedens, was die fokus op die sintese van ‘n 4-hidroksieindool steier as ‘n potensiele NNRTI. Die fokus hier was om ‘n interaksie met Trp229 te kry terselfdetyd as ‘n waterstofbindingsinteraksie met die ruggraat van Lys101, wat by die opening van die bindingssak is. Hierdie was ‘n nuwe konsep vir hierdie klas van middele. Ons het die indool-kern van hierdie molekules suksesvol gesintetiseer deur middel van ‘n Knoevenagel-Hemetsberger metode, maar ongelukkig het hulle geen aktiwiteit teen HIV-1 getoon nie. Laastens het ons gepoog om ‘n nuwe NNRTI te sintetiseer wat effiktiwiteit teen HIV-1 behou, deur te probeer om vorderings te maak op die stabiliteit van ‘n indool-gebaseerde hoof-middel wat al voorheen deur ons navorsingsgroep geraporteer is. Hierdie hoof-middel het ‘n IC50 waarde van 1 nM gelewer, maar was onstabiel in suur medium as gevolg van die teenwoordigheid van ‘n metoksie-groep in die 3-posisie van die indool. Ons het gepoog om hierdie probleem te oorkom deur ‘n gesubtitueerde arielamien in hierdie posisie te plaas. Ons was suksesvol hierin, maar ongelukkig was die middel heelwat minder aktief teen die heel sel HIV-1 as die metoksie-weergawe. Ten spyte hiervan, is ons optimisties dat ons hierdie probleem kan oorkom, aangesien daar verskeie bioisostere is wat die stabilitiet van middel kan verbeter terwyl dit moontlik die effektiwiteit kan behou.

published_or_final_version / abstract / Mechanical Engineering / Doctoral / Doctor of Philosophy

Brackish waters are now considered valuable alternative water resources. Reverse osmosis (RO) membranes are the most promising candidate for drinking water production through desalination. Low recovery (the fraction of influent water that becomes product water) prevents widespread application of RO inland because of the high cost of waste disposal. The recovery of a brackish RO system is limited by sparingly soluble salts that become supersaturated and precipitate on the membrane surface. Precipitation is controlled through pH adjustment and antiscalant addition; however, at high salt supersaturation, antiscalant control is overcome and precipitation occurs. To further increase RO recovery and avoid precipitation, a three-stage process treated the waste stream (concentrate) of a brackish water RO system through antiscalant degradation, salt precipitation, and solid/liquid separation. Ozone (O3) and hydrogen peroxide (H2O2) were used to degrade antiscalants, pH elevation and base (NaOH/NaHCO3) addition were used to precipitate sparingly soluble salts, and microfiltration (0.1 μm) was used to separate precipitated solids from the water. Optimal parameters (pH, ozone dose, H2O2/O3 ratio, antiscalant type and concentration, water composition) for antiscalant oxidation were determined. The influence of antiscalant type and concentration and pH was investigated for the precipitation and filtration stages. Results were obtained for particle size distribution, extent of precipitation, particle morphology, and particle composition. The effect of ozonation on precipitation and filtration was evaluated, with a comparison to two-stage treatment consisting of precipitation and filtration. Antiscalant oxidation is controlled by bivalent cation coordination, while pH and ozone dose significantly affect the extent of oxidation. The addition of antiscalant prior to precipitation caused changes to particle size and morphology, and results varied with water composition and antiscalant type and concentration. Ozonation, even for small times such as one minute, prior to precipitation and filtration increased calcium precipitation and decomposed the antiscalant enough to remove the effect of the antiscalant on particle characteristics. During ozonation, antiscalants were not completely oxidized, but the partial oxidation products did not seem to affect precipitation. Ozonation also reduced the fouling of microfiltration membranes used for solid/liquid separation. Results indicated concentrate treatment can significantly increase the overall recovery of an RO system. / text

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CIVINS / Systems on modern naval vessels are becoming exclusively dependent on electrical power. One example of this is the replacement of distilling and evaporator plants with reverse osmosis units. As the system is in continuous operation, it is critical to have remote real-time monitoring and diagnostic capabilities. The pressure to reduce shipboard manning only adds to the difficulties associated with monitoring such systems. One diagnostic platform that is particularly well suited for use in such an environment is the non-intrusive load monitor (NILM). The primary benefit of the NILM is that it can assess the operational status of multiple electrical loads from a single set of measurements collected at a central point in a ship's power-distribution network. This reduction in sensor count makes the NILM a low cost and highly reliable system. System modeling, laboratory experiments, and field studies have all shown that the NILM can effectively detect and diagnose several critical faults in shipboard fluid systems. For instance, data collected from the reverse osmosis units for two U.S. Coast Guard Medium Endurance Cutters indicate that the NILM can detect micron filter clogging, membrane failures, and several motor-related problems. Field-tested diagnostic indicators have been developed using a combination of physical modeling and laboratory experiments. / CIVINS

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

Photogrammetry and laser scanning are two primary methods used for object reconstruction / reverse engineering. But Laser scanners have much higher initial investment compared to photogrammetry method.<div>Currently, in the industry, researchers have been using 3 different softwares for reverse engineering purposes from photogrammetry/laser scanning.</div><div><br></div><div>This thesis is to showcase research work with following points:</div><div>1. Comparison between laser scanner and photogrammetry tools.</div><div>2. Application of photogrammetry in the industry.</div><div>3. Evaluation of photogrammetry tools based on quality of mesh results and comparison with baseline standard of laser-scanner results.</div><div>4. Proposed integrated methodology with photogrammetry tool for reverse engineering within single environment. </div><div>5. Validation of this integrated methodology with case studies.</div><div>6. Parametric study of tool for accuracy and comparison with actual standard models.</div><div><div><br></div></div>

HINTERGRUND: Langzeitnebenwirkungen der antiretroviralen Therapie werden u.a. auf mitochondriale Toxizität von NRTIs zurückgeführt, wobei es zu einer Dysfunktion der Atmungskette und nachgeschalteter Stoffwechselwege wie der de-novo-Pyrimidinsynthese kommen kann. Experimentell konnte gezeigt werden, dass das Pyrimidinnukleosid Uridin einige dieser Toxizitätseffekte reduzieren konnte. METHODIK: Nach Etablierung einer HPLC-Methodik zur Evaluation von Uridin im menschlichen Serum wurden Uridinspiegel von HIV-positiven Patienten mit Uridinspiegeln einer HIV-negativen Kontrollgruppe verglichen sowie mit Demografie, Nebenwirkungen bzw. Koerkrankungen, antiretroviraler Therapie und Therapiedauer korreliert. ERGEBNISSE: Die 182 HIV-Patienten wiesen signifikant niedrigere Uridinspiegel als die 30 Kontrollpersonen auf, blieben aber noch im physiologischen Normbereich gesunder Erwachsener. Demografische Faktoren sowie Marker der HIV-Infektion zeigten keine signifikante Korrelation mit Uridin. HIV-Patienten mit Begleiterkrankungen boten im Vergleich zu HIV-Patienten ohne Begleiterkrankungen signifikant verminderte Uridinspiegel. Bei Differenzierung nach den einzelnen Befunden zeigte sich, dass nur Patienten mit Diabetes mellitus Typ 2 und chronischen Lebererkrankungen wie Steatosis Hepatis signifikant erniedrigte Spiegel aufwiesen. Im Vergleich von 4 HIV-Patientengruppen mit unterschiedlicher antiretroviraler Therapie verhielten sich die Uridinspiegel analog zur potenziellen mitochondrialen Toxizität des jeweiligen Therapieregimes. Die Patientengruppe mit dideoxy-NRTI-haltiger Therapie wies die niedrigsten Uridinplasmaspiegel auf und zeigte im Vergleich zur Patientenguppe ohne antiretrovirale Therapie signifikant erniedrigte Uridinplasmaspiegel. Mit zunehmender Therapiedauer zeigte sich bei der Therapiegruppe mit dideoxy-haltiger HAART abfallende, bei den anderen 3 Therapiegruppen konstante bis leicht ansteigende Spiegel. SCHLUSSFOLGERUNG: Bei HIV-positiven Patienten scheint die Einnahme von (di)deoxy-NRTIs sowie Koerkrankungen wie Fettleber und Diabetes mellitus Typ 2 mit signifikant niedrigeren Uridinspiegeln assoziiert zu sein. Ob eine Uridinsubstitution zur Vermeidung von NRTI-assoziierten Langzeitnebenwirkungen sinnvoll ist, müssen weitere Untersuchungen zeigen. / BACKGROUND: Long-term side effects of antiretroviral therapy are mainly attributed to mitochondrial toxicity of nucleoside analogue reverse transcriptase inhibitors (NRTI) by causing respiratory chain dysfunction leading to inhibition of downstream metabolic pathways such as a de novo-pyrimidine synthesis. In vitro and in vivo studies indicated that uridine, a pyrimidine precursor, may prevent and reduce long term toxicity of NRTI treatment. METHODS: Uridine plasma levels of HIV-positive patients and HIV-negative controls were assessed by using high-performance liquid chromatography, compared to each other and correlated with demography, markers of HIV, comorbidities respectively long term side effects, antiretroviral therapy and duration of therapy. RESULTS: Uridine plasma levels of 182 patients were significantly decreased compared to HIV-negative controls, although still remaining in lower physiologic range of healthy adults. Demographic factors and markers of HIV-infection both evidenced no correlation with uridine plasma levels. HIV-patients with comorbidities showed significantly decreased uridine plasma levels compared to HIV-positives without comorbidities. This effect was mainly due to chronic liver disease such as fatty liver disease and diabetes mellitus type two. When comparing HIV-patients, divided into four different antiretroviral therapy containing groups, uridine plasma levels correlated with the potential mitochondrial toxicity of each treatment regimen. Furthermore, HIV-positive patients with dideoxy-NRTI containing therapy had statistically significant lower uridine plasma levels compared to HIV-positives without antiretroviral therapy. Regarding the observation period, patients with dideoxy-NRTI containing therapy showed decreasing uridine plasma levels whereas patients without such therapy had constant or slowly increasing levels. CONCLUSION: Dideoxy-NRTI containing HAART or comorbidities such as fatty liver disease and diabetes mellitus may be associated with statistically significant decreased uridine plasma levels in HIV-positive patients. Further investigation is necessary to evaluate whether supplementation of uridine may be beneficial to prevent or reduce NRTI side affects.

Foamyviren (FVs) sind die genetisch stabilsten Viren der Retrovirus-Familie. Dies steht im Gegensatz zur Fehlerrate, die für die rekombinante FV Reverse Transkriptase (RT) gefunden wurde. Um die Genauigkeit der FV Genomreplikation in vivo zu ermitteln, analysierten wir das Auftreten von Mutationen nach FV-Vektortransfer in einer einzigen Replikationsrunde. Die Sequenzanalyse von mehr als 90000 Nukleotiden ergab 39 Mutationen. Dies entspricht einer Fehlerrate von ungefähr 4 x 10-4 pro Base und Replikationszyklus, wobei alle Mutationen Transitionen von G zu A waren. Eine schwache Expression von APOBEC-Enzymen in den vektorproduzierenden Zellen konnte als wahrscheinlichste Ursache für diesen Typ an Mutationen nachgewiesen werden. Das akzessorische FV Bet Protein wirkt APOBEC entgegen. Kotransfektion von Zellen mit einem bet-Expressionsplasmid resultierte in einer signifikanten Reduktion an Mutationen bei über 170000 zusätzlich sequenzierten Basen. Zwei Mutationen konnten nicht der APOBEC-Aktivität zugeschrieben werden, deshalb postulieren wir eine idealisierte FV-Mutationsrate von angenähert 7,5 x 10-6 pro Base und Replikationszyklus. Im Gegensatz zu in vitro-Analysen wurde nur eine einzige Deletion und keine Insertion bei mehr als 260000 sequenzierten Basen identifiziert. Die Analyse der Rekombinationsrate von FV-Vektorgenomen ergab mehr als ein zusätzliches Template-Switching-Ereignis pro reverser Transkription. Wir konnten auch zeigen, dass ein bestimmtes FV-Partikel in der Lage zum Crosstransfer eines heterologen FV-Genoms ist, jedoch mit einer reduzierten Effizienz als bei Verwendung des homologen Vektors. Zusammenfassend zeigen unsere Ergebnisse einerseits, dass das Kopieren des FV-Genoms mit höherer Genauigkeit geschieht als bisher angenommen, auf der anderen Seite ist Rekombination bei FV-Genomen wahrscheinlich. / Foamy viruses (FVs) are the genetically most stable viruses of the retrovirus family. This contrasts to the error rate found for recombinant FV reverse transcriptase (RT). To investigate the accuracy of FV genome copying in vivo we analyzed the occurrence of mutations after a single round of FV vector transfer. Sequence analysis of more than 90,000 nts revealed 39 mutations. This corresponds to an error rate of approx. 4 x 10-4 per site and replication cycle. All mutations were transitions from G to A. A residual expression of APOBEC enzymes in vector producer cells was found to be likely responsible for this type of mutation. The accessory FV Bet protein is implicated to counteract APOBEC. Cotransfection of cells with a bet expression plasmid resulted in a significant drop of mutations among over 170,000 additional sequenced bases. Since two mutations were not correlated to APOBEC activity, we postulate an idealized FV mutation rate of close to 7.5 x 10-6 per site and replication cycle. In contrast to in vitro studies only one deletion and no insertion was identified among the more than 260,000 sequenced bases. Analysis of the recombination frequency of FV vector genomes revealed more than one additional template-switching event per reverse transcription. We also show that a given FV particle is able to cross-transfer a heterologous FV genome, although at reduced efficiency than the homologous vector. Taken together, our results indicate that FV genome copying is of higher accuracy than thought previously. On the other hand recombination among FV genomes appears to be likely.