ABSTRACT The magnitude of the AIDS epidemic is well documented. It has been shown that Africa constitutes about 70 % of people infected with HIV worldwide. Efforts to control the AIDS epidemic have focused heavily on studies pertaining to the biology, biochemistry and structural biology of HIV and on the interactions between HIV proteins and new drugs. One of the most challenging problems in AIDS therapy is that HIV develops drug-resistant variants rapidly. Extensive research has been dedicated to designing resistance-evading drugs for HIV-1 protease (predominantly subtype B), which is crucial for the maturation of viral, structural and enzymatic proteins. There are 10 subtypes of HIV-1 within the major group of the virus, with subtype C accounting for about 95 % of infections in South Africa. Since HIV-1 antiretroviral treatment has been developed and tested against the B subtype, which is prevalent in North America, Western Europe and Australia, an important question relates to the effectiveness of these drugs against the C subtype. At this point, however, little is known about inhibitor-resistant mutations in the subtype C. The study, therefore, looked at the two active site mutations (V82A and V82F/I84V) in the South African HIV-1 subtype C protease (C-SA) emerging from the viral population circulating in patients. These mutations are well-characterized within the framework of the subtype B and are known to cause cross-resistance to most of inhibitors currently in clinical use. Protein engineering techniques were used to generate the V82A and the V82F/I84V variants. Comparative studies with the wild-type HIV-1 C-SA protease were performed. The spectral properties of the V82A and the V82F/I84V variants indicated no changes in the secondary structure in the respective variant proteins. Tryptophan and tyrosine fluorescence indicated a major difference in the intensities at the emission maxima for all three proteins. The fluorescence intensity of the V82F/I84V variant, in particular, was significantly enhanced indicating the occurrence of tertiary structural changes at/near the flap region. Both mutations did not impact significantly upon catalytic function. Both variants also had the same Km values comparable to that of the wild-type enzyme. The catalytic efficiencies and the kinetic constants were lowered 3.6-fold for the V82A mutation and 6-fold for the V82F/I84V mutation relative to the wild-type C-SA protease. Inhibition studies were performed using four inhibitors in clinical use (saquinavir, ritonavir, indinavir and nelfinavir). For the V82A variant, IC50 and Ki values for saquinavir and nelfinavir iv were not affected, whilst those for ritonavir and indinavir were 5- and 9-fold higher than the wild-type C-SA protease, respectively. Against the V82F/I84V variant, however, the inhibition constants were drastically weaker and characterized by IC50 and Ki ratios ranging from 50 to 450. Isothermal titration calorimetry (ITC) was also used to determine the binding energetics of saquinavir, ritonavir, indinavir and nelfinavir to the wild-type C-SA, V82A and V82F/I84V HIV-1 protease. The V82A mutation lowered the Gibbs energy of binding for the respective four clinical inhibitors by 0.4 kcal/mol, 1.3 kcal/mol, 1.5 kcal/mol and 0.6 kcal/mol, respectively, relative to the wild-type C-SA HIV-1 protease. The affinity of V82A HIV-1 protease for saquinavir, ritonavir, indinavir and nelfinavir (Kd = 1.85 nM, 2.00 nM, 12.70 nM and 0.66 nM, respectively, at 25 °C) was in the range of 2- to 13-fold of magnitude weaker than that of the wild-type C-SA protein. The clinical inhibitors exhibited the highest binding affinity to both the wild-type and the V82A enzymes, but were extremely sensitive to the V82F/I84V mutation. The V82F/I84V mutant reduced the binding of saquinavir, ritonavir, indinavir and nelfinavir 117-, 1095-, 474- and 367- fold, respectively. A drop in Kd values obtained for the V82F/I84V in association with saquinavir, ritonavir, indinavir and nelfinavir was consistent with a decrease of between 2.8 - 4.2 kcal/mol in ΔG, which is equivalent to at least 2 to 3 orders of magnitude in binding affinity. Taken together, thermodynamic data indicated that the V82A and V82F/I84V active site mutations in the C-SA subtype lower the affinity of the first-generation inhibitors by making the binding entropy less positive (unfavorable) and making the enthalpy change slightly less favorable.

A thesis presented to the Faculty of Medicine, University of the Witwatersrand, in fulfilment of the requirements for the degree of Doctor of Medicine / Since changes occur in size, shape and haemoglobin content of red cells in disease a classification of the anaemias based on the morphology of the red cell has been widely used ( Wintrobe, 1956 ) . While such classification has a limited usefulness from the diagnostic and therapeutic approach it has two serious defects.. / IT2018

Thesis advisor: Peter Clote / RNA molecules play important roles in living organisms, such as protein translation, gene regulation, and RNA processing. It is known that RNA secondary structure is a scaffold for tertiary structure leading to extensive amount of interest in RNA secondary structure. This thesis is primarily focused on the development of novel algorithms for the analysis of RNA secondary structure evolution and folding kinetics. We describe a software RNAsampleCDS to generate mRNA sequences coding user-specified peptides overlapping in up to six open reading frames. Sampled mRNAs are then analyzed with other tools to provide an estimate of their secondary structure properties. We investigate homology of RNAs with respect to both sequence and secondary structure information as well. RNAmountAlign an efficient software package for multiple global, local, and semiglobal alignment of RNAs using a weighted combination of sequence and structural similarity with statistical support is presented. Furthermore, we approach RNA folding kinetics from a novel network perspective, presenting algorithms for the shortest path and expected degree of nodes in the network of all secondary structures of an RNA. In these algorithms we consider move set MS2 , allowing addition, removal and shift of base pairs used by several widely-used RNA secondary structure folding kinetics software that implement Gillespie’s algorithm. We describe MS2distance software to compute the shortest MS2 folding trajectory between any two given RNA secondary structures. Moreover, RNAdegree software implements the first algorithm to efficiently compute the expected degree of an RNA MS2 network of secondary structures. The source code for all the software and webservers for RNAmountAlign, MS2distance, and RNAdegree are publicly available at http://bioinformatics.bc.edu/clotelab/. / Thesis (PhD) — Boston College, 2018. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

Aptamers are synthetic nucleic acid molecules designed to bind with high specificity and affinity to a selected target. The aptamer selection method, called the systematic evolution of ligands by exponential enrichment (SELEX), was first described in 1990 and has been adapted for the selection of aptamers for a number of applications. One such application is the selective targeting of cells for therapeutic delivery. This thesis explores this application with the selection and characterisation of internalising aptamers specific to the T lymphocyte specific receptor, CD7. The CD7 receptor is expressed on thymus derived progenitor lymphocytes and remains after T cell activation and expression of the CD4 receptor. As such, the CD7 receptor is a noteworthy target for lymphocyte cancers, HIV-1 and other T lymphocyte tropic viruses. A heterogeneous pool of internalising CD7-aptamers was enriched through six rounds of positive selection in a stably transduced CD7-HeLa cell line. Aptamers were selected using a modified whole cell SELEX method that selected specifically for internalising aptamers. Aptamer specificity for CD7-HeLa cells over HeLa cells was screened by flow cytometry. CD7 specific aptamers were screened for binding after blocking CD7-HeLa cells with an anti-CD7 antibody. Eight CD7 specific aptamer clones were selected from CD7-HeLa screening for evaluation in Jurkat cells (T lymphocyte cell line endogenously expressing the CD7 receptor). Three aptamer clones showed high level binding to Jurkat cells by flow cytometry (CSIR 3.14, CSIR 3.37 and CSIR 3.42). Kinetic analysis of aptamer internalisation was analysed using flow cytometry and determined to be within the femtomolar range. Aptamer CSIR 3.14 had a dissociation constant of 2.1 fM and an association rate of 4.7 ± 2.4 × 105 Molar-1minute-1, aptamer CSIR 3.37 had a dissociation constant of 0.23 fM and an association rate of 4.3 ± 3.3 × 106 Molar-1minute-1 and aptamer CSIR 3.42 had a dissociation constant of 1.1 fM with an association rate of 7.9 ± 5.1 × 105 Molar-1minute-1. Aptamer CSIR 3.14 internalisation was tracked by confocal microscopy and the kinetics calculated with an association rate of 6.3 × 104 Molar-1minute-1 and Kd of 13 fM. Deletions within the CSIR 3.14 sequence that altered the predicted structures significantly reduced the aptamer binding. Combined, the data presented in this thesis identifies aptamer CSIR 3.14 as a lymphocyte specific internalising aptamer with potential for therapeutic delivery.

Thesis advisor: Dunwei Wang / Artificial photosynthesis is achieved by placing a semiconductor in water, where photoexcited charges generate a photovoltage at the surface of the semiconductor. However, solar to fuel efficiencies of earth abundant metal oxides and metal nitrides remain limited by their low photovoltages. Many different treatments have been used to improve the photovoltages of semiconductors, such as photocharging, surface regrowths, or the addition of heterogeneous catalysts. However, in these treatments, it remains unclear whether the enhanced photovoltage arises from improved kinetics or energetics. In many of the following studies, the surface kinetics of different semiconductors are measured in order to quantify how surface kinetics are related to the photovoltage of these materials. Different spectroscopic measurements are made along with detailed analysis of the Fermi level and quasi Fermi level in order to corroborate the kinetic data with energetic data. Together, this dissertation explores a multitude of methods and procedures that demonstrate how the photovoltage of semiconductors can be understood and manipulated for photoelectrochemial artificial photosynthesis. / Thesis (PhD) — Boston College, 2018. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

Hon Wan Chee Nicole Wendy. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves 85-87). / Abstracts in English and Chinese. / THESIS COMMITTEE --- p.ii / ABSTRACT (English version) --- p.iii / ABSTRACT (Chinese version) --- p.v / ACKNOWLEDGEMENTS --- p.vii / TABLE OF CONTENTS --- p.viii / LIST OF FIGURES --- p.x / LIST OF TABLES --- p.xiii / Chapter CHAPTER 1. --- General Introduction / Chapter Section 1.1 --- Introduction --- p.1 / Chapter Section 1.2 --- Electronic Structure Calculation --- p.2 / Chapter Section 1.3 --- Molecular Dynamics --- p.10 / Chapter CHAPTER 2. --- Ab Initio Molecular Dynamics Study on Thermal Dissociation of Azomethane / Chapter Section 2.1 --- Introduction / Chapter Section 2.2 --- Computational Method --- p.17 / Chapter Section 2.3 --- Results and Discussion --- p.21 / Chapter Section 2.4 --- Conclusion --- p.47 / Chapter CHAPTER 3. --- "Ab Initio Molecular Dynamics Study on Fluxionality of IF7, TeF7- and iof6-" / Chapter Section 3.1 --- Introduction --- p.49 / Chapter Section 3.2 --- Computational Method --- p.52 / Chapter Section 3.3 --- Analysis --- p.55 / Chapter Section 3.4 --- Results and Discussion --- p.56 / Chapter Section 3.5 --- Conclusion --- p.83 / REFERENCES --- p.85

For sprint events longer than 100 m, more than half the race is run on the bend, yet bend sprinting has received little attention in biomechanics literature. The aim of this thesis was to understand the effect of the bend on maximal effort sprint performance and technique, using bend radii and surfaces typical of outdoor competition. Three empirical studies were undertaken with experienced bend sprinters. Initial 3D kinematics investigations revealed an approximately 5% velocity decrease on the bend compared to the straight. However, step characteristic changes contributing to this reduction were different for the left and right steps. For the left step there were significant decreases in step frequency (p < 0.05), due to increased ground contact time, which agreed with previously proposed theoretical models. For the right step, however, a significantly reduced flight time resulted in a significant reduction in step length (p < 0.05). Maintaining step length and an ‘active touchdown’ were closely related to an athlete’s ability to better maintain straight line velocity on the bend. Generally, velocity decreased as bend radius decreased, with mean differences of up to 2.3% between lanes 8 and 2. However, changes to athletes’ technique due to different lanes were not conclusive. Ground reaction forces revealed between-limb differences during bend sprinting. Furthermore, frontal plane forces were up to 2.6 times larger on the bend than on the straight. Overall, asymmetries were identified between left and right steps for several performance, technique and force variables, suggesting that bend sprinting induces different functional roles between left and right legs, with the left step contributing more to turning to remain on the bend trajectory. The differences in kinematic and kinetic characteristics between the bend and straight, and between-limb asymmetries mean that athletes should apply the principle of specificity to bend sprinting training and conditioning, without sacrificing straight line technique.

Digitized by Kansas Correctional Industries

The application of MIDA boronates (MIDA = N-methyliminodiacetic acid) in Suzuki- Miyaura reactions has increased over the last years. This is mainly because in many cases, the replacement of the boronic acid for the respective MIDA boronate has a positive result in the reaction yield. The key feature that makes MIDA boronates efficient coupling partners is that they can undergo a slow hydrolysis reaction under Suzuki-Miyaura conditions, which generates the boronic acid in situ. The control of the hydrolysis rate is crucial to keep a low concentration of the boronic acid to avoid some of the side reactions frequently observed. The kinetics of the hydrolysis reaction from a range of alkyl, aryl, and heteroaryl MIDA boronates have been determined under different reactions conditions. In addition, competition experiments and computational calculations have resulted in the proposal of three distinct mechanisms for the hydrolysis of MIDA boronates: ‘base-promoted’, ‘water-promoted’, and ‘acid-promoted’. The base and acid mediated processes occur at faster rates than the neutral pathway and involves a rate-limiting addition at the MIDA carbonyl carbon by hydroxide or water, respectively. Whilst the 'neutral' hydrolysis requires neither base nor acid and involves ratelimiting B-N bond cleavage by a water cluster. Under certain conditions the neutral mechanism can operate in parallel with the base or with the acid mediated mechanism; relative rates are easily quantified by 18O incorporation in the MIDA, after this is released from the hydrolysis reaction. This insight is expected to assist informed application and optimisation of MIDA boronates in synthesis as well as the design of new MIDA boronate derivatives.

Bacterial strain Pseudomonas sp. ADP is capable of degrading atrazine via an enzymatic pathway in six sequential steps to yield carbon dioxide and ammonia. Atrazine is a persistent herbicide that frequently contaminates soil, drinking water, and ground water throughout areas of heavy use in the United States. A biological remediation approach using Pseudomonas sp. APD is considered as an effective, cost-efficient, and environmentally conscious method of decreasing atrazine concentration in areas of high contamination. Each enzyme in the degradation pathway is encoded by a corresponding gene, AtzA-AtzF, and is located on a self-transmissible 108-kb plasmid. Due to their prevalence in nature, and their unique genetic and physical characteristics, biofilms are of great interest in the field of bioremediation. Biofilms exhibit high tolerance for harsh environmental stressors/conditions, prodigious potential for recalcitrant compound entrapment via an extracellular polymeric matrix, quorum sensing, and increased horizontal gene transfer compared to their planktonic counterparts. Despite frequent genetic and chemical analyses performed on atrazine-degrading genes on planktonic cells of strain Pseudomonas sp. APD, the genetics and degradation potential of Pseudomonas sp. ADP biofilms is relatively unexplored. Real-time quantitative PCR was used to differentiate the expression of six genes involved in the process of atrazine degradation. Relative expression experiments revealed no statistically significant difference in the expression of atrazine-degrading genes in Pseudomonas sp. ADP cells grown as biofilms relative to Pseudomonas sp. ADP cells grown as planktonic cells. In biofilms alone, the expression of genes AtzDEF was differentiated via temperature of biofilm growth in cells grown at 25, 30, and 37 degrees. Analytical techniques, including GC-MS and HPLC, were used to elucidate atrazine remediation potential of Pseudomonas sp. ADP biofilms and our previously collected genetic data. Stable decreases in atrazine degradation following a first-order kinetic model have been demonstrated for planktonic cells compared to a complex degradation pattern, including transient increases, observed for corresponding biofilm-mediated cells. This is attributed to the unique structure of the biofilm and the potential of atrazine to be entrapped in the substances of the extracellular polymeric matrix and subsequently released into the effluent. Overall, the biodegradation efficiency was higher for Pseudomonas sp. ADP biofilm-mediated cells compared to their planktonic counterparts. A novel methodology of using confocal microscopy and in situ reverse transcription was proposed for optimization to visualize the expression of atrazine-degrading genes in fixed Pseudomonas sp. ADP biofilms. The sugar composition of Pseudomonas sp. ADP was evaluated using fluorescent lectin binding analysis and was determined to exhibit a prominent level of diversity and dependent upon growth medium. The results from these experiments will play a role in application of biofilms grown in bioreactors for atrazine remediation throughout areas of persistent and high contamination throughout the US. The new step in methodology development of an in situ visual gene expression technique can be extended to bioremediation of alternate recalcitrant compounds. The results may also be aid progress in alternate biofilm-related studies in medicine & human health, metallurgy, and engineering.