Replication of HIV-1 is inhibited by azidothymidine (AZT), which leads to chain termination and inhibition of DNA synthesis. Resistance to AZT is frequently the result of mutations that increase the ability of RT to remove the chain-terminating nucleotides after they have been incorporated. It has been proposed that RNase H cleavage of the RNA template can occur when RT is stalled near the site of chain termination and contributes to the inhibition by causing the dissociation of the primer-template before the chain terminator can be excised. Mutations in the connection and RNase H domains of RT have been shown to increase excision. It has long been known that resistance to thymidine analogs is conferred by the mutations M41L, D67N, K70R, L210W, T215F/Y and T219Q/E in RT and that this resistance is suppressed by the additional presence of the M184V mutation. Changes in excision activity on DNA templates have been observed with these mutant RTs, but effects on RNase H cleavage resulting in indirect effects on excision activity is also possible with RNA templates. We used a 5'-labeled -3'-chain-terminated DNA primer annealed to either a DNA or RNA template to evaluate primer rescue activities, a 5'-labeled RNA template to evaluate RNA cleavage activity and a biotin-tagged chain-terminated oligodeoxynucleotide to monitor primer-template dissociation. We first investigated differences between RNA and DNA templates when the primers were chain terminated and observed a correlation between RNase H activities and template/primer (T/P) dissociation. An inverse correlation was observed between excision rescue rates and RNase H cleavages leading to T/P dissociation. We observed that the chain terminator (i.e. AZTMP or ddAMP) affected RNase H cleavages and excision rates with RNA template and dNTPs. When we investigated mutations in the N-terminal domain of RT associated with nucleoside reverse transcriptase inhibitor (NRTI) resistance we found that primer rescue was decreased when M184V was present in combination with thymidine analog mutations (TAMs) and the template was RNA with either ATP or PPi as excision substrate. RNase H cleavage at secondary cleavage sites (-7, -8) was substantially reduced with M41L/T215Y RT in comparison with wild type RT, and primer-template dissociation was decreased. In contrast, when M184V was present, RNase H cleavage at the secondary cleavage sites and dissociation of the primer-template occurred at higher levels and excision rescue was decreased. The ability of RT to rescue an AZT terminated primer in the presence of the 184V mutation was restored when the RNase H activity was inactivated by the RNase H negative mutation E478Q. Electromobility shift assay (EMSA) analysis of AZT-resistant mutant RT with M184V showed an increased Kd for formation of the ternary complex. These results suggest that RNase H-mediated RNA-DNA template-primer dissociation is influenced by mutations associated with thymidine analog resistance, and that suppression of resistance to nucleoside RT inhibitors by M184V may be partly explained by effects on RNase H cleavage that decrease the time available for excision to occur. This is the first time that mutations in the polymerase domain are shown to affect excision rescue through an RNase H-dependent mechanism.

Effective antiretroviral therapy (ART) is essential for viral suppression (VS) in HIV-infected patients. However, there is a lack of nationally representative data on types of ART regimens used and their impact on VS. This thesis used self-reported interview and abstracted medical record from 2009 Medical Monitoring Project (MMP) to study ART regimen type and related health outcomes. Results showed that 88.6% of HIV-infected adults in care was prescribed ART, and about half took regimens designated as ‘preferred’ according to U.S ART guidelines. Among MMP participants prescribed ART, 62.7% achieved durable VS, 77.8% achieved recent VS, 83.5% were 100% dose-adherent, and 17.1% reported side effects. Multivariate regression analyses revealed that although ART was critical for VS, there were minor differences in health outcomes among the major ART classes in the U.S. ART guidelines or six most-commonly used regimens. This study could be potentially useful for future strategic planning of HIV care.

No description available.

Anxiety disorders are one of the most prevalent mental disorders in the world. While normal anxiety serves as an important protective mechanism, pathological anxiety characteristic of an anxiety disorder is both maladaptive and disruptive. The majority of studies have focused on the neurotransmitter systems associated with the actions of known anxiety drugs. This focus may likely limit the exploration of mechanisms underlying anxiety disorders. This project aims to examine changes in gene expression that may underlie higher or lower levels of inherent anxiety. Using a well-established behavior test for anxiety, the elevated plus maze, we identified male Wistar rats exhibiting inherently high- or low-anxiety levels. Brain regions known to mediate anxiety, the amygdala, hippocampus and nucleus accumbens, were dissected and total mRNA isolated. The mRNA was converted to cDNA via reverse transcription-polymerase chain reaction (RT-PCR). Then, the cDNA was used in suppression subtractive hybridization, a technique used to compare two complete populations of cDNAs and identify cDNAs that are upregulated in one population in relation to the other. In this project suppression subtractive hybridization was used to compare high- and low-anxiety cDNA populations. The upregulated cDNAs were amplified in a PCR reaction that enables rare transcripts to be identified. The PCR products from the suppression subtractive hybridization were cloned and used to create two cDNA libraries for high- and low-anxiety related genes. These clones were sequenced to show over 1000 genes upregulated in high- and low-anxiety. The gene list was then subjected to bioinformatic analysis to identify one candidate to be studied in further detail. <p>The prion protein was identified as a potential candidate. Examination of the literature sparked an interest in studying other prion-like proteins, more specifically the cytoplasmic polyadenylation element binding protein (CPEB). The CPEB protein is a potent regulator of mRNA translation in both mature oocytes and the adult brain. While unphosphorylated the CPEB protein keeps specific mRNAs dormant in the cytoplasm. In its phosphorylated form CPEB catalyzes polyadenylation of the mRNA, leading to protein synthesis. p*PCR was used to show the presence of CPEB mRNA transcripts in the rat hippocampus. CPEB protein expression was examined in the brain samples isolated from control, high- and low-anxiety rats. It was found that CPEB was significantly upregulated in high- and low-anxiety rats compared to control. The protein expression of an upstream kinase, Aurora A kinase, and a downstream target, Calcium/Calmodulin Dependent Kinase II (CaMKII), was also investigated. The results from Aurora A kinase were inconclusive. CaMKII, on the other hand, was significantly upregulated in high-anxiety over both control and low-anxiety. These results suggest that CPEB may catalyze increased translation of mRNAs in high-anxiety while acting as a repressor of those same mRNAs in low-anxiety. <p>Recent studies have suggested that CPEB protein plays an important role in synaptic plasticity. The regulation of synaptic plasticity, and its impact on learning and memory, is believed to be a key mechanism behind the maintenance of anxiety disorders. Therefore the results of this study suggest a new molecular mechanism in the development of anxiety disorders.

Sprout control is essential for successful management of stored potatoes. This study examined the effect of dill weed, spearmint and clove essential oils on sprouting of potatoes. Extracts of steam distilled dill weed whole plants containing 41.5-42.7% of S-(+)-carvone and spearmint foliage extracts containing 97.2-97.6% of R-(-)-carvone, were applied to tubers in a series of experiments using either 1-L glass jars or 63-L steel drums. The composition of the essential oils was consistent between years but evaporation rate varied among the oils as dill weed extract evaporated the fastest while clove oil evaporated the slowest under the same conditions. After exposure to essential oil treatments, tuber sprout number and weight were assessed and compared to untreated control and tubers treated with commercially marketed clove oil product (Biox-CTM, containing 78.5-82.3% eugenol). Applications of 32.5 and 47.6 mg L-1 headspace of dill weed oil and 21.5 and 22.3 mg L-1 headspace of spearmint oil achieved 50% reduction in 'Russet Burbank' sprout weight and sprout number respectively, 30 days after the initial treatment. Tubers stored in environments with 60-240 mg L-1 headspace of dill weed or spearmint oils suppressed sprouting at least 5 weeks longer than that of the controls. In 63-L steel drums, repeated dill and spearmint oil vapor treatments effectively and consistently suppressed sprouting of 'Russet Norkotah' and 'Piccolo' tubers for 7-8 months when doses were 25 mg L-1 headspace or higher and when treatments were repeated at least every four weeks. Within this range, sprout suppression was not sensitive to treatment variations, and, therefore, an optimal treatment level could not be determined. Clove oil was less effective in suppressing sprouting, likely due to its slower vaporization compared to dill and spearmint oils. Essential oil treatment effects on seed tuber viability were evaluated on 'Piccolo'. Tubers were planted after exposure to dill or spearmint oil vapor environments ranging from 15-240 mg L-1 headspace for seven days. There were no adverse effects on seed viability at doses less than 120 mg L-1 headspace. Although environments with <120 mg L-1 headspace had no significant adverse impact on sprouting, sprout emergence was delayed at higher doses. Dill weed and spearmint oils could potentially be used as potato sprout inhibitors but further studies are needed to demonstrate its commercial feasibility.

No description available.

There are several advantages to replacing CNC machinery with robotic machine tools and as such robotic machining is emerging into the manufacturing and metal cutting industry. There remain several disadvantages to using robots over CNC stations primarily due to flexibility in robotic manipulators, which severely reduces accuracy when operating under high machining forces. This flexibility is dependent on configuration and thus the configuration can be optimised through posture selection to minimise deflection. In previous work little has been done to account for operating frequency and the additional complications that can arise from frequency dependent responses of robotic machine tools. A Fanuc S-360 manipulator was used to experimentally investigate the benefits of including frequency compensation in posture selection. The robot dynamics first had to be identified and experimental modal analysis was selected due the inherent dependency on frequency characteristics. Specifically, a circle fit operation identified modal parameters and a least squares optimisation generated dynamic parameters for a spatial model. A rigid-link flexible-joint model was selected and a pseudo-joint was used to create an additional DOF to accommodate link flexibility. Posture optimisation was performed using a gradient-descent algorithm that used several starting points to identify a global minimum. The results showed that a subset of modal data that excluded the mode shape vectors could be used to create a model to predict the manipulator vibration response. It was also found that the receptance variation of the manipulator with configuration was insufficient to verify the optimisation throughout the entire selected workspace; however the model was shown to be useful in regions containing multiple peaks where the modelled dynamics were dominant over the highly volatile measured data. Simulations were performed on a redundant planar manipulator to overcome the lack of receptance variation found in the Fanuc manipulator. These simulations showed that there were two mechanisms driving the optimisation; overall amplitude reduction and frequency specific amplitude reduction. Using a stiffness posture measure for comparison, the results of the frequency specific reduction could be separated and were found to be particularly beneficial when operating close to resonant frequencies.

There are several advantages to replacing CNC machinery with robotic machine tools and as such robotic machining is emerging into the manufacturing and metal cutting industry. There remain several disadvantages to using robots over CNC stations primarily due to flexibility in robotic manipulators, which severely reduces accuracy when operating under high machining forces. This flexibility is dependent on configuration and thus the configuration can be optimised through posture selection to minimise deflection. In previous work little has been done to account for operating frequency and the additional complications that can arise from frequency dependent responses of robotic machine tools. A Fanuc S-360 manipulator was used to experimentally investigate the benefits of including frequency compensation in posture selection. The robot dynamics first had to be identified and experimental modal analysis was selected due the inherent dependency on frequency characteristics. Specifically, a circle fit operation identified modal parameters and a least squares optimisation generated dynamic parameters for a spatial model. A rigid-link flexible-joint model was selected and a pseudo-joint was used to create an additional DOF to accommodate link flexibility. Posture optimisation was performed using a gradient-descent algorithm that used several starting points to identify a global minimum. The results showed that a subset of modal data that excluded the mode shape vectors could be used to create a model to predict the manipulator vibration response. It was also found that the receptance variation of the manipulator with configuration was insufficient to verify the optimisation throughout the entire selected workspace; however the model was shown to be useful in regions containing multiple peaks where the modelled dynamics were dominant over the highly volatile measured data. Simulations were performed on a redundant planar manipulator to overcome the lack of receptance variation found in the Fanuc manipulator. These simulations showed that there were two mechanisms driving the optimisation; overall amplitude reduction and frequency specific amplitude reduction. Using a stiffness posture measure for comparison, the results of the frequency specific reduction could be separated and were found to be particularly beneficial when operating close to resonant frequencies.

Anxiety disorders are one of the most prevalent mental disorders in the world. While normal anxiety serves as an important protective mechanism, pathological anxiety characteristic of an anxiety disorder is both maladaptive and disruptive. The majority of studies have focused on the neurotransmitter systems associated with the actions of known anxiety drugs. This focus may likely limit the exploration of mechanisms underlying anxiety disorders. This project aims to examine changes in gene expression that may underlie higher or lower levels of inherent anxiety. Using a well-established behavior test for anxiety, the elevated plus maze, we identified male Wistar rats exhibiting inherently high- or low-anxiety levels. Brain regions known to mediate anxiety, the amygdala, hippocampus and nucleus accumbens, were dissected and total mRNA isolated. The mRNA was converted to cDNA via reverse transcription-polymerase chain reaction (RT-PCR). Then, the cDNA was used in suppression subtractive hybridization, a technique used to compare two complete populations of cDNAs and identify cDNAs that are upregulated in one population in relation to the other. In this project suppression subtractive hybridization was used to compare high- and low-anxiety cDNA populations. The upregulated cDNAs were amplified in a PCR reaction that enables rare transcripts to be identified. The PCR products from the suppression subtractive hybridization were cloned and used to create two cDNA libraries for high- and low-anxiety related genes. These clones were sequenced to show over 1000 genes upregulated in high- and low-anxiety. The gene list was then subjected to bioinformatic analysis to identify one candidate to be studied in further detail. <p>The prion protein was identified as a potential candidate. Examination of the literature sparked an interest in studying other prion-like proteins, more specifically the cytoplasmic polyadenylation element binding protein (CPEB). The CPEB protein is a potent regulator of mRNA translation in both mature oocytes and the adult brain. While unphosphorylated the CPEB protein keeps specific mRNAs dormant in the cytoplasm. In its phosphorylated form CPEB catalyzes polyadenylation of the mRNA, leading to protein synthesis. p*PCR was used to show the presence of CPEB mRNA transcripts in the rat hippocampus. CPEB protein expression was examined in the brain samples isolated from control, high- and low-anxiety rats. It was found that CPEB was significantly upregulated in high- and low-anxiety rats compared to control. The protein expression of an upstream kinase, Aurora A kinase, and a downstream target, Calcium/Calmodulin Dependent Kinase II (CaMKII), was also investigated. The results from Aurora A kinase were inconclusive. CaMKII, on the other hand, was significantly upregulated in high-anxiety over both control and low-anxiety. These results suggest that CPEB may catalyze increased translation of mRNAs in high-anxiety while acting as a repressor of those same mRNAs in low-anxiety. <p>Recent studies have suggested that CPEB protein plays an important role in synaptic plasticity. The regulation of synaptic plasticity, and its impact on learning and memory, is believed to be a key mechanism behind the maintenance of anxiety disorders. Therefore the results of this study suggest a new molecular mechanism in the development of anxiety disorders.

Sprout control is essential for successful management of stored potatoes. This study examined the effect of dill weed, spearmint and clove essential oils on sprouting of potatoes. Extracts of steam distilled dill weed whole plants containing 41.5-42.7% of S-(+)-carvone and spearmint foliage extracts containing 97.2-97.6% of R-(-)-carvone, were applied to tubers in a series of experiments using either 1-L glass jars or 63-L steel drums. The composition of the essential oils was consistent between years but evaporation rate varied among the oils as dill weed extract evaporated the fastest while clove oil evaporated the slowest under the same conditions. After exposure to essential oil treatments, tuber sprout number and weight were assessed and compared to untreated control and tubers treated with commercially marketed clove oil product (Biox-CTM, containing 78.5-82.3% eugenol). Applications of 32.5 and 47.6 mg L-1 headspace of dill weed oil and 21.5 and 22.3 mg L-1 headspace of spearmint oil achieved 50% reduction in 'Russet Burbank' sprout weight and sprout number respectively, 30 days after the initial treatment. Tubers stored in environments with 60-240 mg L-1 headspace of dill weed or spearmint oils suppressed sprouting at least 5 weeks longer than that of the controls. In 63-L steel drums, repeated dill and spearmint oil vapor treatments effectively and consistently suppressed sprouting of 'Russet Norkotah' and 'Piccolo' tubers for 7-8 months when doses were 25 mg L-1 headspace or higher and when treatments were repeated at least every four weeks. Within this range, sprout suppression was not sensitive to treatment variations, and, therefore, an optimal treatment level could not be determined. Clove oil was less effective in suppressing sprouting, likely due to its slower vaporization compared to dill and spearmint oils. Essential oil treatment effects on seed tuber viability were evaluated on 'Piccolo'. Tubers were planted after exposure to dill or spearmint oil vapor environments ranging from 15-240 mg L-1 headspace for seven days. There were no adverse effects on seed viability at doses less than 120 mg L-1 headspace. Although environments with <120 mg L-1 headspace had no significant adverse impact on sprouting, sprout emergence was delayed at higher doses. Dill weed and spearmint oils could potentially be used as potato sprout inhibitors but further studies are needed to demonstrate its commercial feasibility.