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The transcriptional apparatus of higher plant chloroplasts is well characterised and consists of a plastid-encoded polymerase (PEP) and a nuclear encoded polymerase (NEP). PEP is dispensable to cell viability. The situation in green algal species, however, is less clear. Chloroplast genes encoding subunits of the PEP have been cloned and sequenced in the green alga Chlamydomonas reinhardtii and preliminary reverse-genetic studies suggest that PEP is essential to cell viability, which is in contrast to the situation in higher plants. To investigate this further a series of gene knockouts were constructed using the chloroplast gene rpoC2, encoding the " subunit of PEP. Results indicate that PEP is essential to C. reinhardtii cell viability. In addition, inhibitors of PEP have been used in an in vivo transcription assay to try to identify a second RNA polymerase activity in C. reinhardtii chloroplasts. In all higher plant and red algal species so far studied the PEP factor is encoded in the nuclear genome. A C. reinhardtii nuclear gene (rpoD) encoding a putative PEP factor has been cloned and partially sequenced. This is the first factor cloned from a green algal species. A transcript of ~2.9 kb was detected for the rpoD gene by northern analysis. Finally, epitope tagging technology was developed for chloroplast and bacterial gene products. The rpoC2 gene of C. reinhardtii was modified to produce a 6x-histidine tagged polypeptide and an attempt was made to purify this polypeptide from C. reinhardtii cells using IMAC. In addition, a 3x haemagglutinin (HA) epitope tag was codon optimised for use in C. reinhardtii chloroplasts and this epitope was used to tag -galactosidase in E. coli. The protein was detected in a western blot using anti-HA monoclonal antibodies. This epitope will prove useful as a tool to tag C. reinhardtii chloroplast proteins.

Bronchiectasis is a chronic and progressive lung disease associated with cough, sputum, and respiratory infection. The increasing burden on healthcare systems has led to renewed interest in clinical trials and developing therapies for this patient population. Airway clearance techniques (ACTs) are recommended as an integral part of management to facilitate secretion removal, improve symptoms and health-related quality of life (HRQOL). Guidelines recommend that ACTs should be personalised to improve outcomes. Our understanding about ACTs in bronchiectasis however remains limited. Currently there are no definitive guidelines on the optimal prescription of ACTs. Personalised prescription appears to be dependent upon clinical expertise, local training, and workplace culture. Significant knowledge gaps exist in literature regarding optimisation of ACTs for individuals with bronchiectasis. The overarching aim of this thesis was to enhance the personalised prescription of ACTs in bronchiectasis. Four studies were completed. The first study commenced at the airway clearance device itself and the properties of different commonly prescribed devices. The second study summarised outcome measures used in ACT studies and the final two studies examined separately the patient and physiotherapist perspectives of ACTs. Positive expiratory pressure (PEP) devices are a type of ACT device widely used in clinical settings. The comparative performance characteristics of these devices remain unknown. Study One (Chapter Three) was an experimental bench study which compared the performance characteristics (mean PEP, peak PEP, amplitude PEP and oscillation frequency) of six different PEP devices by varying resistance and flow. Results showed the performance characteristics of the devices differed across flows and resistance settings. Some commonly used devices (e.g. Acapella Choice, Acapella DH, Aerobika and Pari PEP S) were flow-dependent, whilst the performance characteristics of other devices (e.g. Acapella DM and Flutter) were independent of flow. Varying flow or resistance typically maintained or increased the production of mean, peak, and amplitude PEP and oscillation frequency. The clinical implications of the small but significant differences in performance characteristics of these PEP devices requires further investigation. Whilst ACTs are recommended for individuals with bronchiectasis, many trials have demonstrated inconsistent benefits or failed to reach their primary outcome. The narrative review of Study Two (Chapter Four) documented the most common clinical and patient-related outcome measures used to evaluate the efficacy of ACTs in bronchiectasis. The review included 27 published studies and 1 abstract. The most common clinical outcome measures were sputum volume (n = 23), lung function (n = 17) and pulse oximetry (n = 9). The most common patient-related outcomes were HRQOL (St George Respiratory Questionnaire, n = 4), cough-related quality of life (QOL) (Leicester Cough Questionnaire, n = 4) and dyspnoea (Borg / modified Borg scale, n = 8). This review highlighted the large heterogeneity in outcome measures used in ACT trials to date and recommended guidance on standardising the most important clinical and patient-related outcome measures for this patient population. With standardised outcome measures in place, the efficacy of different interventions can be compared leading to improved personalisation of ACTs in bronchiectasis. Even though ACTs are recommended in bronchiectasis, data suggests the use of and adherence to ACTs is poor. Study Three (Chapter Five) identified patient perceptions regarding ACTs, the barriers and facilitators of ACTs, and factors affecting adherence. A multi-centre qualitative study using in-depth semi-structured interviews of 24 individuals with bronchiectasis was undertaken. The main facilitators of using ACTs included a perceived health and QOL benefit, a tailored approach to ACTs and the use of self-management strategies. Main barriers to ACTs included lack of time and motivation, a lack of access to resources and, surprisingly, a lack of perceived health benefit. Several suggestions were also made that may help promote adherence including combining and trialling different ACTs, receiving regular ACT reviews and education from physiotherapists, and having good social support. Taking another perspective, Study Four (Chapter Six) examined physiotherapist perceptions of ACTs in both inpatients and outpatients with bronchiectasis. This was a single-centre qualitative study that used in-depth semi-structured interviews with eleven physiotherapists who treated individuals with bronchiectasis. The study revealed that physiotherapists regularly and routinely prescribe ACTs for inpatients and outpatients with bronchiectasis, however the clinical decision-making regarding optimal ACT prescription was regarded as complex. Main themes influencing ACT prescription included organisational factors (workload, scope of service, access to resources / ACTs, financial burden of ACT); patient-related factors (symptom severity, finances, disease-specific knowledge, social commitments, timing of ACT, clinical setting, perceived benefit, and motivation); and physiotherapist / profession-related factors (clinical experience, access to professional support and education, awareness of evidence of ACTs and evaluating ACT effectiveness). In summary, the work presented in this thesis serves to refine the prescription of ACTs for bronchiectasis. There are different ACT devices prescribed by clinicians that have different performance characteristics. Due consideration of these performance characteristics needs to be given when prescribing ACTs. The marked heterogeneity of study designs and outcome measures used to evaluate ACTs in bronchiectasis has made the interpretation of different ACT intervention studies difficult. There is a need for standardisation of outcome measures for ACTs that will enable future intervention studies to be more comparable. Individuals with bronchiectasis and physiotherapists prescribing ACTs describe multiple factors that may impact upon adherence to ACTs. For patients, these barriers and facilitators centre on improving HRQOL. For the prescribing physiotherapist, airway clearance centres on the complexity of the patient and intervention. The results from the four studies in this doctoral program should be considered by physiotherapists to assist the personalised prescription of ACTs with the ultimate outcome of improving adherence and HRQOL for individuals with bronchiectasis.

Mindfulness includes acceptance and awareness subcomponents, and emerging theories imply that cultivating both acceptance and awareness may benefit health by diminishing stress reactivity. Yet, no prior work has manipulated awareness and acceptance simultaneously to begin to test this claim. To address this knowledge gap, 202 participants were enrolled in a 2 x 2 between-subjects experimental design manipulating both awareness (enhanced awareness v. no enhanced awareness) and acceptance (acceptance training v. no acceptance training) of physiological responses to a cold pressor test. Cardiovascular indices were recorded throughout. The awareness manipulation and acceptance training did not independently influence cardiovascular responses, but the combination of enhanced awareness and no acceptance training condition led to higher total peripheral resistance, higher respiratory sinus arrhythmia, and lower cardiac output reactivity than the “enhanced awareness + acceptance training” and the “no enhanced awareness + no acceptance training” conditions. Blood pressure and pre-ejection period were unaffected by the acceptance training or the enhanced awareness manipulation. These results add to a growing body of work suggesting that mindful awareness and acceptance subcomponents interact to influence stress reactivity.

This data-driven journalism project tackles an age-old problem that seems common in virtually every government around the world: governmental officials hiding assets. By applying the theory of social responsibility, this paper uses data-journalism methods to trace the full financial assets of Armenian members of parliament and eventually making those assets and income declarations available via an interactive website to the Armenian public as well as other journalists throughout the country and region.

Plastid genomes of angiosperms are highly conserved in both genome organization and nucleotide substitution rates. Geraniaceae have highly rearranged genomes and elevated nucleotide substitution rates, which provides an attractive system to study nuclear-plastid genome coevolution. My dissertation research has focused on two areas of nuclear-plastid genome coevolution in Geraniaceae. First, I have investigated the correlation of nucleotide substitution rates between nuclear and plastid genes that encode interacting subunits that form the multi-subunit complex of Plastid Encoded RNA Polymerase (PEP). Second, the hypothesis that the unusual changes of plastid genome organization and elevated nucleotide substitution rates of plastid encoded genes is the result of alterations in nuclear encoded DNA replication, recombination and repair (DNA RRR) genes is tested. The second chapter investigates the optimal methods for transcriptome sequencing/assembly. My findings supported the use of transcriptome assemblers optimized for Illumina sequencing platform (Trinity and SOAPdenovo-trans). The third chapter investigated coevolution of nucleotide substitution rates between plastid encoded RNAP (rpoA, rpoB, rpoC1, rpoC2) and nuclear encoded SIG (sig1-6) genes that are part of the multi-subunit complex PEP. Using the transcriptomes of 27 Geraniales species I extracted the PEP genes and performed a systematic correlation test. I detected strong correlations of dN (nonsynonymous substitutions) but not dS (synonymous substitutions) between RNAP and SIG but no correlations were detected for the control genes, which provides a plausible explanation for the cause of plastome-genome incompatibility in Geraniaceae. The fourth chapter investigated the effect of DNA RRR system on the aberrant evolutionary phenomena in Geraniaceae plastid genomes. I extracted DNA RRR and nuclear control genes with different subcellular locations from 27 Geraniales transcriptomes and estimated genome complexity with various measures from plastid genomes of the same species. I detected significant correlations for dN but not dS for three DNA RRR genes, 10 nuclear encoded plastid targeted (NUCP) and three nuclear encoded mitochondrial targeted (NUMT) genes. The findings of a correlation between dN of DNA RRR genes and genome complexity support the hypothesis that changes of plastid genome complexity in Geraniaceae may be caused by dysfunction of DNA RRR systems.

Statement of the Problem: Men who have sex with men (MSM) make up only 2% of the population in the United States, but represent 60% of all HIV infections each year. Pre-exposure prophylaxis (PrEP), a once daily medication to prevent HIV infection, has emerged as a prevention tool for populations most heavily affected by HIV. However, the criteria for PrEP have only been broadly defined and lead many individuals and their medical providers to question if PrEP use is appropriate, given individual risk profile and potential drug side effects. The primary goal of this dissertation is to use risk assessment and HIV testing data to create an HIV risk score for HIV-negative MSM. The secondary goal is to use this HIV risk score to create more targeted criteria for PrEP use and subsequently inform individualized PrEP candidacy. Methods: Behavioral risk assessment and HIV testing data were collected at the Los Angeles LGBT Center from January 2009 to June 2014. Individuals were included in the analysis if they were MSM, had an HIV-negative test result at baseline, and returned for at least one follow-up HIV testing visit (n = 9,981). Bivariate and Multivariate Cox Proportional Hazards Models were used to determine the biological, behavioral, and substance use variables significant in HIV contraction over the 5.5 year follow-up period. Summary of Findings: Self-reported history of gonorrhea (p = 0.03), chlamydia (p < 0.0001), and syphilis (p = 0.01); having receptive anal sex (p < 0.0001), race/ethnicity of partners (p = 0.005), and number of sexual partners in the last three months (p = 0.0003); methamphetamine (p = 0.0008), and nitrate use (p = 0.002) were all significant predictors of HIV infection during follow-up. Age of partners, intimate partner violence, and ecstasy use were not significant. Following risk score creation, approximately 45% of all individuals were above a chosen risk score, which consisted of 76% of all HIV-positives. The use of this targeted strategy is beneficial in that it more accurately outlines PrEP candidacy criteria, subsequently allowing individuals and their medical providers to make a more informed decision before use.

Cooperative relaying is a promising technique to enhance the reliability and data-rate of wireless networks. Among different cooperative relaying schemes, the half-duplex non-orthogonal amplify-and-forward (NAF) protocol is popular due to its low implementation complexity and performance advantages. This thesis investigates precoder design for a cooperative half-duplex single-relay NAF system from an information theoretic point of view using a novel mutual information-based design criterion.The first part of the thesis considers the design of a 2x2 precoder for the NAF half duplex single relay network in the presence of the direct link using mutual information (MI) as the main performance metric. Different from precoder design methods using pairwise error probability (PEP) analysis, which are valid only at high signal-to-noise ratios (SNR), the proposed precoder design can apply to any SNR region, which is of more interest from both information-theoretic and practical points of view. A MI-based criterion is developed for a cooperative frame length of 2, which corresponds to the case of using a 2x2 precoder. The design criterion is established in a closed-form, which can be helpful in finding an optimal precoder. Then it is analytically shown that a good precoder should have all entries that are equal in magnitude, which is different from the optimal precoders obtained thus far using the conventional PEP criterion. Simulation results indicate that the proposed class of precoders outperform the existing precoders in terms of the mutual information performance.The second part of the thesis extends the precoder design to an arbitrary block length of 2T. In general, for this case, a precoder of size 2Tx2T needs to be considered to optimize the MI performance. Similar to the 2 × 2 precoder design, a MI-based design criterion is first established. While the criterion can be expressed in closed form, the design of optimal precoders in this case is not possible, due to the complexity of the optimization problem. As an alternative, a novel grouping technique, which is referred to as symbol grouping, in which a group of only P = 2 information symbols are pre-coded to improve the MI, is proposed. It is then demonstrated that the grouping technique yields a much simpler design criterion and an optimal 2x2 precoder can be developed. Numerical results show that the proposed precoding technique outperforms existing precoder designs while keeping the receiver complexity at a minimum.

The enzymes Escherichia coli DAH7P synthase (phe) and E. coli KDO8P synthase catalyze an unusual condensation between phosphoenolpyruvate (PEP) and a phosphorylated monosaccharide, D-erythrose 4-phosphate (E4P) and D-arabinose 5-phosphate (ASP), respectively. Both enzymatic reactions occur via a nucleophilic attack at the re-face of the carbonyl of the phosphorylated monosaccharide by the si-face of C3PEP even though sequence homology studies indicate sparse sequence homology between both enzymes (18% sequence identity). The catalytic mechanisms and the active site topologies of E. coli DAH7P synthase (phe) and E. coli KDO8P synthase were investigated using site-directed mutagenesis, chemical modification studies, and substrate analogues. Both E. coli KDO8P synthase and E. coli DAH7P synthase (phe) may share a common active site topology as demonstrated utilizing analogues of the respective phosphorylated monosaccharide substrate. The stereochemistry at C2 of the phosphorylated monosaccharide influenced the Km (binding affinity) for both E. coli DAH7P synthase and E. coli KDO8P synthase. Sequence alignment studies between recombinant metallo-KDO8P synthases and a DAH7P synthase cloned from Thermotoga maritima indicate that both enzymes may share a common metal binding motif. This finding provides additional evidence supporting a common active site topology may exist between DAH7P synthases and KDO8P synthases. Both KDO8P synthase and DAH7P synthase (phe) may share a similar mechanism. Site-directed mutagenesis and chemical modification studies of DAH7P synthase (phe) demonstrate that DAH7P synthase (phe) has one essential histidine residue, H268, which is homologous to the residue, H202, in E. coli KDO8P synthase. These two histidine residues occupy analogous positions in the active sites of both enzymes implying that both residues may perform the same role. In addition, studies with substrate analogues of either ASP or E4P which are one-carbon shorter, either E4P for KDO8P synthase or D,L-glyceraldehyde 3-phosphate for DAH7P synthase, demonstrate that both enzymatic reactions may be initiated by a nucleophilic attack of water at C2PEP.

A search was made in e$\sp+$e$\sp-$ annihilations at $\sqrt s$ = 29 GeV with the High Resolution Spectrometer at PEP, for events with an isolated pair of charged tracks e$\sp{\pm}$X$\sp{\mp}$ where X is a muon or meson. Such pairs are a possible signature for the decay of massive neutrinos. The model investigated produces these neutrinos through the virtual neutral weak vector current Z$\sp\circ$ and allows them to decay via the virtual charged weak vector boson W$\sp{\pm}$. The neutrinos were assumed to mix with one of the known neutrinos (electron, muon, or tau), and each case was considered separately. For a given mass and mixing parameter $\vert$U$\vert\sp2$ the production cross section and decay branching ratios are completely determined by the Standard Model. Only one event was found, for an integrated luminosity of 300 pb$\sp{-1}$, satisfying certain selection criteria and with a mass M$\sb{\rm eX}$ $>$ 1.8 GeV/c$\sp2$. Detection efficiencies for events containing at least one e$\sp{\pm}$X$\sp{\mp}$ pair were calculated using Monte Carlo techniques. Limits were first set on the production cross section versus the mass of the neutrino in the limit where the mixing parameter $\vert$U$\vert\sp2$ = 1. Limits were then set on $\vert$U$\vert\sp2$ versus mass of the neutrino for the three cases of mixing. This result places strong constraints on the existence of new neutrinos with masses from 1.8 to 6.7 GeV/c$\sp2$ and with mixing parameters to the known neutrinos in the range 3 $\times$ 10$\sp{-6}$ $