Dissertations / Theses on the topic 'Radio Stalin'

The Visual Staging of Audio Plays explores the directing practice of radio dramas that are staged for viewing purposes rather than their typical solo-auditory purposes. The thesis is comprised of three separate parts: a brief history of theatrical sound, an introduction to radio drama theory and practice, and application. The application portion is a detailed first-person account of my personal experience staging It’s a Wonderful Life: A Live Radio Play by Joe Landry for TheatreVCU’s Mainstage winter special event in 2015. It is also in this section where I integrate history, theory, and practice to formulate technique for directing the genre for stage.

Software radio is a radio that is substantially defined in software and whose physical layer behavior can be significantly altered through changes to its software. As a primary goal of software radio is the ability to support existing and future wireless protocols, software radio necessitates the use of a rapidly reprogrammable baseband processing solution. However third generation wireless protocols represent a significant increase in complexity over second generation protocols. Due to the natural performance sacrifices that must be made when moving an application from an Application Specific Integrated Circuit (ASIC) to a general purpose processor or a digital signal processor, it is feared that reprogrammable processing solutions may not suffice for the emerging wireless protocols, which would significantly hinder the realization of software radio, particularly in the handheld domain where power consumption and chip area are critical. Recently, the Configurable Computing Lab at Virginia Tech developed a new breed of reprogrammable processor which they called â custom computing machinesâ (CCM). Representing a dramatic departure from traditional architectures used for baseband processing solutions, CCMs utilize a large number of optimized and programmable processing cores connected through a programmable mesh. Due to this architectural approach, CCMs have been promoted as supplying a level of processing power and power efficiency similar to ASICs while providing a level of reconfigurability similar to that of a DSP. Subsequently, Dr. Srikathyayani Srikanteswara proposed a new software radio architecture, known as the Layered Radio Architecture, which is intended to facilitate the inclusion of CCMs into a software radio. The primary goal of the research presented in this thesis is to demonstrate how a particular CCM, Stallion, can be used within the Layered Radio Architecture to provide sufficient processing performance, power efficiency, and reconfigurability to meet the constraints of the handheld domain through implementations of a single user adaptive receiver with adaptive complex filtering and a W-CDMA downlink rake receiver. These metrics are measured from a detailed simulation of Stallion and the Configuration Layer of the Layered Radio Architecture using advanced object oriented programming techniques that facilitate the inclusion of statistics gathering routines into normal operation. To provide perspective, these statistics are compared to the performance that could be expected from an implementation on a top-of-the-line DSP.
Master of Science

The membrane proteins signal peptide peptidase, signal peptide peptidase like and presenilin are intramembrane aspartyl proteases located in the endoplasmic reticulum, plasma membrane and organelle. These membrane proteins are able to catalyze a hydrolytic reaction in a hydrophobic space. The downstream consequences of these reactions impact a variety of cellular functions such as cytokine production, inflammatory responses, embryogenesis, and immune system regulation. Additionally, the aspartyl proteases such as signal peptide peptidase and presenilin, a part of the γ-secretase complex, hydrolyze peptides leading to pathogen maturation and Alzheimer’s disease, respectively. Electron crystallography offers the unique aspect of studying membrane proteins in a near native state. Determining the structures of Haloarcula morismortui and Methanoculleus marisnigri JR1 signal peptide peptidases by electron crystallography may provide insight into how a hydrolysis reaction occurs in a hydrophobic environment and how the protein determines which transmembrane signal peptides to cleave. Additionally, structure determination may help answer questions regarding why human presenilin, part of the γ-secretase complex, incorrectly processes amyloid precursor protein into amyloid-beta peptides leading to Alzheimer’s disease. Such structural data may not only shed light on how amyloid precursor protein is processed but how other proteins are processed by signal peptide peptidase leading to immune responses, cell signaling, and pathogen maturation. In addition, structure-function data may have an impact on pharmaceutical drug designs that targets signal peptide peptidase, signal peptide peptidase like, and/or presenilin. To determine the structure of aspartyl proteases, two archaeal signal peptide peptidases were used for two-dimensional crystallization trials to be able to study their structure by electron crystallography. Haloarcula morismortui and Methanoculleus marisnigri JR1 signal peptide peptidases, both human signal peptide peptidase homologues, were recombinantly over-expressed and purified. During dialysis trials, various lipid-to-protein ratios, sodium chloride concentrations, temperatures, detergents and a variety of other variables were tested. Methanoculleus marisnigri JR1 signal peptide peptidase showed the most promising results in terms of crystallinity. Optimizing dialysis conditions, specifically narrowing the lipid to protein ratio, resulted in two-dimensional crystals. Ordered arrays measuring up to 200 nm x 200 nm were observed. These ordered arrays have been shown to be reproducible amongst multiple batches of purified Methanoculleus marisnigri JR1 signal peptide peptidase. Preliminary projection maps of negatively stained ordered arrays show unit cell dimensions of a = 178 Å, b = 160 Å, γ = 92.0 Å and a = 175 Å, b = 167 Å, γ = 92.0 Å. The monomer measurements are approximately 70 Å by 80 Å. This is the first time a signal peptide peptidase homologue has been crystallized by two-dimensional crystallization.

Tese de Doutoramento em Ciências Veterinárias especialidade de Clínica
Recurrent airway obstruction (RAO) is one of the most frequent lung diseases in horses and is similar to human asthma. We characterized equine RAO differential diagnosis (DD) in horses with long term cough and established a parallel between the DD in equine RAO and in human asthma. We correlated clinical, endoscopic, thoracic X-ray and bronchoalveolar lavage fluid scores in horses with RAO to establish relevance of each factor for the characterization of RAO stages in order to suggest a staging method. Cardiovascular effects of RAO were assessed. We also evaluated the response to skin prick tests (SPT) and in vitro allergy tests with common aeroallergens in horses with RAO and characterized RAO in Portugal by identifying relevant allergic factors. The importance of a thorough diagnosis is emphasized, including BAL and respiratory endoscopy, and a DD parallel is made with vocal cord dysfunction in man. A score model for the characterization of RAO stages is suggested. The first ECG and EcoCG values for Lusitano/Lusitano-cross horses are published with subtle changes in the RAO group. In this highly selected population immediate aeroallergen hypersensitivity was significant, allergy being a probable aetiopathogenic mechanism in all RAO group horses.
RESUMO - A Obstrução recorrente das Vias Aéreas (ORVA) é uma das patologias pulmonares mais frequentes no cavalo, semelhante à asma humana. Caracterizámos o diagnóstico diferencial (DD) de ORVA equina em cavalos com tosse de longa duração e avaliamos o paralelismo entre o DD na ORVA equina e na asma humana. Correlacionámos “scores” clínicos, endoscópicos, radiológicos do toráx e da lavagem bronco-alveolar em cavalos com ORVA, para estabelecer a relevância de cada factor na caracterização de estádios de ORVA de forma a sugerir um método de estadiamento. Foram estudados efeitos cardiovasculares da ORVA. Avaliamos a resposta aos testes cutâneos por picada e a testes de alergia in vitro com aeroalergenos comuns em cavalos com ORVA e caracterizamos a ORVA em Portugal. É dada ênfase à importância de efectuar um diagnóstico preciso, envolvendo lavagem bronco-alveolar e endoscopia de aparelho respiratório, e é estabelecido um paralelo do DD com a disfunção das cordas vocais no Homem. É sugerido um modelo de estadiamento da ORVA. São publicados os primeiros valores de ECG e ecocardiograma para cavalos Lusitanos/cruzados de Lusitano, com variações subtis no grupo ORVA. Nesta população altamente seleccionada a hipersensibilidade imediata a aeroalergenos revelou-se significativa, sendo a alergia um mecanismo etiopatogenico provável em todos os cavalos do grupo ORVA.
CIISA (Centro de Investigação Interdisciplinar em Sanidade Animal)

The fundamental business model of banks is based on receiving short-term deposits and giving long-term loans which means that active banks are naturally subject to liquidity risk. During the last financial crisis poor liquidity risk management was seen as one of the main causes which has led to an increased focus on the management of liquidity risk and the introduction of the first minimum requirements for liquidity in banks, through Basel III. As the topic of internal pricing in banks and the effects of the introduction of the liquidity coverage ratio (LCR) and the net stable funding ratio (NSFR) is not extensively covered by existing research, the aim of this thesis is to identify and discuss internal pricing and liquidity cost allocation mechanisms used in practice by Swedish banks. The study also aims to investigate the impact of changes in liquidity requirements on internal pricing and liquidity cost-benefit allocation mechanisms in a Swedish setting. The key findings are that firstly, there are large variations regarding the sophistication of banks funds transfer pricing practices and liquidity cost allocation methods. The banks using less sophisticated methods may be exposed to model risk if they themselves are not aware of the implications of this. Two consequences of using simplified approaches may be distorted assessment of profitability and unwanted maturity transformation. Secondly, the findings indicate that the link between risk management and internal pricing in the banks is rather weak. Lastly, the introduction of LCR and NSFR have had a significant impact on the bank's risk management but the effect on internal pricing practices and methods for allocating liquidity costs is very limited.
Den fundamentala affärsmodellen för en bank baseras på mottagandet av kortfristig inlåning och utgivandet av långfristiga lån vilket innebär att banker är utsatta för likviditetsrisk. I samband med den senaste finansiella krisen sågs undermålig hantering av likviditetsrisk som en av de centrala orsakerna vilket har lett till ett ökat fokus på likviditetsriskhantering samt införandet av det första minimikraven gällande likviditet på banker genom Basel III. Då internprissättning hos banker samt effekten av att likviditetstäckningsgrad (LCR) och stabil nettofinansieringsgrad (NSFR) införts är ämnen som inte i någon större utsträckning täckts av tidigare forskning är syftet med rapporten att identifiera och diskutera internprissättning och metoder för allokering av likviditetskostnader. Vidare är syftet även att undersöka effekten av ändringar och kommande ändringar av regelverk på internprissättning och allokering av likviditetskostnader hos Svenska banker. De mest centrala slutsatserna är för det första, att det finns stora skillnader i hur avancerade bankernas metoder gällande internprissättning och allokering av likviditetskostnader är. Bankerna som använder mindre avancerade metoder kan vara exponerade mot en modell-risk om de inte är medvetna om följderna av att använda en förenklad modell. Två konsekvenser av att använda en förenklade modeller är att bedömningen av lönsamhet kan bli snedvriden samt att det kan uppmuntra till oönskad löptidstransformering. För det andra indikerar resultatet på att kopplingen mellan bankernas riskhantering och internprissättning är relativt svag. Slutligen så indikerar studien att LCR och NSFR har haft en signifikant effekt på bankernas riskhantering men att effekten på internprissättning och allokering av likviditetskostnader är mycket begränsad.

The fundamental business model of banks is based on receiving short-term deposits and giving long-term loans which means that active banks are naturally subject to liquidity risk. During the last financial crisis poor liquidity risk management was seen as one of the main causes which has led to an increased focus on the management of liquidity risk and the introduction of the first minimum requirements for liquidity in banks, through Basel III. As the topic of internal pricing in banks and the effects of the introduction of the liquidity coverage ratio (LCR) and the net stablefunding ratio (NSFR) is not extensively covered by existing research, the aim of this thesis is to identify and discuss internal pricing and liquidity cost allocation mechanisms used in practice bySwedish banks. The study also aims to investigate the impact of changes in liquidity requirements on internal pricing and liquidity cost-benefit allocation mechanisms in a Swedish setting. The key findings are that firstly, there are large variations regarding the sophistication of banks funds transfer pricing practices and liquidity cost allocation methods. The banks using less sophisticated methods may be exposed to model risk if they themselves are not aware of the implications of this. Two consequences of using simplified approaches may be distorted assessment of profitability and unwanted maturity transformation. Secondly, the findings indicate that the link between risk management and internal pricing in the banks is rather weak. Lastly, the introduction of LCR and NSFR have had a significant impact on the bank's risk management but the effect on internal pricing practices and methods for allocating liquidity costs is very limited.
Den fundamentala affärsmodellen för en bank baseras på mottagandet av kortfristig inlåning och utgivandet av långfristiga lån vilket innebär att banker är utsatta för likviditetsrisk. I sambandmed den senaste finansiella krisen sågs undermålig hantering av likviditetsrisk som en av de centrala orsakerna vilket har lett till ett ökat fokus på likviditetsriskhantering samt införandet av det första minimikraven gällande likviditet på banker genom Basel III. Då internprissättning hos banker samt effekten av att likviditetstäckningsgrad (LCR) och stabil nettofinansieringsgrad(NSFR) införts är ämnen som inte i någon större utsträckning täckts av tidigare forskning är syftet med rapporten att identifiera och diskutera internprissättning och metoder för allokering av likviditetskostnader. Vidare är syftet även att undersöka effekten av ändringar och kommande ändringar av regelverk på internprissättning och allokering av likviditetskostnader hos Svenska banker. De mest centrala slutsatserna är för det första, att det finns stora skillnader i hur avancerade bankernas metoder gällande internprissättning och allokering av likviditetskostnader är. Bankerna som använder mindre avancerade metoder kan vara exponerade mot en modell-risk om de inte är medvetna om följderna av att använda en förenklad modell. Två konsekvenser av att använda en förenklade modeller är att bedömningen av lönsamhet kan bli snedvriden sam tatt det kan uppmuntra till oönskad löptidstransformering. För det andra indikerar resultatet på att kopplingen mellan bankernas riskhantering och internprissättning är relativt svag. Slutligen så indikerar studien att LCR och NSFR har haft en signifikant effekt på bankernas riskhantering men att effekten på internprissättning och allokering av likviditetskostnader är mycket begränsad.

Diploma thesis "Radio Stalin as an example of Czech piracy radio broadcast" is aimed to complexly present radio station Radio Stalin which was broadcasting in October 1990 in Prague. Radio Stalin is presented in the context of events of that time and related changes in politics, economy, society and media and in the context of piracy radio broadcast. First of all the emphasis is put on media transformation and changes in legislative framework of radio broadcasting after 1989. Radio Stalin is described through the method of oral history when interviews were held with its several cofounders. This method was chosen because of absence or unavailability of other sources. However, press is used partly. In this thesis is created overall picture of Radio Stalin. It is possible to imagine the situation and conditions in which was this radio created, the period before the start of broadcasting, the broadcasting process, the end of its broadcasting and the continuation of it. The benefit of thesis is the reminder of this often neglected radio which has its own place in the history of Czech radio broadcasting. Radio Stalin is the first private radio station in Czechoslovakia which story is an interplay of many happy coincidences. This radio goal was to fill the missing gap and it enriched in long-term the...

Statins are commonly used for treating the elevation of lipids in the blood stream, also known as hyperlipidemia. Statins are considered to be an economical and effective way to achieve desirable long-term health outcomes for hyperlipdemic patients, however, ensuring adequate adherence to statin medications is often difficult as hyperlipidemia is an asymptomatic condition and patients sometimes fail to recognize the importance of being adherent to their statin medications. The purpose of this study was to evaluate impact of enrollment under Medicaid and Medicare Part D and patient out-of-pocket costs on patient statin adherence, persistence, and mean number of gap days per claim. A retrospective claims database was used in this study to conduct repeated measures analyses on statin prescription claims from independent community pharmacies in Texas. The pre-period in this study extended from January 1, 2005 to September 30, 2005 (Medicaid period) and the post-period extended from January 1, 2006 to September 30, 2006 (Medicare period). The study population consisted of dual-eligible beneficiaries in Texas who had at least two stain claims in the pre and post-periods each. The final study population comprised of 1734 Texas dual-eligible beneficiaries with 6064 statin claims during the pre-period and 7956 claims during the post-period. Patients had an average of 3.49 statin claims during the pre-period and 4.58 statin claims during the post-period. Patients were dispensed an average of 57.34 days of drug supply per claim during the pre-period and 42.02 days of drug supply per claim during the post-period. The results from this study showed that out-of-pocket costs for patients increased from $0.39 per claim under Medicaid to $13.36 per claim under Medicare Part D. Patient adherence to statins was assessed by calculating medication possession ratio (MPR). The results showed that mean patient MPR increased from 75.71 percent under Medicaid to 79.37 percent under Medicare. Results from generalized estimating equations showed that odds of being adherent (i.e., MPR ≥ 80 percent) to statins increased by 36 percent when patients were covered under Medicare Part D. Linear mixed model analysis showed that MPR increased by 3.66 percent when patients were covered under Medicare Part D compared to Medicaid. Also, patient MPR was found to increase by 0.13 percent when patient out-of-pocket payment increased by $1.00. Patient persistence was calculated by measuring gaps in therapy and patients with a gap of 60 or more days were considered to have discontinued therapy. Patients were found to be persistent to their drug therapy for an average of 151.76 days under Medicaid and 159.75 days under Medicare. Linear mixed model analysis showed that patient persistence increased by 7.99 days when patients were enrolled under Medicare Part D compared to Medicaid. Days of persistence was also found to increase by 0.41 days when patient out-of-pocket costs increased by $1.00. Mean number of gap days per claim during the Medicaid period was 11.91 days and decreased to 8.38 days during the Medicare period. Linear mixed model analysis showed that mean number of gap days per claim decreased by 3.52 days when patients were enrolled under Medicare Part D compared to Medicaid. Mean number of gap days in therapy were found to decrease by 0.10 days when patient out-of-pocket costs increased by $1.00. The results of this study showed that implementation of Medicare Part D resulted in an increase in MPR and persistence and a decrease in mean number of gap days per claim for Texas dual-eligible beneficiaries. The results also suggest that increased out-of-pocket costs under Medicare Part D may not have had a negative impact on statin drug utilization by dual-eligible beneficiaries in Texas.
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碩士
長庚大學
醫務管理學系
103
Survivors of stroke is at risk of a recurrent stroke, which is often more severe and disabling than the index event. Diabetes is one of the risk factors for secondary stroke. According to the clinical guidelines, high-intensity statin medication should be added to therapy for diabetic patients with overt ischemic stroke. However, persistence in using statins was decreased that may lead to secondary stroke or subsequent resource utilization. Therefore, it is important to investigate the association of statin medication possession ratio (MPR) with secondary stroke incidence for diabetes patients. This study employs National Health Insurance claim database. The subjective is first-ever ischemic stroke with history of diabetes and used statins in 90 days after discharge from 2001 to 2011, screening out 31,831 patients. The association between of statin MPR and secondary stroke incidence or subsequent resource utilization were analyzed by T-test, ANOVA, Chi-square test, Cox Proportional Hazard Regression, Generalized linear model. The average age of the patients was 65 years old and statin MPR was 0.74, and secondary stroke admission incidence was 10.96%. It found that MPR increases 1% then secondary stroke admission incidence was 2.474 times. The patients had hypertension then it was 1.215. The average expense of readmission was NT 61,795. When the patients had more history of diseases, chronic kidney disease then their expense of secondary stroke was higher. The average of emergency room visits was 2.42, and expense of emergency room was NT 9,036. It found that the patients had higher MPR, history of disease (hypertension, heart disease and chronic kidney disease), higher SSI then their emergency room visits and expense was higher. It showed a positive correlation between MPR and secondary stroke admission incidence, emergency room visit, expense of emergency room. However, it didn’t have the correlation with expense of readmission. Furthermore, higher MPR group that DDDs was lower and SSI was higher. In other words, the patient who has higher MPR implies they take lower intensity of statin, and they have higher severity of disease. Therefore, it is better to follow closely for them.

碩士
國立臺灣大學
獸醫學研究所
91
Ocadia sinensis is a native species of turtles in Taiwan. But the clinicians often have blind spots in therapy for lacking of scientific researches especially in basic hematological information. For this purpose, the study attempt to establish the data of hematology of Ocadia sinensis for clinicians and conservationists in clinical treatments and turtle conservation. There are three parts of this study: health body index, hematologic and biochemical reference values, and the morphology and cytochemical characteristics of blood cells establishments. Three methods were used to establish the health body index: Jackson’s ratio, the equation of logarithm body weight regressed against of logarithm carapace length and condition index (CI). One hundred and eleven Ocadia sinensis are used in this experiment. We found that the adult male turtles have longer and narrower body conformation than adult female and young turtles from the equation and CI. Therefore, we have to study the index of adult male turtles separately. In hematological referene values establishment, fifty nine adult captive Ocadia sinensis included twenty nine males and thirty females were use to the experiment. Blood samples were obtained from jagular vein in November, January, April, and June. Data were analyzed using repeated measures analysis of variance for significant (P<0.05) variation of the basis of sex, seasons and interaction between sex and seasons. Central 95% percentiles were used as reference interval. Significant sex differences were observed for packed cell volume, eosinophils, the ratio of heterophils and monocytes, total protein, albumin, uric acid, aspartate aminotransferase, alanine aminotransferase, triglycerides, cholesterol, and alkaline phosphatase. Marked seasonal variation was noted in most parameters except mean cell hemoglobin, the ratio of monocytes, the number of heterophils, and creatinine. The differences between sex and seasons were primary associated with reproductive cycle and hibernation. In the part of the morphology and cytochemical characteristics, heterophils have strong positive reaction and eosinophils have moderate positive reaction in PER stain. Thrombocytes have positive reaction and monocytes sometimes have weak positive reaction in PAS satin. Besides, we also use modified methods to prepare the blood cells in scanning electron microscope in order to investigate the surface morphology of blood cells. But we find that the white blood cells of Ocadia sinensis lack of obvious and specific characteristic on the surface. The information offered by this experiment can be applied in clinic or related researches directly. We also hope this experiment model can be used in other reptiles in the future.

Dissertação de mestrado em Direito (Ciências Jurídico-Criminais), apresentada à Faculdade de Direito da Universidade de Coimbra
A perseguição persistente, intrusiva e indesejada há muito deixou de se considerar uma realidade exclusiva das celebridades. Na verdade, nunca o foi: o espetro de vítimas do fenómeno do Stalking é abrangente e heterogéneo, tal como o são as formas de atuação dos perpetradores. Resultado dessa perceção tem sido a recente e paulatina criminalização da Perseguição ao redor do mundo, saltando esta realidade dos ecrãs de cinema e dos meios de comunicação social para o catálogo legal de crimes de vários ordenamentos jurídicos. Portugal não ficou de fora dessa tendência: a recente Lei nº 83/2015, de 5 de agosto alterou o Código Penal, aditando-lhe, entre outros, o artigo 154º- A de epígrafe “Perseguição”. Propomo-nos estudar este novo tipo de ilícito e averiguar quais as razões que levaram o nosso legislador penal a optar pela criação ex novo deste crime, tecendo considerações de política-criminal sobre tal opção e, sobretudo, procurando analisar até que ponto a recente criminalização do Stalking no nosso país respeita as apertadas exigências constitucionais inerentes à legitimação de qualquer intervenção do ius puniendi Estadual. Falamos das exigências de dignidade, carência e tutela penal, mediada pelos princípios da subsidiariedade, fragmentariedade, e proporcionalidade aplicados ao Direito Penal. ABSTRACT: The obsessive, intrusive and unwanted attention or harassment is no longer a celebrity exclusive reality. In fact, it never was: victims of stalking belong to a wide and heterogeneous spectrum, as well as the types of stalkers. Its acknowlegment has resulted in a recent and gradual criminalization of harassment all around the world, turning a reality priorly exclusive to movies and media into crimes listed and punished in several countries. Portugal is no exception: the recent Law nr. 83/2015, 5th of August, changed the Criminal Code, appending it, among others, the article 154.º - A, entitled “Perseguição” (harassment). We’ll study this new type of illicit, and ascertain which reasons took our criminal legislator when choosing for the creation of this crime, analyzing this option through a criminal policy point of view, and most of all, try to understand how much Stalking criminalization respects the constitutional requirements, inherent to legitimation of any penal intervention. We speak of criminal dignity, need and custody requirements, mediated by the principles of subsidiarity, proportionality and fragmentary apllied to Criminal Law

Heteropolymeric O antigen (O-Ag)-capped lipopolysaccharide is the principal constituent of the Gram-negative bacterial cell surface. It is assembled via the integral inner membrane (IM) Wzx/Wzy-dependent pathway. In Pseudomonas aeruginosa, Wzx translocates lipid-linked anionic O-Ag subunits from the cytoplasmic to the periplasmic leaflets of the IM, where Wzy polymerizes the subunits to lengths regulated by Wzz1/2. The Wzx and Wzy IM topologies were mapped using random C-terminal-truncation fusions to PhoALacZα, which displays PhoA/LacZ activity dependent upon its subcellular localization. Twelve transmembrane segments (TMS) containing charged residues were identified for Wzx. Fourteen TMS, two sizeable cytoplasmic loops (CL), and two large periplasmic loops (PL3 and PL5 of comparable size) were characterized for Wzy. Despite Wzy PL3–PL5 sequence homology, these loops were distinguished by respective cationic and anionic charge properties. Site-directed mutagenesis identified functionally-essential Arg residues in both loops. These results led to the proposition of a “catch-and-release” mechanism for Wzy function. The abovementioned Arg residues and intra-Wzy PL3–PL5 sequence homology were conserved among phylogenetically diverse Wzy homologues, indicating widespread potential for the proposed mechanism. Unexpectedly, Wzy CL6 mutations disrupted Wzz1-mediated regulation of shorter O-Ag chains, providing the first evidence for direct Wzy–Wzz interaction. Mutagenesis studies identified functionally-important charged and aromatic TMS residues localized to either the interior vestibule or TMS bundles in a 3D homology model constructed for Wzx. Substrate-binding or energy-coupling roles were proposed for these residues, respectively. The Wzx interior was found to be cationic, consistent with translocation of anionic O-Ag subunits. To test these hypotheses, Wzx was overexpressed, purified, and reconstituted in proteoliposomes loaded with I−. Common transport coupling ions were introduced to “open” the protein and allow detection of I− flux via reconstituted Wzx. Extraliposomal changes in H+ induced I− flux, while Na+ addition had no effect, suggesting H+-dependent Wzx gating. Putative energy-coupling residue mutants demonstrated defective H+-dependent halide flux. Wzx also mediated H+ uptake as detected through fluorescence shifts from proteoliposomes loaded with pH-sensitive dye. Consequently, Wzx was proposed to function via H+-coupled antiport. In summary, this research has contributed structural and functional knowledge leading to novel mechanistic understandings for O-Ag biosynthesis in bacteria.
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1.) Canadian Institutes of Health Research (CIHR) Frederick Banting and Charles Best Canada Graduate Scholarship doctoral award, 2.) CIHR Michael Smith Foreign Study Award, 3.) Cystic Fibrosis Canada (CFC) doctoral studentship, 4.) University of Guelph Dean's Tri-Council Scholarship, 5.) Ontario Graduate Scholarship in Science and Technology, 6.) Operating grants to Dr. Joseph S. Lam from CIHR (MOP-14687) and CFC