Diverse Mechanisms of G Protein Regulation by Monoubiquitination

Cell signaling pathways convert information from the extracellular environment into an intracellular response. It is essential that these pathways be turned on and off on the appropriate timescales. Post-translational modifications are one essential mechanism used to maintain proper signaling. One post-translational modification that is emerging as a key regulator of cell signaling is monoubiquitination. Monoubiquitination is dynamic and reversible, making it ideal for temporal and spatial regulation. It has recently become evident that monoubiquitination regulates G proteins, which are the molecular switches that turn signaling pathways on and off. However, the mechanisms by which monoubiquitination acts on these enzymes is not known. We used a chemical ubiquitination approach coupled with biochemical and biophysical assays to elucidate the mechanisms by which two G proteins, the small G protein Ras and the heterotrimeric G protein Gpa1, are regulated by monoubiquitination. Monoubiquitination at one position activates K-Ras by impeding regulator-mediated hydrolysis while monoubiquitination at a distinct site activates H-Ras by increasing intrinsic nucleotide exchange. Together, these results demonstrate that monoubiquitination contributes to isoform-dependent regulation of Ras in a site-specific manner. Furthermore, we found that the site of ubiquitination on Gpa1 was in a unique domain that is essential for trafficking but does not contribute to enzymatic activity. The G protein substrates we chose exhibited diverse mechanisms of regulation by monoubiquitination including altering protein interactions (K-Ras), intrinsic activity (H-Ras), and localization (Gpa1). In summary, our results represent the first mechanistic study of G protein regulation by monoubiquitination and contribute to understanding Ras and Gpa1 regulation specifically as well as regulation of G proteins by monoubiquitination generally. More broadly, these results illustrate the diverse roles for monoubiquitination in the regulation of cell signaling.Doctor of Philosoph

GTP-Cyclohydrolase function in parasitic nematode development

Parasitic nematodes of grazing livestock represent an increasing economic and welfare problem for British agriculture. By investigating specific life-cycle stages of these parasites, it may be possible to identify key molecules or pathways that are required for the survival of the worms, and thus exploit these for future control strategies. It has been shown previously that the third larval stages (L3) of the ovine parasitic nematode Teladorsagia circumcincta produce high levels of transcript for the enzyme GTP-Cyclohydrolase relative to later developmental stages. As the ratelimiting factor in the production of tetrahydrobiopterin, GTP-Cyclohydrolase is required for a number of different biochemical pathways, including those involved in the production of serotonin and melanin. As the L3 do not feed, it can be hypothesised that, if finite resources are being used in the production of transcript encoding this enzyme, then it may be important for survival. In this thesis, a number of approaches were taken to explore the function of GTPCyclohydrolase in the life-cycle development of T. circumcincta. The closely related parasite, Dictyocaulus viviparus, was used as a model organism to explore the role of GTP-Cyclohydrolase and serotonin production with regards to larval arrest, or hypobiosis. This process occurs readily under experimental conditions in D. viviparus, which is not possible with T. circumcincta. Quantitative PCR was used to examine GTP-Cyclohydrolase transcript levels in two different strains of D. viviparus, one that enters larval arrest when exposed to cold conditions and one that does not. No differences were observed between the two strains suggesting that GTP-Cyclohydrolase was unlikely to be involved in hypobiosis. The model nematode, Caenorhabditis elegans, was used to perform functional complementation experiments to assess the role of GTP-Cyclohydrolase in the cuticle, as it has been shown previously that C. elegans GTP-Cyclohydrolase mutants have a ‘leaky cuticle’ and are killed by lower doses of anthelmintics and bleach than the wild-type worms. The T. circumcincta gene for GTP-Cyclohydrolase was able to restore cuticular integrity of C. elegans GTP-Cyclohydrolase-deletion mutants, suggesting that the role played by the protein in both species is similar. In vitro inhibition experiments using a chemical inhibitor of GTP-Cyclohydrolase showed that T. circumcincta larval development was disrupted in the presence of the inhibitor. It was also shown that T. circumcincta L3 that were exposed to sunlight produced melanin, suggesting that the levels of GTP-Cyclohydrolase observed in the preparasitic stages of T. circumcincta may be required for the synthesis of melanin. Together, these data suggest that GTP-Cyclohydrolase is required by the preparasitic stages to survive on pasture. Ultraviolet radiation has been shown previously to be harmful to T. circumcincta L3, so if the melanin production provides protection from this, then it would be crucial for the survival of the pre-parasitic stages

Racial Capitalism and Claims to Space in Post-bankruptcy Detroit

My dissertation examines the roles of land use and property governance in mediating racial and economic inequality in the urban environment. This dissertation is concerned with how gendered and raced subjectivities shape property relations across the urban landscape in the United States. The articles that comprise this manuscript dissertation are based on an extended case method approach utilizing mixed qualitative methods in Detroit, Michigan between 2016 and 2018. These articles explore how the evolving governance of property following Detroits bankruptcy manifested in market formations and legal frameworks that disrupted long-practiced informal relationships to property by residents. Residents voluntary stewardship has revealed the generative capacities of the citys vast stock of vacant properties, the communitys ability to defend themselves against politics of austerity, and how city government has come to depend on residents unpaid labor in the absence of municipal maintenance capacities of fair taxation policies. Detroit is a propagative site for understanding contemporary manifestations of racialization and urban property relations due to the large stock of municipal land holdings, the temporary seizure of democratic representation during the 2013 instatement of emergency management, and this post-bankruptcy moment of imagining how all Detroiters will live together in the increasingly divided city. This is the context in which land justice and housing advocates, including urban farmers are reshaping Detroits narrative of material depravity. By exemplifying how municipal land holdings and foreclosed homes can be harnessed toward ends of racial justice through the redistribution of property back to those whose stewardship has added value to their neighborhoods, Detroiters are working toward a future their elected officials have not yet imagined. These articles address how urban property markets are mobilized toward ends that are increasingly fractured from liberal conceptualizations of the role of city governments. Once thought to hold moral obligations to improve the lives of residents through providing public services, equity in governance, and to advance the human condition via infrastructural development and democratically embedded public process; the volatility of this particular citys government has produced quite the opposite, constructing variegated rather than equitable tiers of citizenship and access to space

Temporal Controls on Dissolved Organic Matter and Lignin Biogeochemistry in a Pristine Tropical River, Democratic Republic of Congo

Dissolved organic carbon (DOC), lignin biomarkers, and the optical properties of dissolved organic matter (DOM) were measured in the Epulu River (northeast Democratic Republic of Congo) with the aim of investigating temporal controls on the quantity and chemical composition of DOM in a tropical rainforest river. Three different periods defined by stages of the hydrologic regime of the region, (1) post dry flushing period, (2) intermediary period, and (3) start of the dry period/post flush, were sampled. Temporal variability in DOM quantity and quality was observed with highest DOC, lignin concentration (Σ8) and carbon‐normalized (Λ8) values during the flushing period attributed to greater surface runoff and leaching of organic‐rich horizons, with lowest values in the dry period/post flush once source materials were well leached. Chromophoric DOM (CDOM) was strongly correlated to DOC and Σ8 (r2 = 0.85 and 0.83, respectively; p \u3c 0.001), and CDOM quality measurements (SUVA254, spectral slope ratio and fluorescence index) were strongly correlated to Λ8 values (r2 = 0.77, 0.69, and 0.75, respectively; p \u3c 0.001), demonstrating the ability to derive DOC and lignin export and to track DOM quality in tropical riverine systems from simple optical measurements. This study demonstrates similar effects in the variability of DOM quantity and quality due to changing hydrologic inputs for a tropical river as has been previously reported for temperate and northern high‐latitude rivers. Therefore, flushing periods in tropical rivers warrant further study, as they are critical toward understanding ecosystem biogeochemistry as maximal export of freshly leached plant material occurs during this time period

Standardised Practice-Based Oral Health Data Collection: A Pilot Study in Different Countries

BACKGROUND: The Oral Health Observatory (OHO), launched in 2014 by FDI World Dental Federation, aims to provide a coordinated approach to international oral health data collection. A feasibility project involving 12 countries tested the implementation of the methodology and data collection tools and assessed data quality from 6 countries. METHODS: National dental associations (NDAs) recruited dentists following a standardised sampling method. Dentists and patients completed paired questionnaires (N = 7907) about patients' demographics, dental attendance, oral health-related behaviours, oral impacts, and clinical measures using a mobile app. In addition, participating dentists (n = 93) completed an evaluation survey, and NDAs completed a survey and participated in workshops to assess implementation feasibility. RESULTS: Feasibility data are presented from the 12 participating countries. In addition, the 6 countries most advanced with data collection as of July 2020 (China, Colombia, India, Italy, Japan, and Lebanon) were included in the assessment of data quality and qualitative evaluation of implementation feasibility. All NDAs in these 6 countries reported interest in collecting standardised, international data for policy and communication activities and to understand service use and needs. Eighty-two percent of dentists (n = 76) reported a patient response rate of between 80% and 100%. More than 70% (n = 71) of dentists were either satisfied or very satisfied with the patient recruitment and data collection methods. There were variations in patient oral health and behaviours across countries, such as self-reporting twice-daily brushing which ranged from 45% in India to 83% in Colombia. CONCLUSIONS: OHO provides a feasible model for collecting international standardised data in dental practices. Reducing time implications, ensuring mobile app reliability, and allowing practitioners to access patient-reported outcomes to inform practice may enhance implementation

Increase Postpartum Gestational Diabetes Screening

Introduction: Diabetic women did not get follow up screening after delivery. Nurses are finding a way to screen more women

Systematic Analysis of Essential Genes Reveals Important Regulators of G Protein Signaling

The yeast pheromone pathway consists of a canonical heterotrimeric G protein and MAP kinase cascade. To identify new signaling components we systematically evaluated 870 essential genes using a library of repressible-promoter strains. Quantitative transcription-reporter and MAPK activity assays were used to identify strains that exhibit altered pheromone sensitivity. Of the 92 newly identified essential genes required for proper G protein signaling, those involved with protein degradation were most highly-represented. Included in this group are members of the SCF (Skp-Cullin-F-Box) ubiquitin ligase complex. Further genetic and biochemical analysis reveals that SCFCdc4 acts together with the Cdc34 ubiquitin conjugating enzyme at the level of the G protein, promotes degradation of the G protein α subunit, Gpa1, in vivo and catalyzes Gpa1 ubiquitination in vitro. These new insights to the G protein signaling network reveal the essential-genome as an untapped resource for identifying new components and regulators of signal transduction pathways

Transient Overexpression of α-Ca2+/Calmodulin-Dependent Protein Kinase II in the Nucleus Accumbens Shell Enhances Behavioral Responding to Amphetamine

Ca2+/calmodulin-dependent protein kinase II (CaMKII) is known to contribute to the expression of psychostimulant sensitization by regulating dopamine (DA) overflow from DA neuron terminals in the nucleus accumbens (NAcc). The present experiments explored the contribution of CaMKII in NAcc neurons postsynaptic to these terminals where it is known to participate in a number of signaling pathways that regulate responding to psychostimulant drugs. Exposure to amphetamine transiently increased αCaMKII levels in the shell but not the core of the NAcc. Thus, HSV (herpes simplex viral) vectors were used to transiently overexpress αCaMKII in NAcc neurons in drug-naive rats, and behavioral responding to amphetamine was assessed. Transiently overexpressing αCaMKII in the NAcc shell led to long-lasting enhancement of amphetamine-induced locomotion and self-administration manifested when αCaMKII levels were elevated and persisting long after they had returned to baseline. Enhanced locomotion was not observed after infection in the NAcc core or sites adjacent to the NAcc. Transient elevation of NAcc shell αCaMKII levels also enhanced locomotor responding to NAcc AMPA and increased phosphorylation levels of GluR1 (Ser831), a CaMKII site, both soon and long after infection. Similar increases in pGluR1 (Ser831) were observed both soon and long after exposure to amphetamine. These results indicate that the transient increase in αCaMKII observed in neurons of the NAcc shell after viral-mediated gene transfer and likely exposure to amphetamine leads to neuroadaptations in AMPA receptor signaling in this site that may contribute to the long-lasting maintenance of behavioral and incentive sensitization by psychostimulant drugs like amphetamine

Effects of Recombinant Protein Expression on Green Fluorescent Protein Diffusion in Escherichia coli

Fluorescence recovery after photobleaching was used to measure the diffusion coefficient of green fluorescent protein (GFP, 27 kDa) in Escherichia coli in the presence or absence of four coexpressed proteins: cytoplasmic maltose binding protein (42 kDa), tau-40 (45 kDa), α-synuclein (14 kDa), or calmodulin (17 kDa). The GFP diffusion coefficient remains constant regardless of the type of coexpresseed protein and whether or not the coexpressed protein was induced. We conclude that expression of these soluble proteins has little to no effect on the diffusion of GFP. These results have implications for the utility of in-cell nuclear magnetic resonance spectroscopy

Enhanced Recovery After Surgery implementation in practice:an ethnographic study of services for hip and knee replacement

Objectives: Enhanced Recovery After Surgery (ERAS) programmes aim to improve care quality by optimising components of the care pathway and programmes for hip and knee replacement exist across the UK. However, there is variation in delivery and outcomes. This study aims to understand processes that influence implementation using the Consolidated Framework for Implementation Research (CFIR) to inform the design and delivery of services. Design: An ethnographic study using observations and interviews with staff involved in service delivery. Data were analysed using a thematic analysis, followed by an abductive approach whereby themes were mapped onto the 31 constructs and 5 domains of the CFIR. Setting: Four hospital sites in the UK delivering ERAS services for hip and knee replacement. Participants: 38 staff participated including orthopaedic surgeons, nurses and physiotherapists. Results: Results showed 17 CFIR constructs influenced implementation in all five domains. Within ‘intervention characteristics’, participants thought ERAS afforded advantages over alternative solutions and guidance was adaptable. In the ‘outer setting’, it was felt ERAS should be tailored to patients and education used to empower them in their recovery. However, there were concerns about postdischarge support and tensions with primary care. Within the ‘inner setting’, effective multidisciplinary collaboration was achieved by transferring knowledge about patients along the care pathway and multidisciplinary working practices. ERAS was viewed as a ‘message’ that had to be communicated consistently. There were concerns about resources and high volumes of patients. Staff access to information varied. At the domain ‘characteristics of individuals’, knowledge and beliefs impacted on implementation. Within ‘process’, involving opinion leaders in development and ‘champions’ who acted as a central point of contact, helped to engage staff. Formal and informal feedback helped to develop services. Conclusions: Findings demonstrate successful implementation involves empowering patients to work towards recovery, providing postdischarge support and promoting successful multidisciplinary team working.</p