Identifying and overcoming barriers to raising funds from individuals for tobacco prevention

Thesis (Master's)--University of Washington, 2016-06

Rabbit CYP4B1 engineered for high-level expression in Escherichia coli: ligand stabilization and processing of the N-terminus and heme prosthetic group

Modifications at the N-terminus of the rabbit CYP4B1 gene resulted in expression levels in Escherichia coli of up to 660 nmol/L. Solubilization of the enzyme from bacterial membranes led to substantial conversion to cytochrome P420 unless alpha-naphthoflavone was added as a stabilizing ligand. Mass spectrometry analysis and Edman sequencing of purified enzyme preparations revealed differential N-terminal post-translational processing of the various constructs expressed. Notably, bacterial expression of CYP4B1 produced a holoenzyme with >98.5% of its heme prosthetic group covalently linked to the protein backbone. The near fully covalently linked hernoproteins exhibited similar rates and regioselectivities of lauric acid hydroxylation to that observed previously for the partially heme processed enzyme expressed in insect cells. These studies shed new light on the consequences of covalent heme processing in CYP4B1 and provide a facile system for future mechanistic and structural studies with the enzyme. (C) 2003 Elsevier Science (USA). All rights reserved.

Partnerships in mental health: Addressing barriers to social inclusion

Current trends in mental health services emphasize working in partnership with consumers and other government and non-government community organizations for improvement in quality of life for service users. People with a mental illness experience social exclusion, thereby limiting their ability to participate fully in community life. Occupational therapists have a substantial role to play in helping service users to overcome barriers to their community inclusion. Partnerships need to be formed to increase access to community resources and participation in activities that are enjoyed by other members of the community. Such partnerships have a health promotion emphasis and foster the relationship between mental-health services and the wider community, thus shifting the focus from direct occupational therapy service delivery to community-based rehabilitation interventions.This article describes the development, implementation and evaluation of an Australian healthy lifestyle course devised to meet the identified rehabilitation goals of people with mental illness to lose weight, get fit, commence vocational study and get a job. The course was run in partnership between mental health consumers, occupational therapists from the St George Mental Health Rehabilitation Service and staff and students from the Sutherland College of Technical and Further Education.

Improved photobiological H-2 production in engineered green algal cells

Oxygenic photosynthetic organisms use solar energy to split water (H2O) into protons (H+), electrons (e(-)), and oxygen. A select group of photosynthetic microorganisms, including the green alga Chlamydomonas reinhardtii, has evolved the additional ability to redirect the derived H+ and e(-) to drive hydrogen (H-2) production via the chloroplast hydrogenases HydA1 and A2 (H(2)ase). This process occurs under anaerobic conditions and provides a biological basis for solar-driven H-2 production. However, its relatively poor yield is a major limitation for the economic viability of this process. To improve H-2 production in Chlamydomonas, we have developed a new approach to increase H+ and e(-) supply to the hydrogenases. In a first step, mutants blocked in the state 1 transition were selected. These mutants are inhibited in cyclic e(-) transfer around photosystem I, eliminating possible competition for e(-) with H(2)ase. Selected strains were further screened for increased H-2 production rates, leading to the isolation of Stm6. This strain has a modified respiratory metabolism, providing it with two additional important properties as follows: large starch reserves ( i.e. enhanced substrate availability), and a low dissolved O-2 concentration (40% of the wild type (WT)), resulting in reduced inhibition of H2ase activation. The H-2 production rates of Stm6 were 5 - 13 times that of the control WT strain over a range of conditions ( light intensity, culture time, +/- uncoupler). Typically, similar to 540 ml of H-2 liter(-1) culture ( up to 98% pure) were produced over a 10-14-day period at a maximal rate of 4 ml h(-1) ( efficiency = similar to 5 times the WT). Stm6 therefore represents an important step toward the development of future solar-powered H-2 production systems.

An Internet training intervention for people with traumatic brain injury: Barriers and outcomes

Primary objective: To test whether people with cognitive-linguistic impairments following traumatic brain injury could learn to use the Internet using specialized training materials. Research design: Pre-post test design. Methods and procedures: Seven participants were each matched with a volunteer tutor. Basic Internet skills were taught over six lessons using a tutor's manual and a student manual. Instructions used simple text and graphics based on Microsoft Internet Explorer 5.5. Students underwent Internet skills assessments and interviews pre- and post-training. Tutors completed a post-training questionnaire. Main outcomes and results: Six of seven participants reached moderate-to-high degrees of independence. Literacy impairment was an expected training barrier; however, cognitive impairments affecting concentration, memory and motivation were more significant. Conclusions: Findings suggest that people with cognitive-linguistic impairments can learn Internet skills using specialized training materials. Participants and their carers also reported positive outcomes beyond the acquisition of Internet skills.

Functional characterisation of an engineered multidomain human P450 E1 by molecular Lego

The human cytochrome P450s constitute an important family of monooxygenase enzymes that carry out essential roles in the metabolism of endogenous compounds and foreign chemicals. We present here results of a fusion between a human P450 enzyme and a bacterial reductase that for the first time is shown does not require the addition of lipids or detergents to achieve wild-type-like activities. The fusion enzyme, P450 2E1-BMR, contains the N-terminally modified residues 22-493 of the human P450 2E1 fused at the C-terminus to residues 473-1049 of the P450 BM3 reductase (BMR). The P450 2E1-BMR enzyme is active, self-sufficient and presents the typical marker activities of the native human P450 2E1: the hydroxylation of p-nitrophenol (K (M)=1.84 +/- 0.09 mM and k (cat) of 2.98 +/- 0.04 nmol of p-nitrocatechol formed per minute per nanomole of P450) and chlorzoxazone (K (M)=0.65 +/- 0.08 mM and k (cat) of 0.95 +/- 0.10 nmol of 6-hydroxychlorzoxazone formed per minute per nanomole of P450). A 3D model of human P450 2E1 was generated to rationalise the functional data and to allow an analysis of the surface potentials. The distribution of charges on the model of P450 2E1 compared with that of the FMN domain of BMR provides the ground for the understanding of the interaction between the fused domains. The results point the way to successfully engineer a variety of catalytically self-sufficient human P450 enzymes for drug metabolism studies in solution.

Interagency collaboration between child protection and mental health services: Practices, attitudes and barriers

Objective: The aim of this paper is to examine some of the factors that facilitate and hinder interagency collaboration between child protection services and mental health services in cases where there is a parent with a mental illness and there are protection concerns for the child(ren). The paper reports on agency practices, worker attitudes and experiences, and barriers to effective collaboration. Method: A self-administered, cross-sectional survey was developed and distributed via direct mail or via line supervisors to workers in statutory child protection services, adult mental health services, child and youth mental health services, and Suspected Child Abuse and Neglect (SCAN) Teams. There were 232 completed questionnaires returned, with an overall response rate of 21%. Thirty-eight percent of respondents were statutory child protection workers. 39% were adult mental health workers, 16% were child and youth mental health workers, and 4% were SCAN Team medical officers (with 3% missing data). Results: Analysis revealed that workers were engaging in a moderate amount of interagency contact, but that they were unhappy with the support provided by their agency. Principle components analysis and multivariate analysis of variance (MANOVA) on items assessing attitudes toward other workers identified four factors, which differed in rates of endorsement: inadequate training, positive regard for child protection workers, positive regard for mental health workers, and mutual mistrust (from highest to lowest level of endorsement). The same procedure identified the relative endorsement of five factors extracted from items about potential barriers: inadequate resources, confidentiality, gaps in interagency processes, unrealistic expectations, and professional knowledge domains and boundaries. Conclusions: Mental health and child protection professionals believe that collaborative practice is necessary; however, their efforts are hindered by a lack of supportive structures and practices at the organizational level. (c) 2005 Published by Elsevier Ltd.