Role of proline residues in the expression and function of the human noradrenaline transporter

The aim was to investigate the roles of proline residues in extracellular loop 2 (P172, P183, P188 and P209) and transmembrane domains 2, 5, 11 and 12 (P108, P270, P526, P551, P552 and P570) in determining noradrenaline transporter (NET) expression and function. Mutants of human NET with these residues mutated to alanine were pharmacologically characterized. Mutation of P108, P270 and P526 disrupted cell surface expression, from [H-3]nisoxetine binding and confocal microscopy data. Mutations of P526, P551 and P570 reduced transporter turnover (V-max of [H-3]noradrenaline uptake/B-max of [H-3]nisoxetine binding) by 1.5-1.7-fold compared with wild-type NET, so these residues might be involved in conformational changes associated with substrate translocation. Conversely, mutations of P172, P183, P188 and P209 increased V-max/B-max by 2-3-fold compared with wild-type, indicating that the presence of these proline residues limits turnover of the NET. The mutations had few effects on apparent affinities of substrates or affinities of inhibitors, except decreases in inhibitor affinities after mutations of the P270 and P570 residues, and increases after mutation of the P526 residue. Hence, proline residues in extracellular loop 2 and in transmembrane domains have a range of roles in determining expression and function of the NET.

Functional properties of multiple isoforms of human divalent metal-ion transporter 1 (DMT1)

DMT1 (divalent metal-ion transporter 1) is a widely expressed metal-ion transporter that is vital for intestinal iron absorption and iron utilization by most cell types throughout the body, including erythroid precursors. Mutations in DMT1 cause severe microcytic anaemia in animal models. Four DMT1 isoforms that differ in their N- and C-termini arise from mRNA transcripts that vary both at their 5'-ends (starting in exon 1A or exon 1B) and at their 3'-ends giving rise to mRNAs containing (+) or lacking (-) the 3'-IRE (iron-responsive element) and resulting in altered C-terminal coding sequences. To determine whether these variations result in functional differences between isoforms, we explored the functional properties of each isoform using the voltage clamp and radiotracer assays in cRNA-injected Xenopus oocytes. 1A/IRE+-DMT1 mediated Fe2+-evoked currents that were saturable (K(0.5)(Fe) approximately 1-2 microM), temperature-dependent (Q10 approximately 2), H+-dependent (K(0.5)(H) approximately 1 muM) and voltage-dependent. 1A/IRE+-DMT1 exhibited the provisional substrate profile (ranked on currents) Cd2+, Co2+, Fe2+, Mn2+>Ni2+, V3+>>Pb2+. Zn2+ also evoked large currents; however, the zinc-evoked current was accounted for by H+ and Cl- conductances and was not associated with significant Zn2+ transport. 1B/IRE+-DMT1 exhibited the same substrate profile, Fe2+ affinity and dependence on the H+ electrochemical gradient. Each isoform mediated 55Fe2+ uptake and Fe2+-evoked currents at low extracellular pH. Whereas iron transport activity varied markedly between the four isoforms, the activity for each correlated with the density of anti-DMT1 immunostaining in the plasma membrane, and the turnover rate of the Fe2+ transport cycle did not differ between isoforms. Therefore all four isoforms of human DMT1 function as metal-ion transporters of equivalent efficiency. Our results reveal that the N- and C-terminal sequence variations among the DMT1 isoforms do not alter DMT1 functional properties. We therefore propose that these variations serve as tissue-specific signals or cues to direct DMT1 to the appropriate subcellular compartments (e.g. in erythroid cells) or the plasma membrane (e.g. in intestine).

Allosteric crosstalk between peptide-binding, transport, and ATP hydrolysis of the ABC transporter TAP

The transporter associated with antigen processing (TAP) is essential for intracellular transport of protein fragments into the endoplasmic reticulum for loading of major histocompatibility complex (MHC) class I molecules. On the cell surface, these peptide–MHC complexes are monitored by cytotoxic T lymphocytes. To study the ATP hydrolysis of TAP, we developed an enrichment and reconstitution procedure, by which we fully restored TAP function in proteoliposomes. A TAP-specific ATPase activity was identified that could be stimulated by peptides and blocked by the herpes simplex virus protein ICP47. Strikingly, the peptide-binding motif of TAP directly correlates with the stimulation of the ATPase activity, demonstrating that the initial peptide-binding step is responsible for TAP selectivity. ATP hydrolysis follows Michaelis–Menten kinetics with a maximal velocity Vmax of 2 μmol/min per mg TAP, corresponding to a turnover number of approximately 5 ATP per second. This turnover rate is sufficient to account for the role of TAP in peptide loading of MHC molecules and the overall process of antigen presentation. Interestingly, sterically restricted peptides that bind but are not transported by TAP do not stimulate ATPase activity. These results point to coordinated dialogue between the peptide-binding site, the nucleotide-binding domain, and the translocation site via conformational changes within the TAP complex.

Membrane associated transporter protein gene (SLC45A2) and the genetic basis of normal human pigmentation variation

This work is concerned with the genetic basis of normal human pigmentation variation. Specifically, the role of polymorphisms within the solute carrier family 45 member 2 (SLC45A2 or membrane associated transporter protein; MATP) gene were investigated with respect to variation in hair, skin and eye colour ― both between and within populations. SLC45A2 is an important regulator of melanin production and mutations in the gene underly the most recently identified form of oculocutaneous albinism. There is evidence to suggest that non-synonymous polymorphisms in SLC45A2 are associated with normal pigmentation variation between populations. Therefore, the underlying hypothesis of this thesis is that polymorphisms in SLC45A2 will alter the function or regulation of the protein, thereby altering the important role it plays in melanogenesis and providing a mechanism for normal pigmentation variation. In order to investigate the role that SLC45A2 polymorphisms play in human pigmentation variation, a DNA database was established which collected pigmentation phenotypic information and blood samples of more than 700 individuals. This database was used as the foundation for two association studies outlined in this thesis, the first of which involved genotyping two previously-described non-synonymous polymorphisms, p.Glu272Lys and p.Phe374Leu, in four different population groups. For both polymorphisms, allele frequencies were significantly different between population groups and the 272Lys and 374Leu alleles were strongly associated with black hair, brown eyes and olive skin colour in Caucasians. This was the first report to show that SLC45A2 polymorphisms were associated with normal human intra-population pigmentation variation. The second association study involved genotyping several SLC45A2 promoter polymorphisms to determine if they also played a role in pigmentation variation. Firstly, the transcription start site (TSS), and hence putative proximal promoter region, was identified using 5' RNA ligase mediated rapid amplification of cDNA ends (RLM-RACE). Two alternate TSSs were identified and the putative promoter region was screened for novel polymorphisms using denaturing high performance liquid chromatography (dHPLC). A novel duplication (c.–1176_–1174dupAAT) was identified along with other previously described single nucleotide polymorphisms (c.–1721C>G and c.–1169G>A). Strong linkage disequilibrium ensured that all three polymorphisms were associated with skin colour such that the –1721G, +dup and –1169A alleles were associated with olive skin in Caucasians. No linkage disequilibrium was observed between the promoter and coding region polymorphisms, suggesting independent effects. The association analyses were complemented with functional data, showing that the –1721G, +dup and –1169A alleles significantly decreased SLC45A2 transcriptional activity. Based on in silico bioinformatic analysis that showed these alleles remove a microphthalmia-associated transcription factor (MITF) binding site, and that MITF is a known regulator of SLC45A2 (Baxter and Pavan, 2002; Du and Fisher, 2002), it was postulated that SLC45A2 promoter polymorphisms could contribute to the regulation of pigmentation by altering MITF binding affinity. Further characterisation of the SLC45A2 promoter was carried out using luciferase reporter assays to determine the transcriptional activity of different regions of the promoter. Five constructs were designed of increasing length and their promoter activity evaluated. Constitutive promoter activity was observed within the first ~200 bp and promoter activity increased as the construct size increased. The functional impact of the –1721G, +dup and –1169A alleles, which removed a MITF consensus binding site, were assessed using electrophoretic mobility shift assays (EMSA) and expression analysis of genotyped melanoblast and melanocyte cell lines. EMSA results confirmed that the promoter polymorphisms affected DNA-protein binding. Interestingly, however, the protein/s involved were not MITF, or at least MITF was not the protein directly binding to the DNA. In an effort to more thoroughly characterise the functional consequences of SLC45A2 promoter polymorphisms, the mRNA expression levels of SLC45A2 and MITF were determined in melanocyte/melanoblast cell lines. Based on SLC45A2’s role in processing and trafficking TYRP1 from the trans-Golgi network to stage 2 melanosmes, the mRNA expression of TYRP1 was also investigated. Expression results suggested a coordinated expression of pigmentation genes. This thesis has substantially contributed to the field of pigmentation by showing that SLC45A2 polymorphisms not only show allele frequency differences between population groups, but also contribute to normal pigmentation variation within a Caucasian population. In addition, promoter polymorphisms have been shown to have functional consequences for SLC45A2 transcription and the expression of other pigmentation genes. Combined, the data presented in this work supports the notion that SLC45A2 is an important contributor to normal pigmentation variation and should be the target of further research to elucidate its role in determining pigmentation phenotypes. Understanding SLC45A2’s function may lead to the development of therapeutic interventions for oculocutaneous albinism and other disorders of pigmentation. It may also help in our understanding of skin cancer susceptibility and evolutionary adaptation to different UV environments, and contribute to the forensic application of pigmentation phenotype prediction.

Soil Carbon Turnover Measurement by Physical Fractionation at a Forest-to-Pasture Chronosequence in the Brazilian Amazon

The effect of conversion from forest-to-pasture upon soil carbon stocks has been intensively discussed, but few studies focus on how this land-use change affects carbon (C) distribution across soil fractions in the Amazon basin. We investigated this in the 20 cm depth along a chronosequence of sites from native forest to three successively older pastures. We performed a physicochemical fractionation of bulk soil samples to better understand the mechanisms by which soil C is stabilized and evaluate the contribution of each C fraction to total soil C. Additionally, we used a two-pool model to estimate the mean residence time (MRT) for the slow and active pool C in each fraction. Soil C increased with conversion from forest-to-pasture in the particulate organic matter (> 250 mu m), microaggregate (53-250 mu m), and d-clay (< 2 mu m) fractions. The microaggregate comprised the highest soil C content after the conversion from forest-to-pasture. The C content of the d-silt fraction decreased with time since conversion to pasture. Forest-derived C remained in all fractions with the highest concentration in the finest fractions, with the largest proportion of forest-derived soil C associated with clay minerals. Results from this work indicate that microaggregate formation is sensitive to changes in management and might serve as an indicator for management-induced soil carbon changes, and the soil C changes in the fractions are dependent on soil texture.

The impact of gender diversity on turnover : the moderating effect of human resource policies and practices

Demography theory suggests that high gender diversity leads to high turnover. As turnover is costly for organizations, we examined whether HR policies and practices influence the expected gender diversity-turnover relationship. Survey data were collected from 198 HR decision makers at publicly listed organizations. We found that HR policies and practices that are supportive of diversity moderate the gender diversity-turnover relationship, such that high gender diversity leads to low turnover in organizations with many diversity supportive policies and practices. Results suggest that organizations can avoid the negative consequences of high gender diversity by implementing diversity supportive HR polices and practices.

Haunted by the past : effects of poor management on employee attitudes and turnover

Change management research has largely ignored the effects of organizational change management history in shaping employee attitudes and behavior. This article develops and tests a model of the effects of poor change management history (PCMH) on employee attitudes (trust, job satisfaction, turnover intentions, change cynicism, and openness to change) and actual turnover. We found that PCMH, through PCMH beliefs, led to lower trust, job satisfaction and openness to change, and higher cynicism and turnover intentions. Also, PCMH beliefs predicted employee turnover over 2 years.

Quality of work life and turnover intention in primary healthcare organisations : a cross-sectional study of registered nurses in Saudi Arabia

Purpose: The purpose of this study was to improve the retention of primary healthcare (PHC) nurses through exploring and assessing their quality of work life (QWL) and turnover intention. Design and methods: A cross-sectional survey design was used in this study. Data were collected using a questionnaire comprising four sections (Brooks’ survey of Quality of Nursing Work Life [QNWL], Anticipated Turnover Intention, open-ended questions and demographic characteristics). A convenience sample was recruited from 143 PHC centres in Jazan, Saudi Arabia. A response rate of 87% (n = 508/585) was achieved. The SPSS v17 for Windows and NVivo 8 were used for analysis purposes. Procedures and tests used in this study to analyse the quantitative data were descriptive statistics, t-test, ANOVA, General Linear Model (GLM) univariate analysis, standard multiple regression, and hierarchical multiple regression. Qualitative data obtained from responses to the open-ended questions were analysed using the NVivo 8. Findings: Quantitative findings suggested that PHC nurses were dissatisfied with their work life. Respondents’ scores ranged between 45 and 218 (mean = 139.45), which is lower than the average total score on Brooks’ Survey (147). Major influencing factors were classified under four dimensions. First, work life/home life factors: unsuitable working hours, lack of facilities for nurses, inability to balance work with family needs and inadequacy of vacations’ policy. Second, work design factors: high workload, insufficient workforce numbers, lack of autonomy and undertaking many non-nursing tasks. Third, work context factors: management practices, lack of development opportunities, and inappropriate working environment in terms of the level of security, patient care supplies and unavailability of recreation room. Finally, work world factors: negative public image of nursing, and inadequate payment. More positively, nurses were notably satisfied with their co-workers. Conversely, 40.4% (n = 205) of the respondents indicated that they intended to leave their current employment. The relationships between QWL and demographic variables of gender, age, marital status, dependent children, dependent adults, nationality, ethnicity, nursing tenure, organisational tenure, positional tenure, and payment per month were significant (p < .05). The eta squared test for these demographics indicates a small to medium effect size of the variation in QWL scores. Using the GLM univariate analysis, education level was also significantly related to the QWL (p < .05). The relationships between turnover intention and demographic variables including gender, age, marital status, dependent children, education level, nursing tenure, organisational tenure, positional tenure, and payment per month were significant (p < .05). The eta squared test for these demographics indicates a small to moderate effect size of the variation in the turnover intention scores. Using the GLM univariate analysis, the dependent adults’ variable was also significantly related to turnover intention (p < .05). Turnover intention was significantly related to QWL. Using standard multiple regression, 26% of the variance in turnover intention was explained by the QWL F (4,491), 43.71, p < .001, with R² = .263. Further analysis using hierarchical multiple regression found that the total variance explained by the model as a whole (demographics and QWL) was 32.1%, F (17.433) = 12.04, p < .001. QWL explained an additional 19% of the variance in turnover intention, after controlling for demographic variables, R squared change =.19, F change (4, 433) = 30.190, p < .001. The work context variable makes the strongest unique contribution (-.387) to explain the turnover intention, followed by the work design dimension (-.112). The qualitative findings reaffirmed the quantitative findings in terms of QWL and turnover intention. However, the home life/work life and work world dimensions were of great important to both QWL and turnover intention. The qualitative findings revealed a number of new factors that were not included in the survey questionnaire. These included being away from family, lack of family support, social and cultural aspects, accommodation facilities, transportation, building and infrastructure of PHC, nature of work, job instability, privacy at work, patients and community, and distance between home and workplace. Conclusion: Creating and maintaining a healthy work life for PHC nurses is very important to improve their work satisfaction, reduce turnover, enhance productivity and improve nursing care outcomes. Improving these factors could lead to a higher QWL and increase retention rates and therefore reinforcing the stabilisation of the nursing workforce. Significance of the research: Many countries are examining strategies to attract and retain the health care workforce, particularly nurses. This study identified factors that influence the QWL of PHC nurses as well as their turnover intention. It also determined the significant relationship between QWL and turnover intention. In addition, the present study tested Brooks’ survey of QNWL on PHC nurses for the first time. The qualitative findings of this study revealed a number of new variables regarding QWL and turnover intention of PHC nurses. These variables could be used to improve current survey instruments or to develop new research surveys. The study findings could be also used to develop and appropriately implement plans to improve QWL. This may help to enhance the home and work environments of PHC nurses, improve individual and organisational performance, and increase nurses’ commitment. This study contributes to the existing body of research knowledge by presenting new data and findings from a different country and healthcare system. It is the first of its kind in Saudi Arabia, especially in the field of PHC. It has examined the relationship between QWL and turnover intention of PHC nurses for the first time using nursing instruments. The study also offers a fresh explanation (new framework) of the relationship between QWL and turnover intention among PHC nurses, which could be used or tested by researchers in other settings. Implications for further research: Review of the extant literature reveals little in-depth research on the PHC workforce, especially in terms of QWL and organisational turnover in developing countries. Further research is required to develop a QWL tool for PHC nurses, taking into consideration the findings of the current study along with the local culture. Moreover, the revised theoretical framework of the current study could be tested in further research in other regions, countries or healthcare systems in order to identify its ability to predict the level of PHC nurses’ QWL and their intention to leave. There is a need to conduct longitudinal research on PHC organisations to gain an in-depth understanding of the determents of and changes in QWL and turnover intention of PHC nurses at various points of time. An intervention study is required to improve QWL and retention among PHC nurses using the findings of the current study. This would help to assess the impact of such strategies on reducing turnover of PHC nurses. Focusing on the location of the current study, it would be valuable to conduct another study in five years’ time to examine the percentage of actual turnover among PHC nurses compared with the reported turnover intention in the current study. Further in-depth research would also be useful to assess the impact of the local culture on the perception of expatriate nurses towards their QWL and their turnover intention. A comparative study is required between PHC centres and hospitals as well as the public and private health sector agencies in terms of QWL and turnover intention of nursing personnel. Findings may differ from sector to sector according to variations in health systems, working environments and the case mix of patients.

Reinvigorating the domestic violence sector : systemically addressing conflict, power and practitioner turnover

The purpose of this exploratory Australian study was to consider methods of retaining skilled and experienced staff within the domestic violence sector. The antecedents that might influence turnover of practitioners were investigated and analysed. Antecedents broadly included the work-related factors, organisational factors and professional factors. The changing nature of the domestic violence sector was also examined, in particular, feminist identity and feminist practice frameworks. It became evident, however, that the primary reasons for the turnover of study participants can be described as parallel power processes. The concept of parallel power processes as developed through this research aims to capture how workplace behaviours can strongly mirror, or parallel, behaviours used by domestic violence perpetrators. As such, it appears that some domestic violence practitioners are experiencing their own abusive relationship, not within the confines of their home, but within their workplace. Additionally, parallel power processes are compounded by ineffective conflict management processes within the workplace. These concepts directly contribute to practitioners leaving their workplace and, sometimes, the sector. This qualitative study utilised a feminist research epistemology and focused strongly on practitioners' stories. Interviews were undertaken with fifteen domestic violence practitioners from three services within South-East Queensland, Australia. Two sets of semi-structured interviews provided in-depth information based on practitioners‘ experiences of working within this specialised sector. Analysis was conducted using a thematic analytical frame, drawing attention to the key themes as mentioned above. From these findings, it is suggested that in order to retain practitioners, domestic violence services must identify and address parallel power processes through effective conflict management processes. In an operational sense, it is recommended that education and training be undertaken within all staffing levels, in particular management committees. Lastly, it is recommended that the sector itself places greater attention on the re-invigoration of the feminist principles and philosophy that has traditionally guided the sector.

The relationship between quality of work life and turnover intention of primary health care nurses in Saudi Arabia

Background Quality of work life (QWL) has been found to influence the commitment of health professionals including nurses. However, reliable information on the QWL and turnover intention of primary health care (PHC) nurses is limited. The aim of this study was to examine the relationship between QWL and turnover intention of PHC nurses in Saudi Arabia. Methods A cross-sectional survey was used in this study. Data were collected using Brooks’ survey of Quality of Nursing Work life (QNWL), the Anticipated Turnover Scale and demographic data questions. A total of 508 PHC nurses in the Jazan region, Saudi Arabia completed the questionnaire (RR = 87%). Descriptive statistics, t-test, ANOVA, General Linear Model (GLM) univariate analysis, standard multiple regression (SMR), and hierarchical multiple regression (HMR) were applied for analysis using SPSS v17 for Windows. Results Findings suggested that the respondents were dissatisfied with their work life, with almost 40% indicating a turnover intention from their current PHC centres. Turnover intention was significantly related to QWL. Using SMR, 26% of the variance in turnover intention was explained by the QWL, p < 0.001, with R² = .263. Further analysis using HMR found that the total variance explained by the model as a whole (demographics and QWL) was 32.1%, p < 0.001. QWL explained an additional 19% of the variance in turnover intention, after controlling for demographic variables. Conclusions Creating and maintaining a healthy work life for PHC nurses is very important to improve their work satisfaction, reduce turnover, enhance productivity and improve nursing care outcomes.

Turnover in entrepreneurial teams : are spousal teams different?

Entrepreneurial teams are increasingly prevalent. An important stage in new venture formation involves the recruitment and retention of team members, characterised by changes in the membership of the ET. ET turnover involves members joining or leaving the team, which could have positive or negative effects on the team and the firm. Spousal Entrepreneurial Teams (SET) are a frequently occurring, yet unique type of ET, with very distinct characteristics and dynamics. In an SET turnover is likely to be have different meanings and effects than in Other Entrepreneurial Teams. This research asks: What influences members joining and leaving entrepreneurial teams? And are SET different? Using data from 561 young firms over X years, we find significant differences between SET and NSET. Specifically, we find X.

Association study of a functional variant in intron 8 of the dopamine transporter gene and migraine susceptibility

Migraine is a common, genetically influenced neurovascular disorder. The dopamine transporter gene is a candidate for migraine association studies. This study tested a functionally linked variable number tandem repeat (VNTR) in intron 8 of the dopamine transporter gene (DATInt8) in 550 migraine cases (401 with aura, 149 without aura) and 550 non-migraine controls. Chi-squared analysis of the DATInt8 revealed that the allele and genotype frequency distributions for migraine cases (including subtype analysis) and controls were not different (P > 0.1). These findings offer no evidence for an association of the DATInt8 with migraine with and without aura and therefore do not implicate the dopamine transporter gene as a modifier of migraine risk.