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Background: Methods are needed to analyze small amounts of samples for variation in disease-causing genes. One means is to couple the sensitivity and multiplexing capability of the ligation detection reaction (LDR) with the use of simple glass slides specifically functionalized with a novel polymer coating to enhance sensitivity. Methods: We developed an array-based genotyping assay based on glass slides coated with copolymer (N,N-dimethylacrylamide, N,N-acryloyloxysuccinimide, and 3-(trimethoxysilyl)propyl methacrylate). The assay consists of an LDR with genomic DNA followed by a universal PCR (U-PCR) of genomic DNA–templated LDR product. The LDR occurs in the presence of 3 primers for each sequence variant under investigation: 2 distinguishing primers (allele specific and perfectly complementary to wild-type and mutant alleles) and 1 common locus-specific primer. The 2 allele-specific primers have different capture sequences for binding different complementary probes on a tag array. The LDR product templated from genomic DNA is made fluorescent during the U-PCR via incorporation of a Cy5-labeled universal primer into all LDR products; detection occurs on the coated glass slides. Results: The assay was designed to detect 7 prevalent mutations in the β-globin gene (HBB, hemoglobin, beta) in a multiplex format, and signals for the different alleles are detected by their fluorescence. The assay was applied to 40 genomic DNA samples from both control individuals and patients with known β-thalassemia mutations. Results show good correspondence between the patients’ genotypes as assessed by DNA sequence analysis and those generated from the LDR assays. Conclusions: The developed technology allows accurate identification of sequence variants in a simple, cost-effective way and offers good flexibility for scaling to other applications with different numbers of single-nucleotide polymorphisms or mutations to be detected.

Background: Mutations in the retina-specific ABC transporter (ABCA4) gene have been associated with several forms of macular degenerations. Because the high complexity of the molecular genotype makes scanning of the ABCA4 gene cumbersome, we describe here the first use of denaturing HPLC (DHPLC) to screen for ABCA4 mutations. Methods: Temperature conditions were designed for all 50 exons based on effective separation of 83 samples carrying 86 sequence variations and 19 mutagenized controls. For validation, samples from 23 previously characterized Stargardt patients were subjected to DHPLC profiling. Subsequently, samples from a cohort of 30 patients affected by various forms of macular degeneration were subjected to DHPLC scanning under the same conditions. Results: DHPLC profiling not only identified all 132 sequence alterations previously detected by double-gradient denaturing gradient gel electrophoresis but also identified 5 sequence alterations that this approach had missed. Moreover, DHPLC scanning of an additional panel of 30 previously untested patients led to the identification of 26 different mutations and 29 polymorphisms, accounting for 203 sequence variations on 29 of the 30 patients screened. In total, the DHPLC approach allowed us to identify 16 mutations that had never been reported before. Conclusions: These results provide strong support for the use of DHPLC for molecular characterization of the ABCA4 gene.

Several microarray systems that have been developed for the detection of human globin gene mutations are appropriate for medium- or high-throughput applications, and the systems that are commercially available include a microelectronic array system, an arrayed primer extension- based system, and systems involving tagged single-base extension with hybridization to either universal glass or flow-through microarrays (7). DNA SAMPLES After we obtained informed consent according to the local Institutional Review Board Committee, we collected genomic DNA samples from control individuals with a wild-type HBB gene and from patients homozygous or heterozygous for a variety of HBB mutations (OMIM accession no. 141900): IVS-I nt 1 (G>A), IVS-I nt 6 (T>C), IVS-I nt 110 (OA), codon 39 (C>T), IVS-II nt 1 (OA), IVS-II nt 745 (C>G), and Hb S ( A>T). Before ligation, we fragmented each sample by heating for 5 min at 99 °C. LIGASE DETECTION REACTION The LDR is performed in a 10-µL reaction containing 200 ng of fragmented genomic DNA, 1 nmol/L of each distinguishing primer (wild-type and mutant), 2 nmol/L common primer, 1x Pfu ligase buffer [20 mmol/L TrisHCl, pH 7.5, 20 mmol/L KCl, 10 mmol/L MgC2, 1 mlVL Igepal, 10 µmol/L ribose ATP, and 1 mmol/L dithiothrcitol (Strategene Cloning Systems)], 40 g/I polyethylene glycol 8000, and 2 U Pfu ligase (Slralagenc Cloning Systems).

This thesis concerns how memory processes and biases may be implicated in substance dependence. The literature review begins by outlining different models of drug dependence and research into cognitive function in addiction. It then outlines the structure of long-term memory, before reviewing and discussing the implications of the limited evidence available for cognitive biases operating in semantic and episodic memory of individuals who are addicted. The review ends by discussing implications for future research and by suggesting additional paradigms that could be used to further investigate the role of memory in addiction. The empirical paper describes a study which investigated semantic priming and verbal learning in current opiate users on a methadone maintenance programme, ex opiate-users in rehabilitation and healthy non-using controls. It is one part of a joint project, the other part having been carried out by a fellow clinical psychology trainee who investigated response inhibition in the same sample population. Both current and ex-users showed preserved semantic priming. Ex-users showed a verbal learning impairment compared with controls, whilst both current and ex-users showed impairment in recalling semantically unrelated words, but intact recall of semantically related words. This may suggest a relative impairment in the ability of opiate users and ex-users to impose structure to unstructured information (e.g. use of mnemonic strategies) and a greater reliance on semantic memory when encoding new information. Providing opiate using clients with highly structured information may be beneficial to intervention. The critical appraisal gives a reflective account of the research process, the study and the treatment implications of the findings.

Background One of the causes of male infertility is associated with altered spermatozoa motility. These sperm features are frequently analyzed by image-based approaches, which, despite allowing the acquisition of crucial parameters to assess sperm motility, they are unable to provide details regarding the flagellar beating forces, which have been neglected until now. Results In this work we exploit Fluidic Force Microscopy to investigate and quantify the forces associated with the flagellar beating frequencies of human spermatozoa. The analysis is performed on two groups divided according to the progressive motility of semen samples, as identified by standard clinical protocols. In the first group, 100% of the spermatozoa swim linearly (100% progressive motility), while, in the other, spermatozoa show both linear and circular motility (identified as 80 − 20% progressive motility). Significant differences in flagellar beating forces between spermatozoa from semen sample with different progressive motility are observed. Particularly, linear motile spermatozoa exhibit forces higher than those with a circular movement. Conclusions This research can increase our understanding of sperm motility and the role of mechanics in fertilization, which could help us unveil some of the causes of idiopathic male infertility.

Purpose Diverse cultures may make people behave differently and this, in turn, can impact project management. While the relationship between culture and project success has been widely explored, there is a need of addressing the gap in the relationship between culture and project management performance outcomes, that is, the performance in implementing project management processes and practices. The purpose of this paper is to investigate this gap by studying the role of cultural dimensions on project management performance. Design/methodology/approach An explorative survey including 200 observations relating to the experiences of project managers with a big experience on projects involving many different national cultures has been conducted to collect primary data on the relationship between the nationality observed and the project management performance outcomes shown. Nationality has been used as a proxy to link individual cultural dimensions and project management performance. Findings The results of this paper show that individualism impacts project dynamics and project control positively. Moreover, masculinity impacts project dynamics positively, and uncertainty avoidance impacts project control negatively. When recognized, different cultural dimensions can drive project management performance outcomes. The increasing awareness on this topic can be a valid instrument to control the cultural effect and take advantage of it to enhance project success. Originality/value This study contributes to the theory of project management by recognizing linkages between cultural dimensions and project management performance. Moreover, this study overcomes the concept of nationality, focusing on individuals and their unique set of cultural dimensions.

To explore perceptions and unconscious psychological processes underlying handwashing behaviours of intensive care nurses, to implement organisational innovations for improving hand hygiene in clinical practice. An action-research intervention was performed in 2012 and 2013 in the intensive care unit of a public hospital in Italy, consisting of: structured interviews, semantic analysis, development and validation of a questionnaire, team discussion, project design and implementation. Five general workers, 16 staff nurses and 53 nurse students participated in the various stages. Social handwashing emerged as a structured and efficient habit, which follows automatically the pattern “cue/behaviour/gratification” when hands are perceived as “dirty”. The perception of “dirt” starts unconsciously the process of social washing also in professional settings. Professional handwashing is perceived as goal-directed. The main concern identified is the fact that washing hands requires too much time to be performed in a setting of urgency. These findings addressed participants to develop a professional “habit-directed” hand hygiene procedure, to be implemented at beginning of workshifts. Handwashing is a ritualistic behaviour driven by deep and unconscious patterns, and social habits affect professional practice. Creating professional habits of hand hygiene could be a key solution to improve compliance in intensive care settings.

To evaluate the long-term effectiveness of an action research intervention aimed at improving hand hygiene in an intensive care unit of a public hospital in Italy. An observational, prospective before-after study was carried out.Compliance with hand hygiene was estimated by measuring the utilization of hand hygiene products before the intervention and four years after the end of the project. Products used were the following: detergent liquid soap, antiseptic liquid soap and alcohol-based hand gel. Endpoints were quantity consumed (in grams) for each product category. Quantitative consumptions per workshift were compared. In 2017 the median consumption of antiseptic liquid soap and alcohol-based hand gel per workshift was significantly higher than in 2012 (111.5 g vs 72.5 g, p = 0.014, and 18.0 g vs 5.0 g, p < 0.001). Odds in favour of a higher value in 2017 were 1.99:1 (CI95%: 1.19:1 to 3.73:1) for antiseptic solution, and 5.39:1 (CI95%: 3.09:1 to 13.61:1) for antiseptic gel. Covariates were not associated with consumption of products, and this made it possible to compare the measurements in the two data collections. Results of this study support the long-term effectiveness of the action research intervention to improve practices of hand hygiene in an intensive care setting.

To establish categories of professionals' attitudes toward incident reporting by analyzing the trends in incident reporting while accounting for general risk indicators. The incident reporting system was evaluated over 6 years. Reporting rates, stratified by year and profession, were estimated using the non-mandatory reported events/full-time equivalent (NM-IR/FTE) rate. Other indicators were collected using the hospital's official database. Staff attitudes toward self-reporting were analyzed. Univariate and multivariable analyses were performed. A 1000-bed Italian academic hospital. Staff of the hospital (over 3200 professionals). None. NM-IT/FTE rates, self-reported rates, patient complaints/praises, work accidents among professionals and 30-day readmissions. The overall reporting rate was 0.44 (95% confidence interval [CI]: 0.42-0.46) among doctors and 0.40 (95% CI: 0.39-0.41) among nurses. Between 2010 and 2015, only the doctors' reporting rate increased significantly (P = 0.04), from 0.29 (95% CI: 0.25-0.34) to 0.67 (95% CI: 0.60-0.73). Patient complaints decreased from 384 to 224 (P < 0.001) and work accidents decreased from 296 to 235 (P = 0.01), while other indicators remained constant. Multivariable logistic regression showed that self-reporting was more likely among nurses than doctors (odds ratio: 1.51; 95% CI: 1.31-1.73) and for severe events than near misses (odds ratio: 1.78; 95% CI: 1.11-2.87). Because the doctors' reporting rates increased during the study period, doctors may be more likely to report adverse events than nurses, although nurses reported more events. Incident reporting trends and other routinely collected risk indicators may be useful to improve our understanding and measurement of patient safety issues.

Bacterial infections are a common complication in patients with decompensated liver cirrhosis. The complex landscape of cirrhosis, characterized by immune paralysis and an exhausted response to exogenous triggers, explains the higher prevalence of such infections, particularly in advanced disease stages. In clinical practice, the onset of a bacterial infection can lead to further deterioration of hepatic and extra-hepatic function, potentially resulting in acute decompensation or acute-on-chronic liver failure. This has significant clinical implications, particularly for patients awaiting a transplant. In this review, we will discuss the latest evidence on the diagnosis and therapy of bacterial infections in patients with decompensated cirrhosis. Additionally, we will analyze the impact of bacterial infections in the context of liver transplantation, discussing debated topics such as the timing of transplantation in patients with infections, potential implications for prioritization, effects on post-operative recovery, grafts, and patient survival.