A mass-conservative control volume-finite element method for solving Richards’ equation in heterogeneous porous media

We present a mass-conservative vertex-centred finite volume method for efficiently solving the mixed form of Richards’ equation in heterogeneous porous media. The spatial discretisation is particularly well-suited to heterogeneous media because it produces consistent flux approximations at quadrature points where material properties are continuous. Combined with the method of lines, the spatial discretisation gives a set of differential algebraic equations amenable to solution using higher-order implicit solvers. We investigate the solution of the mixed form using a Jacobian-free inexact Newton solver, which requires the solution of an extra variable for each node in the mesh compared to the pressure-head form. By exploiting the structure of the Jacobian for the mixed form, the size of the preconditioner is reduced to that for the pressure-head form, and there is minimal computational overhead for solving the mixed form. The proposed formulation is tested on two challenging test problems. The solutions from the new formulation offer conservation of mass at least one order of magnitude more accurate than a pressure head formulation, and the higher-order temporal integration significantly improves both the mass balance and computational efficiency of the solution.

The pursuit of thinness : an outcome study of anorexia nervosa

INTRODUCTION: Anorexia nervosa (AN) is a growing problem among young female Singaporeans. We studied the demographics and follow-up data of AN patients referred to dietitians for nutritional intervention. METHODS: A retrospective nutritional notes review was done on 94 patients seen from 1992 to 2004. All patients were given nutritional intervention, which included individualised counselling for weight gain, personalised diet plan, correction of poor dietary intake and correction of perception towards healthy eating. We collected data on body mass index (BMI), patient demographics and outcome. RESULTS: 96 percent of the patients were female and 86.2 percent were Chinese. The median BMI at initial consultation was 14.7 kilogramme per square metre (range, 8.6-18.8 kilogramme per square metre). 76 percent were between 13 and 20 years old. 83 percent of the patients came back for follow-up appointments with the dietitians in addition to consultation with the psychiatrist. Overall, there was significant improvement in weight and BMI from an average 37 kg to 41 kg and 14.7 kilogramme per square metre to 16.4 kilogramme per square metre, respectively, between the fi rst and fi nal consultations (p-value is less than 0.001). The average duration of followup was about eight months. Among the patients on follow-up, 68 percent showed improvement with an average weight gain of 6 kg. Patients that improved had more outpatient follow-up sessions with the dietitians (4.2 consultations versus 1.6 consultations; p-value is less than 0.05), lower BMI at presentation (14.2 kilogramme per square metre versus 15.7 kilogramme per square metre; p-value is less than 0.01) and shorter duration of disease at presentation (one year versus three years; p-value is less than 0.05) compared with those who did not improve. Seven patients with the disease for more than two years did not show improvement with follow-up. CONCLUSION: We gained valuable understanding of the AN patients referred to our tertiary hospital for treatment, two-thirds of whom improved with adequate follow-up treatment. Patients that had suffered AN longer before seeking help appeared more resistant to improvement.

Influence of culture conditions on the molecular signature of mesenchymal stem cells

Cell based therapies require cells capable of self renewal and differentiation, and a prerequisite is the ability to prepare an effective dose of ex vivo expanded cells for autologous transplants. The in vivo identification of a source of physiologically relevant cell types suitable for cell therapies is therefore an integral part of tissue engineering. Bone marrow is the most easily accessible source of mesenchymal stem cells (MSCs), and harbours two distinct populations of adult stem cells; namely hematopoietic stem cells (HSCs) and bone mesenchymal stem cells (BMSCs). Unlike HSCs, there are yet no rigorous criteria for characterizing BMSCs. Changing understanding about the pluripotency of BMSCs in recent studies has expanded their potential application; however, the underlying molecular pathways which impart the features distinctive to BMSCs remain elusive. Furthermore, the sparse in vivo distribution of these cells imposes a clear limitation to their in vitro study. Also, when BMSCs are cultured in vitro there is a loss of the in vivo microenvironment which results in a progressive decline in proliferation potential and multipotentiality. This is further exacerbated with increased passage number, characterized by the onset of senescence related changes. Accordingly, establishing protocols for generating large numbers of BMSCs without affecting their differentiation potential is necessary. The principal aims of this thesis were to identify potential molecular factors for characterizing BMSCs from osteoarthritic patients, and also to attempt to establish culture protocols favourable for generating large number of BMSCs, while at the same time retaining their proliferation and differentiation potential. Previously published studies concerning clonal cells have demonstrated that BMSCs are heterogeneous populations of cells at various stages of growth. Some cells are higher in the hierarchy and represent the progenitors, while other cells occupy a lower position in the hierarchy and are therefore more committed to a particular lineage. This feature of BMSCs was made evident by the work of Mareddy et al., which involved generating clonal populations of BMSCs from bone marrow of osteoarthritic patients, by a single cell clonal culture method. Proliferation potential and differentiation capabilities were used to group cells into fast growing and slow growing clones. The study presented here is a continuation of the work of Mareddy et al. and employed immunological and array based techniques to identify the primary molecular factors involved in regulating phenotypic characteristics exhibited by contrasting clonal populations. The subtractive immunization (SI) was used to generate novel antibodies against favourably expressed proteins in the fast growing clonal cell population. The difference between the clonal populations at the transcriptional level was determined using a Stem Cell RT2 Profiler TM PCR Array which focuses on stem cell pathway gene expression. Monoclonal antibodies (mAb) generated by SI were able to effectively highlight differentially expressed antigenic determinants, as was evident by Western blot analysis and confocal microscopy. Co-immunoprecipitation, followed by mass spectroscopy analysis, identified a favourably expressed protein as the cytoskeletal protein vimentin. The stem cell gene array highlighted genes that were highly upregulated in the fast growing clonal cell population. Based on their functions these genes were grouped into growth factors, cell fate determination and maintenance of embryonic and neural stem cell renewal. Furthermore, on a closer analysis it was established that the cytoskeletal protein vimentin and nine out of ten genes identified by gene array were associated with chondrogenesis or cartilage repair, consistent with the potential role played by BMSCs in defect repair and maintaining tissue homeostasis, by modulating the gene expression pattern to compensate for degenerated cartilage in osteoarthritic tissues. The gene array also presented transcripts for embryonic lineage markers such as FOXA2 and Sox2, both of which were significantly over expressed in fast growing clonal populations. A recent groundbreaking study by Yamanaka et al imparted embryonic stem cell (ESCs) -like characteristic to somatic cells in a process termed nuclear reprogramming, by the ectopic expression of the genes Sox2, cMyc and Oct4. The expression of embryonic lineage markers in adult stem cells may be a mechanism by which the favourable behaviour of fast growing clonal cells is determined and suggests a possible active phenomenon of spontaneous reprogramming in fast growing clonal cells. The expression pattern of these critical molecular markers could be indicative of the competence of BMSCs. For this reason, the expression pattern of Sox2, Oct4 and cMyc, at various passages in heterogeneous BMSCs population and tissue derived cells (osteoblasts and chondrocytes), was investigated by a real-time PCR and immunoflourescence staining. A strong nuclear staining was observed for Sox2, Oct4 and cMyc, which gradually weakened accompanied with cytoplasmic translocation after several passage. The mRNA and protein expression of Sox2, Oct4 and cMyc peaked at the third passage for osteoblasts, chondrocytes and third passage for BMSCs, and declined with each subsequent passage, indicating towards a possible mechanism of spontaneous reprogramming. This study proposes that the progressive decline in proliferation potential and multipotentiality associated with increased passaging of BMSCs in vitro might be a consequence of loss of these propluripotency factors. We therefore hypothesise that the expression of these master genes is not an intrinsic cell function, but rather an outcome of interaction of the cells with their microenvironment; this was evident by the fact that when removed from their in vivo microenvironment, BMSCs undergo a rapid loss of stemness after only a few passages. One of the most interesting aspects of this study was the integration of factors in the culture conditions, which to some extent, mimicked the in vivo microenvironmental niche of the BMSCs. A number of studies have successfully established that the cellular niche is not an inert tissue component but is of prime importance. The total sum of stimuli from the microenvironment underpins the complex interplay of regulatory mechanisms which control multiple functions in stem cells most importantly stem cell renewal. Therefore, well characterised factors which affect BMSCs characteristics, such as fibronectin (FN) coating, and morphogens such as FGF2 and BMP4, were incorporated into the cell culture conditions. The experimental set up was designed to provide insight into the expression pattern of the stem cell related transcription factors Sox2, cMyc and Oct4, in BMSCs with respect to passaging and changes in culture conditions. Induction of these pluripotency markers in somatic cells by retroviral transfection has been shown to confer pluripotency and an ESCs like state. Our study demonstrated that all treatments could transiently induce the expression of Sox2, cMyc and Oct4, and favourably affect the proliferation potential of BMSCs. The combined effect of these treatments was able to induce and retain the endogenous nuclear expression of stem cell transcription factors in BMSCs over an extended number of in vitro passages. Our results therefore suggest that the transient induction and manipulation of endogenous expression of transcription factors critical for stemness can be achieved by modulating the culture conditions; the benefit of which is to circumvent the need for genetic manipulations. In summary, this study has explored the role of BMSCs in the diseased state of osteoarthritis, by employing transcriptional profiling along with SI. In particular this study pioneered the use of primary cells for generating novel antibodies by SI. We established that somatic cells and BMSCs have a basal level of expression of pluripotency markers. Furthermore, our study indicates that intrinsic signalling mechanisms of BMSCs are intimately linked with extrinsic cues from the microenvironment and that these signals appear to be critical for retaining the expression of genes to maintain cell stemness in long term in vitro culture. This project provides a basis for developing an “artificial niche” required for reversion of commitment and maintenance of BMSC in their uncommitted homeostatic state.

Heat strain, hydration status, and symptoms of heat illness in surface mine workers

The aim of the research program was to evaluate the heat strain, hydration status, and heat illness symptoms experienced by surface mine workers. An initial investigation involved 91 surface miners completing a heat stress questionnaire; assessing the work environment, hydration practices, and heat illness symptom experience. The key findings included 1) more than 80 % of workers experienced at least one symptom of heat illness over a 12 month period; and 2) the risk of moderate symptoms of heat illness increased with the severity of dehydration. These findings highlight a health and safety concern for surface miners, as experiencing symptoms of heat illness is an indication that the physiological systems of the body may be struggling to meet the demands of thermoregulation. To illuminate these findings a field investigation to monitor the heat strain and hydration status of surface miners was proposed. Two preliminary studies were conducted to ensure accurate and reliable data collection techniques. Firstly, a study was undertaken to determine a calibration procedure to ensure the accuracy of core body temperature measurement via an ingestible sensor. A water bath was heated to several temperatures between 23 . 51 ¢ªC, allowing for comparison of the temperature recorded by the sensors and a traceable thermometer. A positive systematic bias was observed and indicated a need for calibration. It was concluded that a linear regression should be developed for each sensor prior to ingestion, allowing for a correction to be applied to the raw data. Secondly, hydration status was to be assessed through urine specific gravity measurement. It was foreseeable that practical limitations on mine sites would delay the time between urine collection and analysis. A study was undertaken to assess the reliability of urine analysis over time. Measurement of urine specific gravity was found to be reliable up to 24 hours post urine collection and was suitable to be used in the field study. Twenty-nine surface miners (14 drillers [winter] and 15 blast crew [summer]) were monitored during a normal work shift. Core body temperature was recorded continuously. Average mean core body temperature was 37.5 and 37.4 ¢ªC for blast crew and drillers, with average maximum body temperatures of 38.0 and 37.9 ¢ªC respectively. The highest body temperature recorded was 38.4 ¢ªC. Urine samples were collected at each void for specific gravity measurement. The average mean urine specific gravity was 1.024 and 1.021 for blast crew and drillers respectively. The Heat Illness Symptoms Index was used to evaluate the experience of heat illness symptoms on shift. Over 70 % of drillers and over 80 % of blast crew reported at least one symptom. It was concluded that 1) heat strain remained within the recommended limits for acclimatised workers; and 2) the majority of workers were dehydrated before commencing their shift, and tend to remain dehydrated for the duration. Dehydration was identified as the primary issue for surface miners working in the heat. Therefore continued study focused on investigating a novel approach to monitoring hydration status. The final aim of this research program was to investigate the influence dehydration has on intraocular pressure (IOP); and subsequently, whether IOP could provide a novel indicator of hydration status. Seven males completed 90 minutes of walking in both a cool and hot climate with fluid restriction. Hydration variables and intraocular pressure were measured at baseline and at 30 minute intervals. Participants became dehydrated during the trial in the heat but maintained hydration status in the cool. Intraocular pressure progressively declined in the trial in the heat but remained relatively stable when hydration was maintained. A significant relationship was observed between intraocular pressure and both body mass loss and plasma osmolality. This evidence suggests that intraocular pressure is influenced by changes in hydration status. Further research is required to determine if intraocular pressure could be utilised as an indirect indicator of hydration status.

Natural convection flow with combined buoyancy effects due to thermal and mass diffusion in a thermally stratified media

We present here a numerical study of laminar doubly diffusive free convection flows adjacent to a vertical surface in a stable thermally stratified medium. The governing equations of mass, momentum, energy and species are non-dimensionalized. These equations have been solved by using an implicit finite difference method and local non-similarity method. The results show many interesting aspects of complex interaction of the two buoyant mechanisms that have been shown in both the tabular as well as graphical form.

Malnutrition and clinical outcomes in elderly patients from a Singapore acute hospital

Older adults, especially those acutely ill, are vulnerable to developing malnutrition due to a range of risk factors. The high prevalence and extensive consequences of malnutrition in hospitalised older adults have been reported extensively. However, there are few well-designed longitudinal studies that report the independent relationship between malnutrition and clinical outcomes after adjustment for a wide range of covariates. Acutely ill older adults are exceptionally prone to nutritional decline during hospitalisation, but few reports have studied this change and impact on clinical outcomes. In the rapidly ageing Singapore population, all this evidence is lacking, and the characteristics associated with the risk of malnutrition are also not well-documented. Despite the evidence on malnutrition prevalence, it is often under-recognised and under-treated. It is therefore crucial that validated nutrition screening and assessment tools are used for early identification of malnutrition. Although many nutrition screening and assessment tools are available, there is no universally accepted method for defining malnutrition risk and nutritional status. Most existing tools have been validated amongst Caucasians using various approaches, but they are rarely reported in the Asian elderly and none has been validated in Singapore. Due to the multiethnicity, cultural, and language differences in Singapore older adults, the results from non-Asian validation studies may not be applicable. Therefore it is important to identify validated population and setting specific nutrition screening and assessment methods to accurately detect and diagnose malnutrition in Singapore. The aims of this study are therefore to: i) characterise hospitalised elderly in a Singapore acute hospital; ii) describe the extent and impact of admission malnutrition; iii) identify and evaluate suitable methods for nutritional screening and assessment; and iv) examine changes in nutritional status during admission and their impact on clinical outcomes. A total of 281 participants, with a mean (+SD) age of 81.3 (+7.6) years, were recruited from three geriatric wards in Tan Tock Seng Hospital over a period of eight months. They were predominantly Chinese (83%) and community-dwellers (97%). They were screened within 72 hours of admission by a single dietetic technician using four nutrition screening tools [Tan Tock Seng Hospital Nutrition Screening Tool (TTSH NST), Nutritional Risk Screening 2002 (NRS 2002), Mini Nutritional Assessment-Short Form (MNA-SF), and Short Nutritional Assessment Questionnaire (SNAQ©)] that were administered in no particular order. The total scores were not computed during the screening process so that the dietetic technician was blinded to the results of all the tools. Nutritional status was assessed by a single dietitian, who was blinded to the screening results, using four malnutrition assessment methods [Subjective Global Assessment (SGA), Mini Nutritional Assessment (MNA), body mass index (BMI), and corrected arm muscle area (CAMA)]. The SGA rating was completed prior to computation of the total MNA score to minimise bias. Participants were reassessed for weight, arm anthropometry (mid-arm circumference, triceps skinfold thickness), and SGA rating at discharge from the ward. The nutritional assessment tools and indices were validated against clinical outcomes (length of stay (LOS) >11days, discharge to higher level care, 3-month readmission, 6-month mortality, and 6-month Modified Barthel Index) using multivariate logistic regression. The covariates included age, gender, race, dementia (defined using DSM IV criteria), depression (defined using a single question “Do you often feel sad or depressed?”), severity of illness (defined using a modified version of the Severity of Illness Index), comorbidities (defined using Charlson Comorbidity Index, number of prescribed drugs and admission functional status (measured using Modified Barthel Index; MBI). The nutrition screening tools were validated against the SGA, which was found to be the most appropriate nutritional assessment tool from this study (refer section 5.6) Prevalence of malnutrition on admission was 35% (defined by SGA), and it was significantly associated with characteristics such as swallowing impairment (malnourished vs well-nourished: 20% vs 5%), poor appetite (77% vs 24%), dementia (44% vs 28%), depression (34% vs 22%), and poor functional status (MBI 48.3+29.8 vs 65.1+25.4). The SGA had the highest completion rate (100%) and was predictive of the highest number of clinical outcomes: LOS >11days (OR 2.11, 95% CI [1.17- 3.83]), 3-month readmission (OR 1.90, 95% CI [1.05-3.42]) and 6-month mortality (OR 3.04, 95% CI [1.28-7.18]), independent of a comprehensive range of covariates including functional status, disease severity and cognitive function. SGA is therefore the most appropriate nutritional assessment tool for defining malnutrition. The TTSH NST was identified as the most suitable nutritional screening tool with the best diagnostic performance against the SGA (AUC 0.865, sensitivity 84%, specificity 79%). Overall, 44% of participants experienced weight loss during hospitalisation, and 27% had weight loss >1% per week over median LOS 9 days (range 2-50). Wellnourished (45%) and malnourished (43%) participants were equally prone to experiencing decline in nutritional status (defined by weight loss >1% per week). Those with reduced nutritional status were more likely to be discharged to higher level care (adjusted OR 2.46, 95% CI [1.27-4.70]). This study is the first to characterise malnourished hospitalised older adults in Singapore. It is also one of the very few studies to (a) evaluate the association of admission malnutrition with clinical outcomes in a multivariate model; (b) determine the change in their nutritional status during admission; and (c) evaluate the validity of nutritional screening and assessment tools amongst hospitalised older adults in an Asian population. Results clearly highlight that admission malnutrition and deterioration in nutritional status are prevalent and are associated with adverse clinical outcomes in hospitalised older adults. With older adults being vulnerable to risks and consequences of malnutrition, it is important that they are systematically screened so timely and appropriate intervention can be provided. The findings highlighted in this thesis provide an evidence base for, and confirm the validity of the current nutrition screening and assessment tools used among hospitalised older adults in Singapore. As the older adults may have developed malnutrition prior to hospital admission, or experienced clinically significant weight loss of >1% per week of hospitalisation, screening of the elderly should be initiated in the community and continuous nutritional monitoring should extend beyond hospitalisation.

Chlorzoxazone, an SK-type potassium channel activator used in humans, reduces excessive alcohol intake in rats

Background Alcoholism imposes a tremendous social and economic burden. There are relatively few pharmacological treatments for alcoholism, with only moderate efficacy, and there is considerable interest in identifying additional therapeutic options. Alcohol exposure alters SK-type potassium channel (SK) function in limbic brain regions. Thus, positive SK modulators such as chlorzoxazone (CZX), a US Food and Drug Administration–approved centrally acting myorelaxant, might enhance SK function and decrease neuronal activity, resulting in reduced alcohol intake. Methods We examined whether CZX reduced alcohol consumption under two-bottle choice (20% alcohol and water) in rats with intermittent access to alcohol (IAA) or continuous access to alcohol (CAA). In addition, we used ex vivo electrophysiology to determine whether SK inhibition and activation can alter firing of nucleus accumbens (NAcb) core medium spiny neurons. Results Chlorzoxazone significantly and dose-dependently decreased alcohol but not water intake in IAA rats, with no effects in CAA rats. Chlorzoxazone also reduced alcohol preference in IAA but not CAA rats and reduced the tendency for rapid initial alcohol consumption in IAA rats. Chlorzoxazone reduction of IAA drinking was not explained by locomotor effects. Finally, NAcb core neurons ex vivo showed enhanced firing, reduced SK regulation of firing, and greater CZX inhibition of firing in IAA versus CAA rats. Conclusions The potent CZX-induced reduction of excessive IAA alcohol intake, with no effect on the more moderate intake in CAA rats, might reflect the greater CZX reduction in IAA NAcb core firing observed ex vivo. Thus, CZX could represent a novel and immediately accessible pharmacotherapeutic intervention for human alcoholism. Key Words: Alcohol intake; intermittent; neuro-adaptation; nucleus accumbens; SK potassium channel

Loss-of-function fibroblast growth factor receptor-2 mutations in melanoma

We report that 10% of melanoma tumors and cell lines harbor mutations in the fibroblast growth factor receptor 2 (FGFR2) gene. These novel mutations include three truncating mutations and 20 missense mutations occurring at evolutionary conserved residues in FGFR2 as well as among all four FGFRs. The mutation spectrum is characteristic of those induced by UV radiation. Mapping of these mutations onto the known crystal structures of FGFR2 followed by in vitro and in vivo studies show that these mutations result in receptor loss of function through several distinct mechanisms, including loss of ligand binding affinity, impaired receptor dimerization, destabilization of the extracellular domains, and reduced kinase activity. To our knowledge, this is the first demonstration of loss-of-function mutations in a class IV receptor tyrosine kinase in cancer. Taken into account with our recent discovery of activating FGFR2 mutations in endometrial cancer, we suggest that FGFR2 may join the list of genes that play context-dependent opposing roles in cancer.