Dissertations / Theses on the topic 'Deprivation'

This thesis is concerned with rights-based justifications for redistribution. Orthodox views are critically examined in three of the chapters. The case against fundamental moral rights to welfare, not derived from other more fundamental moral rights or principles, is pressed in chapter three. Chapter five distinguishes between rights-based and equality-based justifications for redistribution and argues that Ronald Dworkin's idea of a right to equal respect and concern is best understood as an equality-based justification. The enabling model of rights and deprivation is introduced in chapter six. This model says that liberty rights require that others ensure that the right-holder enjoys the means to do what he or she has the right to do as well as not interfere with him or her doing what he or she has the right to do. It is found to break down because it is unable to accommodate the right to do wrong. The other four chapters are concerned with defending an alternative model of rights and deprivation. The groundwork for this alternative model - the development model of rights and deprivation - is laid in chapters two and four. Chapter two presents a person-affecting theory of rights. The two principal conclusions of the development model of rights and deprivation are defended in chapter seven. It is argued, first, that from both of the abstract moral rights to liberty introduced in chapter four flow certain derivative rights against others to have one's needs met and, second, that the state is required to promote and protect particular forms of culture as well as to meet certain sorts of personal needs including special needs, collective needs, and the unmet personal needs that arise when the prevailing methods of meeting those needs breaks down. The final chapter discusses two general issues relating to the development model of rights and deprivation.

1- ROLE OF SIRT1 IN THE REGULATION OF FATTY ACID OXIDATION AND KETOGENESIS UNDER DIFFERENT NUTRIONAL CHANGES. The homolog of the yeast silencing information regulator2 (SIRT1) has been implicated in several aspects of food limitation and caloric restriction in mammals. We have observed that there were no important changes, between wild type (WT) and SIRT1 liver-specific knockout (LKO) mice subjected to either CR or high fat diet (HFD), in the mRNA expression of Cpt1a, Cpt2 and Hmgcs2. SIRT1 had been shown to control hepatic glyconeogenic/glycolytic pathways in response to nutrients (Rodgers et al., 2005). So, we have hypothesized that SIRT1 could have a role in the metabolic adaptation to the changes of nutrient of weaning, when milk is replaced by the adult diet which contains less fat and more carbohydrate. Neither fatty acid oxidation (Cpt1a), ketogenesis (Hmgcs2), nor gluconeogenic (Pck1) liver pathways were significantly affected by the liver-specific knockdown of SIRT1, in both suckling and post-weaning conditions. If SIRT1 is involved in the response to aging, old LKO mice might be more susceptible to age-associated diseases as obesity, type 2 diabetes, hypertension, etc. To test this hypothesis we first weight and performed a glucose tolerance test in 7 months-old mice. There were no differences in weight neither in glucose tolerance between WT and LKO mice. Using a cell system, PPARα induced the expression of its known target genes CPT1A, HMGCS2, and FGF21 in HepG2 cells. SIRT1 overexpression by itself had almost no effect, although it increased PPARα induction of its target genes. We have interfered SIRT1 in these cells, and PPARα-induced expression of PEPCK and FGF21 was SIRT1-dependent. We have fasted WT and LKO mice for 15h and we found that the expression of Pck1 in liver was moderately but significantly induced in SIRT1-LKO mice after fasting, consistent with an increase in glucose levels. However, neither G6pase nor Pgc1α mRNA levels were affected. As expected, liver mRNA levels of Cpt1a, Hmgcs2, and Fgf21 were also induced upon fasting. However, Cpt1a and Hmgcs2 mRNA transcripts were comparable in fasted WT versus LKO mice, while Fgf21 expression was reduced around 40% in LKO mice liver. This result was consistent with the fact that fasting induction of FGF21 serum levels was also impaired in LKO mice. 2- ROLE OF SIRT1 IN THE HMGCS2 REGULATION OF FGF21 EXPRESSION. (Article 1: Human HMGCS2 regulates fatty acid oxidation and FGF21 expression in HepG2 cells. Vilà-Brau et al., 2011) Recently, our group has seen that HMGCS2 expression stimulates FGF21 expression and that these events are dependent on HMGCS2 activity. A catalytic dead mutant (C166A) failed to induce either fatty acid β-oxidation or FGF21 expression, whereas acetoacetate (an oxidized form of ketone bodies) could stimulate FGF21 mRNA expression in a dose-dependent manner. Because ketone bodies production implies the reduction of acetoacetate to β- hydroxybutyrate with the concomitant generation of NAD+ (Hegardt et al, 1999), and SIRT1 is a NAD+-dependent deacetylase enzyme, this specificity could explain why FGF21 fasting induction was affected in LKO-SIRT1 mice liver, while other PPARα target genes were not. We have treated HepG2 cells with the oxidizing (acetoacetate) partner of ketone bodies, and endogenous SIRT1 was knockdown by a specific siRNA. FGF21 induction was dependent on SIRT1 expression, since knocking down impaired acetoacetate response. 3- ACTIVATING TRANSCRIPTION FACTOR 4-DEPENDENT INDUCTION OF FGF21 DURING AMINO ACID DEPRIVTION (Article 2: De Sousa-Coelho et al., 2012) Considering the central role of PPARα in the regulation of metabolic homeostasis we sought to investigate how the turnover of PPARα affected the expression of its target genes. HepG2 cells were infected with PPARα and exposed to DMSO or to the 26S proteasome inhibitor MG132. As expected, MG132-treatment blocked the PPARα-dependent expression of HMGCS2, indicating that the transcriptional activity of PPARα is increased by protein degradation (Blanquart et al, 2004). Contrary to what we had predicted, the expression of FGF21 was strongly increased by the MG132 treatment. We hypothesized that proteasome inhibition in HepG2 could decrease the pool of free amino acids. We treated HepG2 cells with histidinol (HisOH) a potent and reversible inhibitor of protein synthesis, (Hansen et al, 1972). Amino acid deprivation produced a time-dependent induction of FGF21 mRNA. To test whether this induction was due to an increase in the FGF21 gene transcription, we measured the FGF21 primary transcript (hnRNA) levels; HisOH treatment clearly induced FGF21 hnRNA levels in a time-dependent manner. As expected, HisOH induced an increase in the ATF4 protein levels after 2h treatment. By analyzing the sequence of the 5’-flanking region of the human FGF21 gene, we found two putative ATF4 response elements (AARE) starting at positions -152 and -610 upstream of the transcription start site. HepG2 cells were transfected with pGL3b-hFGF21 promoter-luciferase constructs and an expression vector for human ATF4. The expression of ATF4 induced the WT reporter in a concentration dependent manner. This induction was totally obliterated either when the AARE1 was mutated or when both elements were deleted. Induction was diminished when AARE2 was mutated. To further analyze the functionality of this sequence we tested the binding of ATF4 by an EMSA, where ATF4 bound as a C/EBPβ heterodimer to both AARE sequence elements. We also confirmed the in vivo binding by ChIP experiments. The chromatin binding of ATF4 was greatly increased in both ATF4 responsive sequences in HisOH treated cells. To confirm if the induction of FGF21 produced by amino acid starvation was mediated by ATF4, we treated siCtl and siATF4 HepG2 cells with HisOH. FGF21 mRNA levels after HisOH treatment were significantly lower when ATF4 was depleted. To analyze the effect of amino acid deprivation on FGF21 expression in vivo, we fed mice with a leucine-deficient [(-)leu] diet or a control (Ctl, nutrionally complete) diet for 7 days. Fgf21 mRNA levels were greatly increased in liver from mice fed a (-)leu diet compared to control. The circulating FGF21 levels were also increased in the serum of leucine deprived animals, paralleling hepatic gene expression. 4- LEUCINE DEPRIVATION SIGNALING UNDER FASTING CONDITIONS. We were interested to know whether FGF21 induction by a (-)leu diet would affect, or be affected by, the adaptive fasting response. We have fed mice for 7 days within a Ctl or (-)leu diet. Weights and food intake were recorded daily. Then, mice were randomly separated in a total of 4 groups, where 2 groups (one from each diet) were fasted overnight. Leucine deprivation affected the levels of free fatty acids and ketone bodies in serum in the fed state, while it does not upon fasting. Although no changes between groups were observed in the fed state, after fasting the β-oxidation, ketogenesis and gluconeogenesis keygenes were further up-regulated in the (-)leu diet group compared to control. The highest induction was seen in the Pgc1α gene, a known coactivator on these processes. The fasting activation of FGF21 was impaired in mice fed with (-)leu diet, underlying a crosstalk between the fasting and amino acid deprivation signalling. 5- ROLE OF FGF21 IN THE LEUCINE DEPRIVATION PHENOTYPE IN MICE (Article 3: De Sousa-Coelho et al., in preparation). According with our previously reported results (De Sousa-Coelho et al., 2012) mice maintained on a leucine-deficient [(-)leu] diet show a dramatic increase in FGF21 circulating levels. To check its origin we analyzed the Fgf21 gene expression in liver, where Fgf21 mRNA levels paralleled those in serum; brown adipose tissue (BAT), where it were unchanged; and in epididymal white adipose tissue (eWAT), where unexpectedly it were significantly decreased in wild type mice maintained in (-)leu diet. Because upon (-)leu diet, mice undergo rapid weight loss (Cheng et al., 2010), we wanted to investigate whether this phenotype is FGF21-dependent. For this purpose, WT and FGF21-KO mice were fed a Ctl or (-)leu diet for 7 days. Weight loss was diminished in FGF21-KO, while food intake decrease by (-)leu was unchanged between genotypes. Histological analysis of WAT showed that leucine deprivation resulted in a reduction in adipocyte volume compared with mice fed a control diet, while it was only slightly reduced in (-)leu FGF21-KO mice. It has been previously described that leucine deprivation increases lipolysis in WAT (Cheng et al., 2010). Consistent with changes in body weight, lack of FGF21 significantly decreased levels of phosphorylated (P)-HSL in WAT, indicating an impaired lipolysis. Gene expression analysis revealed reduction in the mRNA levels of the lipogenic genes Fas, Srebp1c and Acc1 in the WAT of mice maintained in (-)leu diet. These changes were impaired in FGF21-KO. Consistent with previous results (Cheng et al., 2010), leucine deprivation increased levels of Ucp1 mRNA in WT mice BAT. This increase was not observed in the FGF21-KO mice. mRNA levels of Pgc1α, which regulates the expression of Ucp1 (Handschin and Spiegelman, 2006), were also increased, although did not differ between WT and FGF21-KO mice under either control or (-)leu diet conditions. It has been recently proposed a link between FGF21 and SREBP1c during lipogenesis in HepG2 cells (Zhang et al., 2011). We examined levels of Fas, Srebp1c and Acc1 mRNA in liver of WT and FGF21-KO. As expected (Guo and Cavener, 2007), lipogenic program was decreased upon (-)leu diet, but this reduction was blocked in FGF21-KO mice. However, the amino acid response program was correctly initiated in these mice as shown by the increased levels of ATF4 protein and the increase in mRNA levels of Asns, a prototypical ATF4 target gene. The liver staining showed a decreased lipid accumulation under (-)leu in WT animals that is not produced in the FGF21-KO mice. These results demonstrate an important role of FGF21 in the regulation of lipid metabolism during amino acid starvation. References: Blanquart C, Mansouri R, Fruchart JC, Staels B, & Glineur C (2004) Different ways to regulate the PPARalpha stability. Biochem Biophys Res Commun 319, 663-70. Cheng, Y., Meng, Q., Wang, C., Li, H., Huang, Z., Chen, S., Xiao, F., and Guo, F. (2010). Leucine deprivation decreases fat mass by stimulation of lipolysis in white adipose tissue and upregulation of uncoupling protein 1 (UCP1) in brown adipose tissue. Diabetes 59, 17-25. Guo, F., and Cavener, D.R. (2007). The GCN2 eIF2alpha kinase regulates fatty-acid homeostasis in the liver during deprivation of an essential amino acid. Cell Metab 5, 103-14. Handschin, C., and Spiegelman, B.M. (2006). Peroxisome proliferator-activated receptor gamma coactivator 1 coactivators, energy homeostasis, and metabolism. Endocr Rev 27, 728-735. Hansen BS, Vaughan MH, & Wang L (1972) Reversible inhibition by histidinol of protein synthesis in human cells at the activation of histidine. J Biol Chem 247, 3854-3857. Hegardt FG (1999) Mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase: a control enzyme in ketogenesis. Biochem J. 338, 569-582. Hotta, Y., Nakamura, H., Konishi, M., Murata, Y., Takagi, H., Matsumura, S., Inoue, K., Fushiki, T., and Itoh, N. (2009). Fibroblast growth factor 21 regulates lipolysis in white adipose tissue but is not required for ketogenesis and triglyceride clearance in liver. Endocrinology 150, 4625-4633. Rodgers JT, Lerin C, Haas W, Gygi SP, Spiegelman BM, Puigserver P (2005) Nutrient control of glucose homeostasis through a complex of PGC-1alpha and SIRT1. Nature 434, 113-8 Zhang, Y., Lei, T., Huang, J.F., Wang, S.B., Zhou, L.L., Yang, Z.Q., and Chen, X.D. (2011). The link between fibroblast growth factor 21 and sterol regulatory element binding protein 1c during lipogenesis in hepatocytes. Mol Cell Endocrinol 342, 41-47.
Durante su vida un individuo se somete a diversos cambios nutricionales. La capacidad de detectar la disponibilidad de nutrientes y regular la homeostasis energética es un proceso fundamental. SIRT1 es un regulador clave en el metabolismo energético. SIRT1 puede modular la expresión génica en tejidos metabólicamente activos en respuesta a la restricción calórica o el ayuno. La dependencia de la actividad deacetilasa de SIRT1 en los niveles de NAD+ constituye un vínculo fundamental entre el estado metabólico celular y la regulación de genes. FGF21 es una hormona que se induce en el ayuno y que afecta al metabolismo de los carbohidratos y de los lípidos. Recientemente se han demostrado sus efectos benéficos en la protección de la obesidad inducida por la dieta y en la mejoría en la resistencia a la insulina. En este trabajo hemos demostrado que en células hepáticas en cultivo, SIRT1 desempeña un papel importante en la activación por PPARα de la expresión de FGF21, CPT1A, HMGCS2, y PEPCK. También, que la actividad de SIRT1 regula los niveles de glicemia y la expresión de PCK1 en hígado, en la respuesta al ayuno. Aún así, hemos visto que la actividad de SIRT1 no afecta la expresión de los genes de la oxidación de los ácidos grasos o la cetogénesis en el hígado, en respuesta a diferentes cambios nutricionales, como la restricción calórica, la transición de la lactancia/destete, y el ayuno. Adicionalmente, hemos demostrado que la activación de FGF21 por SIRT1 depende de la actividad HMGCS2. También hemos descrito que FGF21 se induce por la privación de aminoácidos, de manera dependiente de ATF4, y hemos identificado dos elementos de respuesta funcionales en la región promotora del gene humano, altamente conservados entre las especies. Además, hemos demostrado que FGF21 interviene en la regulación del metabolismo de los lípidos en el hígado y el tejido adiposo blanco, y de la termogénesis en el tejido adiposo marrón, durante la privación de aminoácidos. De todas formas, hemos visto que la privación de leucina afecta a los niveles de ácidos grasos libres y cuerpos cetónicos en suero en el estado de alimentación, mientras que no lo hace en el ayuno; y la activación de FGF21 en el ayuno está afectada en los ratones alimentados con esta dieta, desvelando un “crosstalk” entre la señalización del ayuno y la privación de aminoácidos.

Socioeconomic deprivation (SED) is inversely associated with mortality. The most deprived are at a higher risk of all cause mortality and cardiovascular mortality. However, only limited study of the relationship between SED and non-fatal cardiovascular disease has been previously undertaken. In those studies that have examined the relationship between SED and non-fatal cardiovascular disease, analyses have been limited to one form of cardiovascular disease (CVD), such as myocardial infarction or stroke and often prevalent disease. Furthermore, these studies have often failed to examine the association between SED and CVD whilst adjusting analyses for cardiovascular risk factors which are more prevalent in the most deprived. The aim of this work was to examine the association between SED and a number of cardiovascular outcomes after adjusting for the traditional cardiovascular risk factors of age, sex, smoking, blood pressure, diabetes mellitus and cholesterol. To determine is SED is in fact a risk factor for CVD after adjustment for these other risk factors, the relationship between SED and a number of fatal and non-fatal cardiovascular outcomes was examined. A number of forms of CVD were examined, including all coronary heart disease, myocardial infarction, stroke and heart failure A cohort of over 15,000 men and women who participated in the Renfrew Paisley cohort study was examined. These individuals were enrolled between 1974 and 1976 and underwent comprehensive screening for cardiorespiratory risk factors. They have since been followed for hospitalisations and deaths for 28 years. SED was measured using the Registrar General’s social class system and the Carstairs Morris index of deprivation. Rates of fatal and non-fatal outcomes were calculated, as were a number of composite outcomes. Adjusted analyses using multivariable regression were conducted to account for the risk factors of age, sex, smoking, blood pressure, diabetes and cholesterol. Further adjustment for the risk factors of lung function as measured by forced expiratory volume in 1 second, cardiomegaly on chest x-ray, body mass index, and a history of bronchitis was also made. The association between SED and the risk of recurrent cardiovascular hospitalisations, the burden of cardiovascular disease, as well as mortality and premature mortality was assessed for SED. I found that SED was associated with higher rates of hospitalisation for CVD disease in men and women irrespective of the measure of SED, either social class or the area based score of the Carstairs Morris index. This association persisted after adjustment for the traditional cardiovascular risk factors of age, sex, smoking, systolic blood pressure and diabetes and cholesterol. Further adjustment for lung function, the presence of bronchitis, body mass index and cardiomegaly on a chest x-ray did not explain the relationship between SED and each outcome. This risk was long lasting and persisted to the end of follow up. The strength of association of SED with coronary heart disease, myocardial infarction and stroke and all cause mortality was similar. The risk of a recurrent CVD hospitalisation was not higher in the most deprived after adjustment for CVD risk factors. However, I observed that SED was associated with higher mortality following an admission to hospital with CVD, before and after adjustment for cardiovascular risk factors of age, sex, smoking, systolic blood pressure, cholesterol and diabetes and after adjusting for the year of first developing cardiovascular disease. All cause mortality and cardiovascular mortality was highest in the most deprived. Again this association persisted after adjustment for cardiovascular risk factors. The most deprived also experienced longer hospital stays than the least deprived for a number of cardiovascular diseases including myocardial infarction and stroke. As a result the costs associated with cardiovascular disease admissions to hospital were highest in the most deprived despite their higher risk of dying during follow up. The cost differential was also explained by the finding that the most deprived experienced a higher number of admissions per person. Finally, the population attributable risk associated with SED is comparable to that of other traditional cardiovascular risk factors. In conclusion, I have found that the risk of CVD in the most deprived is higher even after adjustment for a number of cardiovascular risk factors. The numbers of hospitalisations, costs and mortality are also highest in the most deprived. Efforts are required to redress this imbalance. This can be achieved at the level of the individual through health care interventions to reduce the absolute burden of cardiovascular risk factors and to treat disease. However, societal level interventions are also required to tackle this problem as SED exerts complex effects on health that seem to also be independent of risk factors.

Mestrado em Econometria Aplicada e Previsão
O objetivo deste trabalho é identificar os determinantes socio-económicos e demográficos da Intensidade da Privação Matéria, e investigar as suas alterações quando se considera diferentes contextos macroeconómicos - crescimento moderado versus recessão (Economia Portuguesa, anos 2004 e 2012) e os grupos complementares - Economicamente Pobres e Não-Pobres. Este trabalho usa os 9 indicadores de Privação Material presentemente usados pelo Eurostat nos 27 Estados Membros da União Europeia para modelar o Score de Privação Material. Dado que a nossa variável de interesse é uma variável de contagem, vamos aplicar métodos para modelação de dados de contagem, nomeadamente, o modelo de Poisson e o modelo Zero-Inflated Poisson. Os resultados mostram quais são os principais determinantes da Intensidade da Privação Material, e que estes não variam, de forma significativa, face aos diferentes cenários macroeconómicos e grupos considerados.
The aim of this dissertation is to determine the socio-economic and demographic determinants of Material Deprivation Intensity, and investigate their changes when considering different macroeconomic backgrounds - moderate growth versus recession (Portuguese economy, years 2004 and 2012), and the additional groups - Income and Non-Income Poor. This work uses the 9 Material Deprivation indicators currently in use by Eurostat in the 27 European Union Member States to model the Material Deprivation Score. Given that the interest variable is a count variable, we will apply methodological framework for count data modeling, more particularly, Poisson and Zero-Inflated Poisson models. The results have shown what are the key determinants of the Material Deprivation Intensity, and also that they don?t change, in a significant way, in face of the considered macroeconomic backgrounds and groups.

My aim in this thesis is to examine whether there are some mitigating factors, i.e. reasons to punish an offender less for his crime than an otherwise similar offender (other than that the offender suffered from mental disorder or disturbance or other forms of irrationality at the time of offence), that are more applicable to socially deprived offenders than to non-socially deprived offenders. I will answer the thesis question through a critical examination of twelve arguments for claiming that there is a mitigating factor that is more applicable to socially deprived offenders, each proposing a different mitigating factor. My conclusions are as follows: (1) Most of the arguments that I examine fail, i.e. they either fail to highlight a genuine mitigating factor, or we do not have much evidence that the mitigating factor highlighted by the argument has a greater applicability to socially deprived offenders than to non-socially deprived offenders. (2) However, one argument, which can be called the no violation of natural duties argument, is successful. (3) Moreover, the improvement of the worst off argument, an argument that is not often discussed in the literature, is particularly noteworthy. If my discussion about that argument is correct, then even if, as I will argue, the mitigating factor highlighted by that argument may not be more applicable to socially deprived offenders than to non-socially deprived offenders, the remaining parts of that argument would still have profound influence on punishment in our unjust societies.

Synaptic connectivity is highly plastic in early development and undergoes extensive remodeling in response to changes in neuronal activity and sensory experience. Microglia, the resident central nervous system macrophages, participate in shaping mature neuronal circuits by dynamically surveying the brain parenchyma and pruning away less active synaptic connections. However, it is unknown how changes in neuronal activity regulates microglial pruning within circuits and whether this activity-dependent pruning is necessary to achieve plasticity. Using the rodent somatosensory circuit, I identified that microglia engulf and eliminate synapses in the cortex following early postnatal (P4) unilateral removal of mouse whiskers. I found this early life microglial synaptic remodeling requires specific chemokine signaling between neurons and microglia. Mice that lack expression of either the neuronal chemokine CX3CL1 (fractalkine), or its microglial receptor CX3CR1, have significantly reduced microglial synapse engulfment and fail to eliminate synapses following whisker removal. To gain more insight into how this signaling is regulated, I performed both single-cell RNA sequencing of the primary somatosensory cortex as well as microglia-specific Translating Ribosome Affinity Purification (TRAP) sequencing. I identified that the majority of central nervous system (CNS) cell populations in the somatosensory cortex, including microglia, undergo transcriptional changes following whisker removal. Further, the transcriptional changes in microglia after whisker cauterization require expression of the receptor CX3CR1. Importantly, I also found that Adam10, a gene encoding the metalloprotease known to post-translationally cleave CX3CL1 into a soluble chemokine, is upregulated in the deprived cortex after whisker ablation. Pharmacological inhibition of ADAM10 inhibits microglia-mediated removal of synapses in the deprived cortex. These data support a mechanism by which cleavage of membrane-bound CX3CL1 by ADAM10 is necessary for neuronal signaling to microglia via CX3CR1 to induce transcriptional changes within microglia upstream of synaptic engulfment and elimination following sensory deprivation.

Since the Second World War a range of policies have been implemented by central and local government agencies, with a view to improving accessibility to facilities, housing and employment opportunities within rural areas. It has been suggested that a lack of reasonable access to a range of such facilities and opportunities constitutes a key aspect of deprivation or disadvantage for rural residents. Despite considerable interest, very few attempts have been made to assess the nature and incidence of this disadvantage or the reaction of different sections of the population of rural areas to it. Moreover, almost all previous assessments have relied on so-called 'objective' measures of accessibility and disadvantage and failed to consider the relationship between such measures and 'subjective' measures such as individual perceptions. It is this gap in knowledge that the research described in this thesis has addressed. Following a critical review of relevant literature the thesis describes the way in which data on 'objective' and 'subjective' indicators of accessibility and behavioural responses to accessibility problems was collected, in six case study areas in Shropshire. Analysis of this data indicates that planning and other government policies have failed to significantly improve rural resident's accessibility to their basic requirements, and may in some cases have exacerbated it, and that as a result certain sections of the rural population are relatively disadvantaged. Moreover, analysis shows that .certain aspects of individual subjective' assessments of such accessibility disadvantage are significantly associated with more easily-obtained 'objective' measures. By using discriminant analysis the research demonstrates that it is possible to predict the likely levels of satisfaction with access to facilities from a range of 'objective' measures. The research concludes by highlighting the potential practical applications of such indicators in policy formulation, policy appraisal and policy evaluation.

In order to evaluate arginine deprivation as a putative anticancer therapy this study investigated arginine metabolism at the molecular, cellular and whole animal levels. Firstly, a murine model for arginine deprivation was developed using the catabolic enzyme arginase to reduce the blood arginine levels in vivo. However, although the arginine levels were limiting to both host and tumour, no prolongation in survival time was noted between treated and control groups. Analysis of the L1210 lymphocytic leukaemia cells used in this study revealed their ability to recycle arginine from its precursor molecule citrulline even in the presence of the enzyme arginase indicating that the citrulline level may be as important as the arginine level to the generation of deprivation conditions in vivo. Secondly, at the cellular level a number of different immortal cell lines were analysed for their ability to utilise urea cycle intermediates in lieu of arginine in order to identify those tumour types susceptible to the therapy. Most cell lines could be classified as one of the following: Type a - can only be rescued from arginine deprivation conditions with arginine alone; Type b - can utilise arginine and argininosuccinate but not citrulline; Type c can utilise arginine and citrulline but not the intermediate argininosuccinate. This data in addition to the radiolabelled tracer data presented in this thesis supports the theory of metabolite channelling and indicates that the differential utilisation of urea cycle intermediates is not due to metabolite availability within the cell. Finally, since argininosuccinate synthetase (ASS) is the rate limiting step in the conversion of citrulline to arginine the 5' untranslated region of the ASS gene was analysed for differential expression in cell types a, b and c in order to determine if the phenotype differnces noted between the three classes were due to a genotypic difference in the expression of this gene. The region chosen for this study is the site of alternative splicing and although no quantitative or qualitative differnces were observed in either variant 1 or variant 2, three novel bands were also amplified in type c cells but not in type b cells.

This thesis details the development of a child-derived index of child material deprivation. Whilst child poverty has come to the fore in academic and policy circles in recent decades, definitions and measures have tended to draw on adult-derived understandings of poverty. The aims of this thesis are to test whether children’s own perceptions of poverty can be used to form a scientifically robust and practically useful measurement tool, and to demonstrate the use of such a tool. The research draws on Mack and Lansley’s (1985) consensual approach to poverty measurement. Focus groups and surveys with children were used to produce a child-derived index of material deprivation. Analysis indicates that this index, whilst open to development and improvement, is a useful tool in measuring child poverty and in understanding the relationship between child poverty and children’s subjective well-being. It can also be used to compare children’s and adults’ conceptions and reports of poverty. Findings indicate that commonly used indicators of poverty such as income, receipt of free school meals and adults in paid work appear to make much more sense to adult conceptions of poverty than they do to children’s conceptions. These findings reinforce the view that children’s conceptions of their needs can be used to further our understandings of child poverty and its impacts. The work is split into four parts: a literature and data review, providing the rationale and justification for the work; a methodological section detailing the development of the child-derived index of material deprivation; a substantive section providing examples of uses of the index and exploring what it can contribute to understandings of child poverty; and a conclusion detailing limitations, drawing together findings, and making recommendations for research and policy.

Mestrado em Economia e Políticas Públicas
The purpose of this dissertation is to study the material deprivation indicators applied to the most recent conceptions of poverty and social exclusion, namely in the context of the European Union enlargement towards Eastern Europe. Social indicators are useful instruments for monitoring and evaluating a country’s level of development, providing means to assess the results and impacts of the politics pursued. In the framework of Poverty there has been an effort to introduce a wider and more complete concept of poverty in the construction of the indicators, pointing out the multidimensional nature of poverty, in opposition to the single use of monetary indicators, based on inputs, such as the measures of income. In recent years, with the generalization of the use of deprivation indicators, there has been a complementary approach to poverty measurement, focused on non-monetary measures that are concerned with the capacity of individuals to reach certain patterns of life and well-being. Deprivation indicators currently used allow us to measure the lack of material goods, but also to evaluate restrictions on the access to social life. Consistent Poverty is defined as the situation in which the incomes of an individual are inferior to a predetermined threshold, and simultaneously, there is a marginalization towards another dimension of daily life, such as having financial difficulties (to solve debt) or housing degradation. Through an empirical analysis, we will observe the way in which income indicators combine with deprivation indicators, in order to identify and suggest the adequate indicators and methodology for the measurement of poverty and social exclusion, in Portugal and in the European Union.
O objectivo desta dissertação é estudar os indicadores de privação aplicados às concepções de pobreza e exclusão social mais recentes, nomeadamente no contexto do alargamento da União Europeia a leste. Os indicadores sociais são instrumentos indispensáveis na avaliação e monitorização do nível de desenvolvimento de um país, permitindo estimar os resultados e impactos das políticas prosseguidas. No âmbito da Pobreza, tem sido desenvolvido um trabalho no sentido de introduzir conceitos mais amplos e completos de pobreza na construção dos indicadores, salientando a sua natureza multidimensional, em contraste com o simples uso dos indicadores monetários baseados em “inputs”, como são, por exemplo, as medidas de rendimento. Nos últimos anos, a generalização do uso dos indicadores de privação tem levado à implementação de uma abordagem complementar na medição da pobreza baseada em medidas não monetárias relacionadas com a capacidade dos indivíduos em atingir determinados padrões de vida e bem-estar. Os indicadores de privação usados actualmente permitem, de uma forma geral, medir a capacidade de aquisição de bens e serviços, mas também, apreciar restrições no acesso à vida em sociedade. A Pobreza Consistente é definida como a situação em que os rendimentos de um indivíduo são inferiores a um determinado patamar e, simultaneamente, se verifica uma marginalização em relação a outras dimensões da vida quotidiana, como sejam as dificuldades financeiras (em pagar empréstimos) ou a degradação habitacional. Por meio de uma análise empírica, iremos observar de que forma se ajustam e combinam os indicadores de rendimentos e os indicadores de privação, no intuito de identificar as metodologias e os indicadores mais adequados na medição da pobreza e exclusão social, em Portugal e na União Europeia.

The hypocretin peptides are two hypothalamic peptides known to be involved in both sleeping and feeding behavior, however their specific roles in these domains are not well understood. The present study sought to determine the effect of chronic (72-hour) sleep deprivation and (48-hour) food deprivation on preprohypocretin (PPH), which is the precursor for the hypocretin peptides. PPH levels were visualized and quantified via in situ hybridization. A three-factor ANOVA ( group x dorsal/ventral x medial/lateral) revealed a significant effect of subregion, specifically dorsal/middle and ventral/medial exhibited elevated PPH levels, however there was no effect of group. A between group one-way ANOVA revealed no effect of group on PPH levels. It is theorized that four possible domains may be responsible for these results: presence of hypothalamic neuronal subpopulations, role of circadian rhythm, role of hypocretins in locomotive behavior and inextricably confounded variables. These are discussed at length.

Over the past three decades there has been a significant increase in adiposity - prevalence of accumulation of excess fat around some human organs - globally. This has been characterised by an increase of body mass index (BMI) among men and women. In Sub-Sahara Africa, South Africa has one of the highest prevalence of obesity and the country currently experiences some epidemiological transitions. Excess adiposity is a major risk factor for a number of non-communicable diseases creating a burden for individuals, families, the health care system and society at large (Colditz, 1999). Therefore, there are both direct and indirect costs that can be averted by effectively controlling the obesity epidemic. Still this can only be achieved when there is a good understanding of its determinants. This study sought to investigate association between neighbourhood deprivation and adult adiposity (a combination of body mass index and waist circumference), the association of neighbourhood deprivation and body mass index and waist circumference individually and to examine individual and household level determinants impacting adult adiposity. The study utilised the South African National Income Dynamic Survey (NIDS) 2012 (wave 3) and the ward level South African Index of Multiple Deprivation 2011 (SAIMD 2011) produced by Southern Africa Labour and Development Research Unit (SALDRU) and the Southern African Social Policy Research Institute/Insights (SASPRI) respectively. Individuals with high body mass index (BMI ≥ 25kg/m²) and an expanded waist circumference (WC ≥ 102cm for men and WC ≥ 88cm for women) were considered as having high adiposity. Multilevel logistic regression was used for data analysis due to hierarchical nature of the data to allow simultaneous examination of the impact of some socio-economic factors influencing adiposity. The results showed that individuals that were living in districts that are in quintile 3 (OR= 0.659; 95% CI 0.461, 0.942) of the multiple deprivation score had significantly lower odds of having high adiposity as compared to those living in the least deprived districts. Those living in districts that are in quintiles 3 (OR= 0.652; 95% 0.449, 0.945) and 4 (OR= 0.621; 95% 0.393, 0.983) of the multiple deprivation score were at significantly lower odds of having high BMI as compared to those living in the least deprived districts. When the analysis was stratified by gender the results showed that women living in districts in that are in quintiles 3 (OR= 0.654; 95% 0.450, 0.951) and 4 (OR= 0.624; 95% 0.394, 0.986) of the multiple deprivation score were at lower odds of having high adiposity as compared to women living in the least deprived district. The results for men on the other hand showed no association between adiposity and district level deprivation. Our results show that individual level characteristics and neighbourhood level deprivation regardless of how far distal has an impact on adiposity. Neighbourhood affluence seems to be a buffer that promotes weight gain. The impact of neighbourhood deprivation on adiposity is stronger among women as compared to men. However, further studies that employ a smaller area metric of analysis (preferably ward level) are required to better inform policy prescriptions of neighbourhood deprivation and adiposity.

Sleep has become less important in western society during modern times, where many have the habit of prioritizing productive activities instead of sufficient sleep. However, recent studies have indicated the importance of sleep for emotional processing. A crucial finding in literature regarding sleep and emotions has been the enhancement of negative emotions after sleep deprivation. The aim of this systematic literature review was to investigate the neural basis of the effects of sleep deprivation on emotional reactivity. In order to conduct this review, three databases were used to obtain relevant articles. Out of the total 1041 articles, 11 fulfilled the inclusion criteria and were included in the review. The selected articles exclusively contained results regarding reactivity to visual emotional stimuli. Results showed that total and partial sleep deprivation result in enhanced amygdala activity in response to negative stimuli. Enhanced amygdala activity was also found in response to positive and neutral stimuli after sleep deprivation. The insula was another brain region that displayed enhanced activity toward all types of valenced stimuli after sleep deprivation. Moreover, weaker connectivity between the amygdala and prefrontal areas (specifically the medial prefrontal cortex) was found after total and partial sleep loss. Together, these results suggest that sleep deprivation induces hyperreactivity toward emotional stimuli and disrupts top-down regulation of emotional reactivity.

Alcoholism is a persistent substance abuse disorder that is associated with negative health, social, and economic outcomes. Treatment strategies for alcohol use disorders are limited, and only three drugs have been approved by the FDA for treatment. Although behavioral therapy and drug combination strategies improve abstinence outcomes, the majority of those in treatment will not achieve long-term abstinence. Therefore, better treatment strategies are needed. While much progress has been made toward understanding the neurobiology of alcoholism, this knowledge has not been effectively translated into treatment strategies. Animal models of alcohol drinking have been crucial to this research effort, but until recently there have been few procedures that effectively model alcoholism by producing binge-like drinking, withdrawal, and relapse behavior. In the last five years the intermittent alcohol access (IAA) model, which uses repeated cycles of scheduled alcohol deprivation and reinstatement to elevate drinking, has been established as such a procedure, with substantial evidence that escalation of drinking produced by IAA is mediated by similar mechanisms as in human alcoholics, which include transcriptional regulation that alters functioning of mesolimbocortical reward pathways. The IAA model. The studies reported herein characterize changes in gene expression in mesolimbocortical brain regions associated with development of maladaptive binge-like alcohol drinking due to scheduled abstinence, particularly in the nucleus accumbens, which regulates motivated behavior. Furthermore the IAA model is characterized with regard to effectiveness in 2 ethanol-preferring C57BL/6 inbred mouse strains, and the influence of concurrent access to multiple alcohol concentrations is examined. Finally, the potential of naltrexone and novel mu-opioid receptor-selective antagonist NAQ to modulate alcohol drinking under continuous access and intermittent access procedures is reported. Microarray analysis is used to analyze the transcriptome in prefrontal cortex, nucleus accumbens, and ventral midbrain of C57BL/6NCrl mice after alcohol deprivation, and to identify differentially expressed genes and gene co-expression networks in C57BL/6J mice during continuous access, as well as after six cycles of IAA. Differentially expressed genes, network hub genes, and regulation mechanisms represent high priority targets for further study in binge-like drinking behavior, with the goal of translating this knowledge to treatment strategies for alcoholism.

Background: The high availability of palatable, calorie dense and nutrient poor foods promote hedonic eating, defined as the drive to eat to obtain pleasure in the absence of an energy deficit. Poor sleep and hedonic eating, independently, drive obesity at the societal level. However, it is unknown whether the combination of sleep loss and access to palatable food synergistically increases weight gain. Objective: The purpose was to test whether chronic partial sleep deprivation by a method that increases weight gain also increases hedonic eating and exacerbates weight gain in rodents. We hypothesized that 1) type of diet, 2) preference for the diets and 3) sex would moderate the effect of sleep loss on calorie intake and weight gain in sleep deprived rodents. Subjects/Methods: Three-month old male and female Sprague-Dawley rats (n = 58 males and n = 10 females) were acclimated to hedonic diets for 7-d and then exposed to pre-recorded environmental noise (8h/d for 9-d). Body weight was measured every other day, unless mentioned otherwise. Food intake, corrected for uneaten food, was measured daily. Results: Noise exposure did not affect body weight gain and total calorie intake among male rats who had access to both a high and a low-fat diet. The effect of noise exposure on chocolate intake differed between male rats who were classified as high or low preference for chocolate. Initial preference for chocolate was greater in females compared to male rats. Conclusions: These results highlight the importance of preference and sex when investigating the effects of partial sleep deprivation on hedonic eating and obesity.

Gestational weight gain outside the recommended ranges puts women at risk for pregnancy complications and adverse birth outcomes. Food insecurity and environmental factors including neighborhood deprivation may influence gestational weight gain. This research 1) examines the impact of neighborhood deprivation on gestational weight gain, 2) identifies if the association varies by selected maternal characteristics, 3) examines the relationship between food insecurity and gestational weight gain, 4) determines if stress mediates the relationship between food insecurity and gestational weight gain, and 5) examines whether selected maternal characteristics mediate this relationship. The research was conducted through the analysis of the Pregnancy Risk Assessment Monitoring System, the American Community Survey and Florida Vital Statistics. Bivariate analyses, logistic regression and multilevel logistic regression were conducted to examine the associations. Results indicate that neighborhood deprivation and food insecurity are important risk factors for gestational weight gain that vary by stress and maternal characteristics.

College students are some of the most sleep deprived people in the nation. They have some of the worst sleep hygiene behaviors compared to other adult groups. Most people benefit from at least 7 to 8 hours of sleep each night, which is an adequate amount of time for a person to complete a regular sleep cycle. When students lose sleep, they disrupt their sleep cycles and their bodies respond by decreasing their ability to concentrate and complete complex tasks. This paper investigates the factors that contribute to sleep loss, and the correlating effects that it has on college students' academic performance.

This Thesis has been divided into three different chapters, the regulation of Fsp27 during the fasting adaptation, the transcriptional regulation of Fsp27 in liver and finally the role of FSP27 as a lipid-droplet protein in PPARalpha signaling, with the purpose to achieve the following objectives: 1) To describe the expression pattern of FSP27 during fasting adaptation in different tissues and different fasting times. 2) To define the transcriptional mechanisms responsible for hepatic expression of FSP27 during fasting specially the ones that cause the fall of Fsp27beta expression during late fasting. 3) To identify the role of Fsp27beta in PPARalpha signaling by studying its ability to control the store/release of specific endogenous ligands of PPARalpha from lipid droplets. The results of this work show that Fsp27beta is the main isoform expressed in tissues that actively oxidize fatty acids such as liver and BAT, but also in small intestine. Fsp27beta is a target gene of CREBH, but not of PPARaplha and there is no cross talk between both transcription factors in the regulation of hepatic expression of Fsp27beta. Nonetheless, Fsp27beta expression depends on the level of CREBH acetylation. It has been also described that FSP27beta expression is necessary for the hepatic accumulation of TAG in liver and plays a fundamental role in the development of the physiological hepatic steatosis that occurs during fasting during. Finally, this work demonstrates that the hepatic expression of FSP27beta is necessary for the correct signaling of PPARalpha during fasting at least in part because FSP27beta plays a key role in the storage/release of phospholipids proposed as endogenous ligands of PPARalpha form the liver lipid droplets. In vitro, Fsp27beta interferes with the PPARalpha signaling as it was determined by the use of a TK-luciferase reporter under the control of three PPRE. Lack of Fsp27beta expression Increases the concentration of PPARalpha endogenous ligands in serum, which triggers the PPARalpha signaling of in peripheral tissues such as BAT, while decreasing its signaling in the liver. Concretely, Fsp27beta plays a role in the down-regulation of PPARaplha target genes in BAT during fasting. As a conclusion we propose that during early fasting, fatty acids delivered by WAT will induce, through CREBH-FSP27beta axis, the formation of LDs in liver, needed to produce a transitory steatosis and, in addition, avoid the release of endogenous PPARalpha ligands from the liver. Conversely, during the late fasting, SIRT1 activity, also through CREBH modulation, will mediate FSP27 clearance from a liver that is already prepared to oxidize fatty acids. In turn, the increased FAO capacity from liver could alleviated ER stress contributing to CREBH downregulation. During fed states, the absence of Fsp27beta in liver will allow the new synthetized fat to leave the liver and actuate as PPARalpha agonist in extrahepatic tissues like BAT.
Esta Tesis está dividida en tres capítulos, la regulación de la expresión de las isoformas de Fsp27 durante la adaptación al ayuno, la regulación transcripcional de Fsp27b en hígado y el papel de FSP27 como proteína lipídica en la señalización de PPARalfa, teniendo los siguientes objetivos: 1. Estudiar el patrón de expresión de las isoformas de Fsp27 durante la adaptación al ayuno en diferentes tejidos y tiempos de ayuno; 2. Definir los mecanismos transcipcionales responsables por la expresión hepática de Fsp27beta durante el ayuno; 3. Identificar el papel de FSP27beta en la señalización de PPARalfa. Como conclusiones, se ha observado que Fsp27beta es la principal isoforma expresada en el hígado, BAT, y también en intestino delgado. Fsp27beta es un gen diana de CREBH pero no de PPARalfa, y su expresión depende del nivel de acetilación de CREBH. La expresión de FSP27 es necesaria para la acumulación de los TAG en hígado , y tiene un papel fundamental en el desarrollo de la esteatosis hepática durante el ayuno. In vitro, Fsp27beta interfiere con la señalización de PPARalfa, determinado por la utilización de un reportero TK-luciferasa bajo el control de los tres PPRE. La ausencia de la expresión de Fsp27alfa aumenta la concentración de los ligandos endógenos de PPARbeta en suero, activando su señalización en los tejidos periféricos, como el BAT, y bajando su señalización en el hígado. Nuestra modelo de trabajo propone que durante el ayuno temprano los ácidos grasos que llegan al WAT inducen, a través del eje CREBH-FSP27beta, la formación de gotas lipídicas en hígado, necesarias para producir una esteatosis transitoria y evitar la liberación de los ligandos endógenos de PPARalfa en el hígado. Durante el ayuno tardío, la actividad de SIRT1, a través de la modulación de CREBH, va a mediar la desaparición de FSP27beta del hígado, ya preparado para oxidar los ácidos grasos. La capacidad de oxidar ácidos grasos puede mejorar el stress del ER contribuyendo a la disminución de la regulación de CREBH. Durante los estados alimentados, la ausencia de FSP27beta en hígado permite a la nueva grasa sintetizada dejar el hígado y actuar como una agonista de PPARalfa en tejidos extra hepáticos como el BAT.

Oestrogen deprivation strategies, notably aromatase inhibitors, are of increasing value in hormone sensitive breast cancer. Unfortunately, however, oestrogen deprivation, like all other antihormones, is subject to acquisition of resistance. Further understanding of resistance is required to design approaches to effectively treat this state. This project aimed to delineate and target the underlying autocrine signalling mechanisms promoting this resistant phenotype, using a unique severely oestrogen and growth factor deprived in vitro breast cancer model, MCF-7X. The MCF-7X model revealed breast cancer cells are readily able to survive oestrogen deprivation, but are not oestrogen hypersensitive and lack input from classical growth factor receptors under conditions of parallel exogenous growth factor deprivation, contrasting previous models derived in the presence of stripped serum. However, there was a retained importance of oestrogen receptor (ERa) signalling, supporting use of the pure anti-oestrogen faslodex, which reduces ERa level, AF-1 phosphorylation at serine 118 (via an unknown kinase) and ERa-regulated transcriptional activity in MCF-7X cells. Furthermore, intracellular kinase signalling, primarily PI3K/AKT, contributed in MCF-7X cells, again driving transcriptional and growth-promoting activity of ERa, in this instance via ERa serine 167 phosphorylation. Critically, individual/dual targeting with faslodex and/or PI3K inhibition, while initially partially inhibitory of ERa phosphorylation and growth, ultimately supported emergence of resistance. This was invariably associated with gain of the growth factor receptors EGFR/HER2 and IGF1R and kinase-promoted re-activation of ERa phosphorylation/function. However, triple treatment using faslodex, PI3K and MAPK blockade to completely eliminate ERa phosphorylation substantially improved anti-tumour response and prevented resistance. Clearly, intelligent design of combination treatments of faslodex with targeted therapies to totally deplete ERa activity is needed to maximally inhibit oestrogen deprivation resistance. In contrast, the project showed sequential use of such agents may translate into poorer prognosis, since faslodex resistant cells were more aggressive (potentially driven by HER2).

There is a known link between relative deprivation and street crime among market liberal countries. Although some research has been done with cross-national data, there is none to suggest that the observed link is as valid in a country well known for its extended welfare and generous social expenditure like Sweden. The following study uses longitudinal data to see if this recognized link between relative deprivation and street crime also stands in a country with almost a century long tradition of striving for social-, economic- and cultural equality. Even though Sweden, over the last decade, has been subjected to a series of deregulations due to policy changes at national level, and with a following rise in inequality, it is still considered to be one of the most equal countries in the west. It is therefore not unlikely that the high degree of social security will reduce the negative impact of relative deprivation on social relations among its citizens, perhaps enough so that the correlation will be significantly weakened. The results indicate that the negative impact of relative deprivation on social relations still remains evident, even with a high degree of social security, suggesting that an extensive welfare does not significantly reduce the negative effect of relative deprivation on social relations.

Kyoto University (京都大学)
0048
新制・課程博士
博士(農学)
甲第15450号
農博第1835号
新制||農||982(附属図書館)
学位論文||H22||N4549(農学部図書室)
27928
京都大学大学院農学研究科応用生命科学専攻
(主査)教授 間藤 徹, 教授 矢﨑 一史, 教授 梅澤 俊明
学位規則第4条第1項該当

Adenosine is a multi-functional physiological molecule found abundantly in the body. It is one of the important components of ATP cellular energy metabolism. Adenosine has diverse actions as a ligand on many different types of cells and tissues acting via specific receptors. Currently, four subtypes of adenosine receptors are described, namely, the A1, A2A, A2B and A3 receptors. Neuroblastoma, mostly found in young children, is a malignant tumor derived from peripheral neurons in the body. Several different types of neuroblastoma cell lines of human origin have been established and contributed to the studies of neuroblastoma itself, neuronal differentiation, neurotransmitters, alcoholism, Alzheimer's disease and other neuronal diseases and disorders. In 1987, it was shown by Abbracchio et al. that a human neuroblastoma cell line, IMR32, could be induced to differentiate into cells that have a more neuronal morphology, with long neurites, by an adenosine receptor agonist 5'-N-ethylcarboxamideadenosine (NECA) 2. 'Neuronal differentiation' is expected to be a new alternative to the conventional clinical therapies, such as surgery, chemotherapy and radiotherapy. Unlike IMR32, PC12 cells, a rat adrenal pheochromocytoma cell line, resembling human neuroblastoma cell lines and also expressing the A2 subtype of adenosine receptors, was shown not to differentiate under stimulation of the A2A subtype of adenosine receptors 3. Moreover, adenosine inhibited neuronal differentiation in mouse dorsal root ganglion cells presumably via the A1 subtype 4. The mechanism(s) of these confusing effects of adenosine on neuronal differentiation require examination. First, a detection method for each of the adenosine receptor subtypes was developed using reverse transcriptase polymerase chain reaction (RT-PCR). This provided a sensitive, non-radioactive, analytical tool. Subtype-specific, four pairs of PCR primers, corresponding to the A1, A2A, A2B and A3 receptors, were designed and synthesized. The RT-PCR study revealed the presence of adenosine A1, A2A and A2B receptor mRNAs in untreated SH-SY5Y cells. These PCR primers were also designed so that they would allow multiplex PCR. Optimization of conditions for multiplex PCR was conducted, allowing it to detect several adenosine receptor subtypes simultaneously, and it was proven to be partially successful. In the study of differentiation, the use of the designed PCR primers was not quantitative to measure the levels of adenosine receptors due to variations of the expressions levels of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene, a house-keeping gene commonly used as the internal control in PCR or northern blot analysis. An adequate neuronal differentiation model system was established in order to study the possible role(s) of adenosine in neuronal differentiation. Nerve growth factor (NGF), a well-known inducer of differentiation of rat PC12 cells, did not show any apparent differentiation effects on human neuroblastoma SH-SY5Y cells. All-trans retinoic acid (50 µM) induced distinct neuronal differentiation in SH-SY5Y cells, however ethanol, used as a vehicle for retinoic acid, was also shown to have effects on this cell line causing morphological changes. Adenosine (100 µM) alone also did not induce marked differentiation in this cell line probably due to the presence of adenosine in serum. Adenosine deaminase-resistant, synthetic adenosine analogues were used and demonstrated enhancement of differentiation. A serum deprivation-induced differentiation in SH-SY5Y was found to be a consistent and useful model to evaluate the effects of other factors on differentiation in this cell line. This serum deprivation-induced differentiation was also found to accompany a substantial rise in the expression of neurofilament-H (NF-H), one of the marker proteins for neuronal differentiation, at the protein level. Using this model, the possible involvement of adenosine signaling via its receptors was investigated. Treatment of cells with selective adenosine analogues for the A1 and A2A subtypes, 2-chloro-N6-cyclopentyladenosine (CCPA, 100 nM) and 2-[4-(2-carboxylethyl)phenylamino]-5'-N-ethylcarboxamido (CGS21680, 30 and 100 nM), respectively, enhanced the differentiation induced by serum deprivation at day 7 by approximately 60% and 70%, respectively. These enhancing effects of agonists were blocked by selective antagonists, 8-cyclophenyl-1,3-dipropylxanthine (DPCPX) and 9-chloro-2-(2-furyl)[1,2,4]triazolo[1,5-c]quinalzolin-5-amine (CGS15943), respectively. Simultaneous co-stimulation of the A1 and A2A subtypes with these agonists gave no further effects compared to the enhancing effects exerted by CCPA or CGS21680 alone. Signal transduction pathways were examined using various protein kinase inhibitors. A selective protein kinase A (PKA) inhibitor N-(2-[p-bromocinnamylamino]ethyl)-5-isoquinolinesulfonamide hydrochloride (H-89, 100 nM) alone greatly enhanced the differentiation induced by serum deprivation in this cell line. No additive or synergistic effects of 10 nM H-89 with either the A1 or A2A receptor agonist were seen. A selective mitogen-activated protein kinase kinase (MAPKK) inhibitor 2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one (PD098,059) showed a similar pattern to H-89: 100 nM PD098,059 alone caused enhanced differentiation in serum deprivation-induced SH-SY5Y cells. The combination of PD098,059 and adenosine agonists did not show any further enhancement of differentiation. On the contrary, a selective protein kinase C (PKC) inhibitor, chelerythrine, suppressed the differentiation (by 51%) by serum deprivation at 1 uM, and at 100 nM, chelerythrine suppressed the enhancement of differentiation caused by CCPA and CGS21680 with no effect on the basic level of differentiation, indicating the possible involvement of PKC both in the differentiation induced by serum deprivation and the adenosine receptor-induced potentiation. Surprisingly, contrary to the assumption that the stimulation of PKA induces or assists neuronal differentiation, H-89 (20 uM) alone exerted a prompt differentiation (44% at day 2) in SH-SY5Y cells in the presence of the normal serum concentration (10%). This data suggests that the previously assumed role of PKA in differentiation must be re-evaluated. This H-89-induced differentiation model was shown to have a different differentiation mechanism to the previous serum deprivation-induced differentiation. Establishment of these new differentiation study models will add further options to explore neuronal differentiation, especially, of human type.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Science
Full Text

Thesis (M.S. in Operations Research)--Naval Postgraduate School, Sept. 2004.
Thesis Advisor(s): Nita Lewis Miller, J. Jeffrey Crowson. Includes bibliographical references (p. 77-79). Also available online.

Gradients in area-level socioeconomic deprivation have been associated with variations in the delivery of healthcare services and outcomes across geographical areas. This thesis assesses the impact of deprivation on prescribing rates for non-prescription medicines and prescription medicines indica. ted for the management ... of conditions affected by socioeconomic variables including cardiovascular disease and diabetes. Th~ research also examined the influence ofthe Quality and Outcomes Framework (QOF) in minimisfng . prescribing variations and outcomes in cardiovascular disease. Northern Ireland demographic, mapping, prescribing and area-level deprivation data were collected retrospectively from 2000 to 2005. The first stage ofthe investigation revealed that the levels of General Practitioner prescribing for non-prescription medicines were high (30%). These prescribing rates increased as proportions of practice populations living in areas of highest deprivation increased, particularly in urban locations, and were closely correlated to income and employment deprivation. Measures of deprivation developed for application in NI appeared to be better at predicting these variations in rural areas than other extant indices of deprivation. Prescribing rates for codeine-containing and non-steroidal anti-inflammatory drugs (NSAIDs) analgesics together with benzodiazepines and, most notably, antidepressants were positively associated with increased proportions of patients living in areas associated with highest levels of deprivation. Inconclusive findings in relation to trends in Hormone Replacement Therapy (HRT) and methylphenidate prescribing may have been accounted for by lower observed rates of prescribing volume. The final study observed cardiovascular and diabetes prescribing trends and demonstrated annual increases in growth of prescribed items which appeared to respond to the implementation ofthe QOF. A time series analysis, employing a refined methodology, confirmed that significant increases in statin doses were observed after the second quarter of2004 immediately post-QOF and that the increase responded positively to rising levels of practice deprivation. However, no impact on the reduction of Coronary Heart Disease admissions to hospital was observed over the course of this investigation. Overall the research has tested the consistent influence of deprivation on prescribing trends and has identified how the employment of area-based deprivation measures may be used to target prescribing resources. Policy and planning arrangements should take into account the impact of multiple deprivation in driving demand for healthcare services and associated prescribing resources. Healthcare strategies should consider how these demands can be equitably managed and met. Pharma.ceutical public health and medicines management interventions should be evaluated and commissioned.