FAS is highly expressed in the germinal center but is not required for regulation of the B-cell response to antigen.

In establishing the memory B-cell population and maintaining self-tolerance during an immune response, apoptosis mediates the removal of early, low-affinity antibody-forming cells, unselected germinal center (GC) cells, and, potentially, self-reactive B cells. To address the role of the apoptosis-signaling cell surface molecule FAS in the B-cell response to antigen, we have examined the T-cell-dependent B-cell response to the carrier-conjugated hapten (4-hydroxy-3-nitrophenyl)acetyl (NP) in lpr mice in which the fas gene is mutated. High levels of FAS were expressed on normal GC B cells but the absence of FAS did not perturb the progressive decline in numbers of either GC B cells or extrafollicular antibody-forming cells. Furthermore, the rate of formation and eventual size of the NP-specific memory B-cell population in lpr mice were normal. The accumulation of cells with affinity-enhancing mutations and the appearance of high-affinity anti-NP IgG1 antibody in the serum were also normal in lpr mice. Thus, although high levels of FAS are expressed on GC B cells, FAS is not required for GC selection or for regulation of the major antigen-specific B-cell compartments. The results suggest that the size and composition of B-cell compartments in the humoral immune response are regulated by mechanisms that do not require FAS.

Novel multiple sclerosis susceptibility loci implicated in epigenetic regulation.

We conducted a genome-wide association study (GWAS) on multiple sclerosis (MS) susceptibility in German cohorts with 4888 cases and 10,395 controls. In addition to associations within the major histocompatibility complex (MHC) region, 15 non-MHC loci reached genome-wide significance. Four of these loci are novel MS susceptibility loci. They map to the genes L3MBTL3, MAZ, ERG, and SHMT1. The lead variant at SHMT1 was replicated in an independent Sardinian cohort. Products of the genes L3MBTL3, MAZ, and ERG play important roles in immune cell regulation. SHMT1 encodes a serine hydroxymethyltransferase catalyzing the transfer of a carbon unit to the folate cycle. This reaction is required for regulation of methylation homeostasis, which is important for establishment and maintenance of epigenetic signatures. Our GWAS approach in a defined population with limited genetic substructure detected associations not found in larger, more heterogeneous cohorts, thus providing new clues regarding MS pathogenesis.

Population ageing and the implications for finanical markets

How will financial institutions respond to the transactions and asset management needs of both the ageing population and their carers? The ageing of the population has generated increased interest from both government and business, including banking and financial services, in the sorts of services that will be required by older people, and how their money and property will be managed. This article examines the trends and implications for banking practice of this increasing population of customers and their carers.

Comment on "On the regulation of populations of mammals, birds, fish, and insects" II

Sibly et at. (Reports, 22 July 2005, p. 607) recently estimated the relationship between population size and growth rate for 1780 time series of various species. I explain why some aspects of their analysis are questionable and, therefore, why their results and estimation procedure should be used with care.

Spring Phenology of Butterflies : The role of seasonal variation in life-cycle regulation

Animals and plants in temperate regions must adapt their life cycle to pronounced seasonal variation. The research effort that has gone into studying these cyclical life history events, or phenological traits, has increased greatly in recent decades. As phenological traits are often correlated to temperature, they are relevant to study in terms of understanding the effect of short term environmental variation as well as long term climate change. Because of this, changes in phenology are the most obvious and among the most commonly reported responses to climate change. Moreover, phenological traits are important for fitness as they determine the biotic and abiotic environment an individual encounters. Fine-tuning of phenology allows for synchronisation at a local scale to mates, food resources and appropriate weather conditions. On a between-population scale, variation in phenology may reflect regional variation in climate. Such differences can not only give insights to life cycle adaptation, but also to how populations may respond to environmental change through time. This applies both on an ecological scale through phenotypic plasticity as well as an evolutionary scale through genetic adaptation. In this thesis I have used statistical and experimental methods to investigate both the larger geographical patterns as well as mechanisms of fine-tuning of phenology of several butterfly species. The main focus, however, is on the orange tip butterfly, Anthocharis cardamines, in Sweden and the United Kingdom. I show a contrasting effect of spring temperature and winter condition on spring phenology for three out of the five studied butterfly species. For A. cardamines there are population differences in traits responding to these environmental factors between and within Sweden and the UK that suggest adaptation to local environmental conditions. All populations show a strong negative plastic relationship between spring temperature and spring phenology, while the opposite is true for winter cold duration. Spring phenology is shifted earlier with increasing cold duration. The environmental variables show correlations, for example, during a warm year a short winter delays phenology while a warm spring speeds phenology up. Correlations between the environmental variables also occur through space, as the locations that have long winters also have cold springs. The combined effects of these two environmental variables cause a complex geographical pattern of phenology across the UK and Sweden. When predicting phenology with future climate change or interpreting larger geographical patterns one must therefore have a good enough understanding of how the phenology is controlled and take the relevant environmental factors in to account. In terms of the effect of phenological change, it should be discussed with regards to change in life cycle timing among interacting species. For example, the phenology of the host plants is important for A. cardamines fitness, and it is also the main determining factor for oviposition. In summary, this thesis shows that the broad geographical pattern of phenology of the butterflies is formed by counteracting environmental variables, but that there also are significant population differences that enable fine-tuning of phenology according to the seasonal progression and variation at the local scale.

Impact of diet composition on blood glucose regulation

Abstract Nutritional management of blood glucose levels is a strategic target in the prevention and management of type 2 diabetes mellitus (T2DM). To implement such an approach it is essential to understand the effect of food on glycaemic regulation and on the underlying metabolic derangements. This comprehensive review summarises the results from human dietary interventions exploring the impact of dietary components on blood glucose levels. Included are the major macronutrients; carbohydrate, protein and fat, micronutrient vitamins and minerals, non-nutrient phytochemicals and additional foods including low-calorie sweeteners, vinegar and alcohol. Based on the evidence presented in this review, it is clear that dietary components have significant and clinically relevant effects on blood glucose modulation. An integrated approach that includes reducing excess body weight, increased physical activity along with a dietary regime to regulate blood glucose levels will not only be advantages in T2DM management, but will benefit the health of the population and limit the increasing worldwide incidence of T2DM.

Catching transcriptional regulation by thermostatistical modeling

Gene expression is frequently regulated by multiple transcription factors (TFs). Thermostatistical methods allow for a quantitative description of interactions between TFs, RNA polymerase and DNA, and their impact on the transcription rates. We illustrate three different scales of the thermostatistical approach: the microscale of TF molecules, the mesoscale of promoter energy levels and the macroscale of transcriptionally active and inactive cells in a cell population. We demonstrate versatility of combinatorial transcriptional activation by exemplifying logic functions, such as AND and OR gates. We discuss a metric for cell-to-cell transcriptional activation variability known as Fermi entropy. Suitability of thermostatistical modeling is illustrated by describing the experimental data on transcriptional induction of NF?B and the c-Fos protein.

Social and environmental regulation of signal plasticity and signal reliability in the electric fish Brachyhypopomus gauderio

The balance between the costs and benefits of conspicuous signals ensures that the expression of those signals is related to the quality of the bearer. Plastic signals could enable males to maximize conspicuous traits to impress mates and competitors, but reduce the expression of those traits to minimize signaling costs, potentially compromising the information conveyed by the signals. ^ I investigated the effect of signal enhancement on the information coded by the biphasic electric signal pulse of the gymnotiform fish Brachyhypopomus gauderio. Increases in population density drive males to enhance the amplitude of their signals. I found that signal amplitude enhancement improves the information about the signaler's size. Furthermore, I found that the elongation of the signal's second phase conveys information about androgen levels in both sexes, gonad size in males and estrogen levels in females. Androgens link the duration of the signal's second phase to other androgen-mediated traits making the signal an honest indicator of reproductive state and aggressive motivation. ^ Signal amplitude enhancement facilitates the assessment of the signaler's resource holding potential, important for male-male interactions, while signal duration provides information about aggressive motivation to same-sex competitors and reproductive state to the opposite sex. Moreover, I found that female signals also change in accordance to the social environment. Females also increase the amplitude of their signal when population density increases and elongate the duration of their signal's second phase when the sex ratio becomes female-biased. Indicating that some degree of sexual selection operates in females. ^ I studied whether male B. gauderio use signal plasticity to reduce the cost of reproductive signaling when energy is limited. Surprisingly, I found that food limitation promotes the investment in reproduction manifested as signal enhancement and elevated androgen levels. The short lifespan and single breeding season of B. gauderio diminishes the advantage of energy savings and gives priority to sustaining reproduction. I conclude that the electric signal of B. gauderio provides reliable information about the signaler, the quality of this information is reinforced rather than degraded with signal enhancement.^

Regulation of Bone Marrow Stem Cells through Oscillatory Shear Stresses - A Heart Valve Tissue Engineering Perspective

Heart valve disease occurs in adults as well as in pediatric population due to age-related changes, rheumatic fever, infection or congenital condition. Current treatment options are limited to mechanical heart valve (MHV) or bio-prosthetic heart valve (BHV) replacements. Lifelong anti-coagulant medication in case of MHV and calcification, durability in case of BHV are major setbacks for both treatments. Lack of somatic growth of these implants require multiple surgical interventions in case of pediatric patients. Advent of stem cell research and regenerative therapy propose an alternative and potential tissue engineered heart valves (TEHV) treatment approach to treat this life threatening condition. TEHV has the potential to promote tissue growth by replacing and regenerating a functional native valve. Hemodynamics play a crucial role in heart valve tissue formation and sustained performance. The focus of this study was to understand the role of physiological shear stress and flexure effects on de novo HV tissue formation as well as resulting gene and protein expression. A bioreactor system was used to generate physiological shear stress and cyclic flexure. Human bone marrow mesenchymal stem cell derived tissue constructs were exposed to native valve-like physiological condition. Responses of these tissue constructs to the valve-relevant stress states along with gene and protein expression were investigated after 22 days of tissue culture. We conclude that the combination of steady flow and cyclic flexure helps support engineered tissue formation by the co-existence of both OSS and appreciable shear stress magnitudes, and potentially augment valvular gene and protein expression when both parameters are in the physiological range.

microRNA Regulation of Cellular Immunity

Immunity is broadly defined as a mechanism of protection against non-self entities, a process which must be sufficiently robust to both eliminate the initial foreign body and then be maintained over the life of the host. Life-long immunity is impossible without the development of immunological memory, of which a central component is the cellular immune system, or T cells. Cellular immunity hinges upon a naïve T cell pool of sufficient size and breadth to enable Darwinian selection of clones responsive to foreign antigens during an initial encounter. Further, the generation and maintenance of memory T cells is required for rapid clearance responses against repeated insult, and so this small memory pool must be actively maintained by pro-survival cytokine signals over the life of the host.

T cell development, function, and maintenance are regulated on a number of molecular levels through complex regulatory networks. Recently, small non-coding RNAs, miRNAs, have been observed to have profound impacts on diverse aspects of T cell biology by impeding the translation of RNA transcripts to protein. While many miRNAs have been described that alter T cell development or functional differentiation, little is known regarding the role that miRNAs have in T cell maintenance in the periphery at homeostasis.

In Chapter 3 of this dissertation, tools to study miRNA biology and function were developed. First, to understand the effect that miRNA overexpression had on T cell responses, a novel overexpression system was developed to enhance the processing efficiency and ultimate expression of a given miRNA by placing it within an alternative miRNA backbone. Next, a conditional knockout mouse system was devised to specifically delete miR-191 in a cell population expressing recombinase. This strategy was expanded to permit the selective deletion of single miRNAs from within a cluster to discern the effects of specific miRNAs that were previously inaccessible in isolation. Last, to enable the identification of potentially therapeutically viable miRNA function and/or expression modulators, a high-throughput flow cytometry-based screening system utilizing miRNA activity reporters was tested and validated. Thus, several novel and useful tools were developed to assist in the studies described in Chapter 4 and in future miRNA studies.

In Chapter 4 of this dissertation, the role of miR-191 in T cell biology was evaluated. Using tools developed in Chapter 3, miR-191 was observed to be critical for T cell survival following activation-induced cell death, while proliferation was unaffected by alterations in miR-191 expression. Loss of miR-191 led to significant decreases in the numbers of CD4+ and CD8+ T cells in the periphery lymph nodes, but this loss had no impact on the homeostatic activation of either CD4+ or CD8+ cells. These peripheral changes were not caused by gross defects in thymic development, but rather impaired STAT5 phosphorylation downstream of pro-survival cytokine signals. miR-191 does not specifically inhibit STAT5, but rather directly targets the scaffolding protein, IRS1, which in turn alters cytokine-dependent signaling. The defect in peripheral T cell maintenance was exacerbated by the presence of a Bcl-2YFP transgene, which led to even greater peripheral T cell losses in addition to developmental defects. These studies collectively demonstrate that miR-191 controls peripheral T cell maintenance by modulating homeostatic cytokine signaling through the regulation of IRS1 expression and downstream STAT5 phosphorylation.

The studies described in this dissertation collectively demonstrate that miR-191 has a profound role in the maintenance of T cells at homeostasis in the periphery. Importantly, the manipulation of miR-191 altered immune homeostasis without leading to severe immunodeficiency or autoimmunity. As much data exists on the causative agents disrupting active immune responses and the formation of immunological memory, the basic processes underlying the continued maintenance of a functioning immune system must be fully characterized to facilitate the development of methods for promoting healthy immune function throughout the life of the individual. These findings also have powerful implications for the ability of patients with modest perturbations in T cell homeostasis to effectively fight disease and respond to vaccination and may provide valuable targets for therapeutic intervention.

Quantifying Eukaryotic Gene Regulation in Hormone Response and Disease.

Quantifying the function of mammalian enhancers at the genome or population scale has been longstanding challenge in the field of gene regulation. Studies of individual enhancers have provided anecdotal evidence on which many foundational assumptions in the field are based. Genome-scale studies have revealed that the number of sites bound by a given transcription factor far outnumber the genes that the factor regulates. In this dissertation we describe a new method, chromatin immune-enriched reporter assays (ChIP-reporters), and use that approach to comprehensively test the enhancer activity of genomic loci bound by the glucocorticoid receptor (GR). Integrative genomics analyses of our ChIP-reporter data revealed an unexpected mechanism of glucocorticoid (GC)-induced gene regulation. In that mechanism, only the minority of GR bound sites acts as GC-inducible enhancers. Many non-GC-inducible GR binding sites interact with GC-induced sites via chromatin looping. These interactions can increase the activity of GC-induced enhancers. Finally, we describe a method that enables the detection and characterization of the functional effects of non-coding genetic variation on enhancer activity at the population scale. Taken together, these studies yield both mechanistic and genetic evidence that provides context that informs the understanding of the effects of multiple enhancer variants on gene expression.

Using Action Research to Develop My Solution-Focused Practice in Collaboration with Year 6 And 7 Pupils When Discussing Their Behavioural Self-Regulation at School

This action research study aimed to develop the researcher's use of solutionfocused techniques when working with Year 6 and 7 pupils’ self-regulation. A systematic literature review highlighted an evidence base that demonstrated the efficacy of solution-focused methods when working with this population. The researcher’s intention was to add to the body of Educational Psychology practice-based evidence in this area. The researcher recruited eight participants from primary and secondary school provisions. Solution-focused techniques were systematically trialled in partnership with the pupils and were modified through an action research cycle. Semistructured interviewing provided participants the opportunity to critically evaluate the researcher’s solution-focused practice. Thematic Analysis was used to assess feedback in order to adapt the delivery of solution-focused techniques. Developments to practice explored within this study included modifications to the use of the six core components of Solution Focused Brief Therapy. Adaptations have the potential to inform the use of these solution-focused approaches with other educational practitioners.

Regulation of osteoclast activation and autophagy through altered protein kinase pathways in Paget's disease of bone

Résumé : La maladie osseuse de Paget (MP) est un désordre squelettique caractérisé par une augmentation focale et désorganisée du remodelage osseux. Les ostéoclastes (OCs) de MP sont plus larges, actifs et nombreux, en plus d’être résistants à l’apoptose. Même si la cause précise de la MP demeure inconnue, des mutations du gène SQSTM1, codant pour la protéine p62, ont été décrites dans une proportion importante de patients avec MP. Parmi ces mutations, la substitution P392L est la plus fréquente, et la surexpression de p62P392L dans les OCs génère un phénotype pagétique partiel. La protéine p62 est impliquée dans de multiples processus, allant du contrôle de la signalisation NF-κB à l’autophagie. Dans les OCs humains, un complexe multiprotéique composé de p62 et des kinases PKCζ et PDK1 est formé en réponse à une stimulation par Receptor Activator of Nuclear factor Kappa-B Ligand (RANKL), principale cytokine impliquée dans la formation et l'activation des OCs. Nous avons démontré que PKCζ est impliquée dans l’activation de NF-κB induite par RANKL dans les OCs, et dans son activation constitutive en présence de p62P392L. Nous avons également observé une augmentation de phosphorylation de Ser536 de p65 par PKCζ, qui est indépendante d’IκB et qui pourrait représenter une voie alternative d'activation de NF-κB en présence de la mutation de p62. Nous avons démontré que les niveaux de phosphorylation des régulateurs de survie ERK et Akt sont augmentés dans les OCs MP, et réduits suite à l'inhibition de PDK1. La phosphorylation des substrats de mTOR, 4EBP1 et la protéine régulatrice Raptor, a été évaluée, et une augmentation des deux a été observée dans les OCs pagétiques, et est régulée par l'inhibition de PDK1. Également, l'augmentation des niveaux de base de LC3II (associée aux structures autophagiques) observée dans les OCs pagétiques a été associée à un défaut de dégradation des autophagosomes, indépendante de la mutation p62P392L. Il existe aussi une réduction de sensibilité à l’induction de l'autophagie dépendante de PDK1. De plus, l’inhibition de PDK1 induit l’apoptose autant dans les OCs contrôles que pagétiques, et mène à une réduction significative de la résorption osseuse. La signalisation PDK1/Akt pourrait donc représenter un point de contrôle important dans l’activation des OCs pagétiques. Ces résultats démontrent l’importance de plusieurs kinases associées à p62 dans la sur-activation des OCs pagétiques, dont la signalisation converge vers une augmentation de leur survie et de leur fonction de résorption, et affecte également le processus autophagique.

Yellow Perch Population Assessment in Southwestern Lake Michigan, Including Evaluation of Sampling Techniques and Identification of the Factors that Determine Yellow Perch Year-Class Strength April 1, 2007 – March 31, 2008

Division of Fisheries, Illinois Department of Natural Resources Grant/Contract No: F-123 R-14

The regulation of cancer cell invasive behaviour by Chloride Interacellular Channel 3 (CLIC3) and Transglutaminasae 2

A study into the role of secreted CLIC3 in tumour cell invasion. The initiation and progression of cancers is thought to be linked to their relationship with a population of activated fibroblasts, which are associated with tumours. I have used an organotypic approach, in which plugs of collagen I are preconditioned with fibroblastic cells, to characterise the mechanisms through which carcinoma-associated fibroblasts (CAFs) influence the invasive behaviour of tumour cells. I have found that immortalised cancer-associated fibroblasts (iCAFs) support increased invasiveness of cancer cells, and that this is associated with the ability of CAFs to increase the fibrillar collagen content of the extracellular matrix (ECM). To gain mechanistic insight into this phenomenon, an in-depth SILAC-based mass proteomic analysis was conducted, which allowed quantitative comparison of the proteomes of iCAFs and immortalised normal fibroblast (iNFs) controls. Chloride Intracellular Channel Protein 3 (CLIC3) was one of the most significantly upregulated components of the iCAF proteome. Knockdown of CLIC3 in iCAFs reduced the ability of these cells to remodel the ECM and to support tumour cell invasion through organotypic plugs. A series of experiments, including proteomic analysis of cell culture medium that had been preconditioned by iCAFs, indicated that CLIC3 itself was a component of the iCAF secretome that was responsible for the ability of iCAFs to drive tumour cell invasiveness. Moreover, addition of soluble recombinant CLIC3 (rCLIC3) was sufficient to drive the extension of invasive pseudopods in cancer cell lines, and to promote disruption of the basement membrane in a 3D in vitro model of the ductal carcinoma in situ (DCIS) to invasive ductal carcinoma (IDC) transition. My investigation into the mechanism through which extracellular CLIC3 drives tumour cell invasiveness led me to focus on the relationship between CLIC3 and the ECM modifying enzyme, transglutaminase-2 (TG2). Through this, I have found that TG2 physically associates with CLIC3 and that TG2 is necessary for CLIC3 to drive tumour cell invasiveness. These data identifying CLIC3 as a key pro-invasive factor, which is secreted by CAFs, provides an unprecedented mechanism through which the stroma may drive cancer progression.