The degradation of gel-spun poly(beta-hydroxybutyrate) fibrous matrix

Poly(��-hydroxybutyrate), (PHB), is a biologically produced, biodegradable thennoplastic with commercial potential. In this work the qualitative and quantitative investigations of the structure and degradation of a previously unstudied, novel, fibrous form of PHB, were completed. This gel-spun PHB fibrous matrix, PHB(FM), which has a similar appearance to cotton wool, possesses a relatively complex structure which combines a large volume with a low mass and has potential for use as a wound scaffolding device. As a result of the intrinsic problems presented by this novel structure, a new experimental procedure was developed to analyze the degradation of the PHB to its monomer hydroxybutyric acid, (HBA). This procedure was used in an accelerated degradation model which accurately monitored the degradation of the undegraded and degraded fractions of a fibrous matrix and the degradation of its PHB component. The in vitro degradation mechanism was also monitored using phase contrast and scanning electron microscopy, differential scanning calorimetry, fibre diameter distributions and Fourier infra-red photoacoustic spectroscopy. The accelerated degradation model was used to predict the degradation of the samples in the physiological model and this provided a clearer picture as to the samples potential biodegradation as medical implantation devices. The degradation of the matrices was characterized by an initial penetration of the degradative medium and weakening of the fibre integrity due to cleavage of the ester linkages, this then led to the physical collapse of the fibres which increased the surface area to volume ratio of the sample and facilitated its degradation. Degradation in the later stages was reduced due to the experimental kinetics, compaction and degradation resistant material, most probably the highly crystalline regions of the PHB. The in vitro degradation of the PHB(FM) was influenced by blending with various polysaccharides, copolymerizing with poly(~-hydroxyvalerate), (PHV), and changes to the manufacturing process. The degradation was also detennined to be faster than that of conventional melt processed PHB based samples. It was concluded that the material factors such as processing, sample size and shape affected the degradation of PHB based samples with the major factor of sample surface area to volume ratio being of paramount importance in determining the degradation of a sample.

The development of collaborative skills in young children

This thesis examines young children's early collaborative development when engaged in joint tasks with both a peer and a parent. It begins by examining how the term "collaborative" has been applied and researched in previous literature. As collaboration is found to usually require dialogue, and intersubjectivity is seen as an important component in the construction of both collaboration and dialogue, the ability to construct intersubjectivity is the subject of the rest of the chapter. The chapter concludes by introducing the research questions that underpin the experiments that follow. A number of experiments are then described. Experiments 1 and 2 investigate age differences in interaction styles and the communication strategies used by similar aged dyads. Experiments 3 and 4 investigate differences due to the age of the child and/or the status of the information giver (either parent or child) in the styles of interaction and the communication strategies used by parent and child dyads. Experiment 5 investigates the benefits of collaborating with a parent, and finally, Experiment 6 examines the collaborative ability of pre-schools. The thesis identifies a series of skills required for successful collaboration. These include recognition of a joint goal and the need to suppress individual desires, the ability to structure joint interaction, moving from role-based to a negotiating style, and communicative skills, for example, asking for clarification. Other reasons for children's failure in collaborative tasks involve task-related skills, such as the development of spatial terms, and failure to recognise the need for accuracy. The findings support Vygotsky's theory that when working with an adult, children perform at a higher level than when working with a peer. Evidence was also found of parents scaffolding the interaction for their children. However, further research is necessary to establish that such scaffolding skills affect the child's development of collaborative interactive skills.

Structural behaviour and design of load-bearing falsework

Tne object of this research was to investigate the behaviour of birdcage scaffolding as used in falsework structures, assess the suitability of existing design methods and make recommendations for a set of design rules. Since excessive deflection is as undesirable in a structure as total collapse, the project was divided into two sections. These were to determine the ultimate vertical and horizontal load-carrying capacity and also the deflection characteristics of any falsework. So theoretical analyses were developed to ascertain the ability of both the individual standards to resist vertical load, and of the bracing to resist horizontal load.Furthermore a model was evolved which would predict the horizontal deflection of a scaffold under load using strain energy methods. These models were checked by three series of experiments. The first was on individual standards under vertical load only. The second series was carried out on full scale falsework structures loading vertically and horizontally to failure. Finally experiments were conducted on scaffold couplers to provide additional verification of the method of predicting deflections. This thesis gives the history of the project and an introduction into the field of scaffolding. It details both the experiments conducted and the theories developed and the correlation between theory and experiment. Finally it makes recommendations for a design method to be employed by scaffolding designers.

In vitro hydrolytic degradation of centrifugally spun polyhydroxybutyrate-pectin composite fibres

BACKGROUND: Centrifugal spinning is a novel fibre-forming process that readily permits the incorporation of additives while avoiding the thermal damage often associated with conventional melt spinning. Centrifugal spinning of a viscous solution of poly(3-hydroxybutyrate) (PHB) mixed with pectin was used to fabricate a range of fibres containing different concentrations of this biologically active agent. The influence of this blending on fibre morphology and in vitro degradation in an accelerated hydrolytic model at 70 ?C and pH of 10.6 is reported. RESULTS: Blending influenced the physiochemical properties of the fibres, andthis significantly affected thedegradation profile of both the fibre and its PHB constituent. A greater influence on degradation was exerted by the type of pectin and its degree of esterification than by variations in its loading. CONCLUSION: Centrifugal spinning permits the fabrication of composite fibrous matrices from PHB and pectin. Incorporation of the polysaccharide into the fibres can be used to manipulate degradation behaviour and demonstrates a model for doping of matrices with active biological constituents. The unique features of the centrifugal spinning process, as illustrated by the structure of the fibres and the degradation profiles, suggest possible applications of centrifugally spun biopolymers as wound scaffolding devices and in tissue engineering.

Cognitive Bias in Knowledge Engineering Course

The paper presents experience in teaching of knowledge and ontological engineering. The teaching framework is targeted on the development of cognitive skills that will allow facilitating the process of knowledge elicitation, structuring and ontology development for scaffolding students��� research. The structuring procedure is the kernel of ontological engineering. The 5-steps ontology designing process is described. Special stress is put on ���beautification��� principles of ontology creating. The academic curriculum includes interactive game-format training of lateral thinking, interpersonal cognitive intellect and visual mind mapping techniques.

Facilitating intuitive-guided learning in a serious game through integration with a learning content management system

Increased global uptake of entertainment gaming has the potential to lead to high expectations of engagement and interactivity from users of technology-enhanced learning environments. Blended approaches to implementing game-based learning as part of distance or technology-enhanced education have led to demonstrations of the benefits they might bring, allowing learners to interact with immersive technologies as part of a broader, structured learning experience. In this article, we explore how the integration of a serious game can be extended to a learning content management system (LCMS) to support a blended and holistic approach, described as an 'intuitive-guided' method. Through a case study within the EU-Funded Adaptive Learning via Intuitive/Interactive, Collaborative and Emotional Systems (ALICE) project, a technical integration of a gaming engine with a proprietary LCMS is demonstrated, building upon earlier work and demonstrating how this approach might be realized. In particular, how this method can support an intuitive-guided approach to learning is considered, whereby the learner is given the potential to explore a non-linear environment whilst scaffolding and blending provide guidance ensuring targeted learning objectives are met. Through an evaluation of the developed prototype with 32 students aged 14-16 across two Italian schools, a varied response from learners is observed, coupled with a positive reception from tutors. The study demonstrates that challenges remain in providing high-fidelity content in a classroom environment, particularly as an increasing gap in technology availability between leisure and school times emerges.

GPCR-styrene maleic acid lipid particles (GPCR-SMALPs):their nature and potential

G-protein-coupled receptors (GPCRs) form the largest class of membrane proteins and are an important target for therapeutic drugs. These receptors are highly dynamic proteins sampling a range of conformational states in order to fulfil their complex signalling roles. In order to fully understand GPCR signalling mechanisms it is necessary to extract the receptor protein out of the plasma membrane. Historically this has universally required detergents which inadvertently strip away the annulus of lipid in close association with the receptor and disrupt lateral pressure exerted by the bilayer. Detergent-solubilized GPCRs are very unstable which presents a serious hurdle to characterization by biophysical methods. A range of strategies have been developed to ameliorate the detrimental effect of removing the receptor from the membrane including amphipols and reconstitution into nanodics stabilized by membrane scaffolding proteins (MSPs) but they all require exposure to detergent. Poly(styrene-co-maleic acid) (SMA) incorporates into membranes and spontaneously forms nanoscale poly(styrene-co-maleic acid) lipid particles (SMALPs), effectively acting like a 'molecular pastry cutter' to 'solubilize' GPCRs in the complete absence of detergent at any stage and with preservation of the native annular lipid throughout the process. GPCR-SMALPs have similar pharmacological properties to membrane-bound receptor, exhibit enhanced stability compared with detergent-solubilized receptors and being non-proteinaceous in nature, are fully compatible with downstream biophysical analysis of the encapsulated GPCR.

The Role of PP2A Regulatory Subunit B56 in Dictyostelium Discoideum Motility

Protein Phosphatase 2A, PP2A, is a heterotrimeric threonine/serine phosphatase system that is involved in a variety of cellular processes. This phosphatase is composed ofthree subunits: a catalytic subunit (C subunit), a scaffolding subunit (A subunit), and a regulatory subunit (B subunit). The regulatory subunit B is divided into four subclasses, B, B' (B56), B'' and B'' '. Studies showed that PP2A/B56 complexes regulate development of Dictyostelium and other metazoan cells. In addition to development, our experimental data suggest that PP2A/B56 complex also plays an important role in Dictyostelium cell motility. Cells lacking B56 was generated previously in our laboratory (Lee et al., 2008). Further studies showed that b56- cells are compromised in random cell motility compared to the wild type (AX3) cells. In contrast, b56 cells with re-introduced B56 displayed wild-type like motilities. Furthermore, one of the colleagues in our laboratory found that one of the Dictyostelium Ras species, RasG, associates with PP2A/B56 complex and RasG activation is compromised in b56- cells. Considering that Ras proteins are central in cellular motility regulation, PP2A/B56 complex may modulate cell motility through regulating Ras. We propose to determine if an introduction of constitutive active RasG proteins improves compromised b56- cell motility.

Investigating the Effects of Cognitive Apprenticeship-Based Instructional Coaching on Science Teaching Efficacy Beliefs

The overall purpose of this collected papers dissertation was to examine the utility of a cognitive apprenticeship-based instructional coaching (CAIC) model for improving the science teaching efficacy beliefs (STEB) of preservice and inservice elementary teachers. Many of these teachers perceive science as a difficult subject and feel inadequately prepared to teach it. However, teacher efficacy beliefs have been noted as the strongest indicator of teacher quality, the variable most highly correlated with student achievement outcomes. The literature is scarce on strong, evidence-based theoretical models for improving STEB. This dissertation is comprised of two studies. STUDY #1 was a sequential explanatory mixed-methods study investigating the impact of a reformed CAIC elementary science methods course on the STEB of 26 preservice teachers. Data were collected using the Science Teaching Efficacy Belief Instrument (STEBI-B) and from six post-course interviews. A statistically significant increase in STEB was observed in the quantitative strand. The qualitative data suggested that the preservice teachers perceived all of the CAIC methods as influential, but the significance of each method depended on their unique needs and abilities. STUDY #2 was a participatory action research case study exploring the utility of a CAIC professional development program for improving the STEB of five Bahamian inservice teachers and their competency in implementing an inquiry-based curriculum. Data were collected from pre- and post-interviews and two focus group interviews. Overall, the inservice teachers perceived the intervention as highly effective. The scaffolding and coaching were the CAIC methods portrayed as most influential in developing their STEB, highlighting the importance of interpersonal relationship aspects in successful instructional coaching programs. The teachers also described the CAIC approach as integral in supporting their learning to implement the new inquiry-based curriculum. The overall findings hold important implications for science education reform, including its potential to influence how preservice teacher training and inservice teacher professional development in science are perceived and implemented. Additionally, given the noteworthy results obtained over the relatively short durations, CAIC interventions may also provide an effective means of achieving improvements in preservice and inservice teachers��� STEB more expeditiously than traditional approaches.

Scaffold-free, Human Mesenchymal Stem Cell-Based Tissue Engineered Blood Vessels.

Tissue-engineered blood vessels (TEBV) can serve as vascular grafts and may also play an important role in the development of organs-on-a-chip. Most TEBV construction involves scaffolding with biomaterials such as collagen gel or electrospun fibrous mesh. Hypothesizing that a scaffold-free TEBV may be advantageous, we constructed a tubular structure (1���mm i.d.) from aligned human mesenchymal cell sheets (hMSC) as the wall and human endothelial progenitor cell (hEPC) coating as the lumen. The burst pressure of the scaffold-free TEBV was above 200���mmHg after three weeks of sequential culture in a rotating wall bioreactor and perfusion at 6.8 dynes/cm(2). The interwoven organization of the cell layers and extensive extracellular matrix (ECM) formation of the hMSC-based TEBV resembled that of native blood vessels. The TEBV exhibited flow-mediated vasodilation, vasoconstriction after exposure to 1�����M phenylephrine and released nitric oxide in a manner similar to that of porcine femoral vein. HL-60 cells attached to the TEBV lumen after TNF-�� activation to suggest a functional endothelium. This study demonstrates the potential of a hEPC endothelialized hMSC-based TEBV for drug screening.

Characterizing the Role of the Previously Undescribed Protein Caskin2 in Vascular Biology

<p>Maintenance of vascular homeostasis is an active process that is dependent on continuous signaling by the quiescent endothelial cells (ECs) that line mature vessels. Defects in vascular homeostasis contribute to numerous disorders of significant clinical impact including hypertension and atherosclerosis. The signaling pathways that are active in quiescent ECs are distinct from those that regulate angiogenesis but are comparatively poorly understood. Here we demonstrate that the previously uncharacterized scaffolding protein Caskin2 is a novel regulator of EC quiescence and that loss of Caskin2 in mice results in elevated blood pressure at baseline. Caskin2 is highly expressed in ECs from various vascular beds both in vitro and in vivo. When adenovirally expressed in vitro, Caskin2 inhibits EC proliferation and migration but promotes survival during hypoxia and nutrient deprivation. Likewise, loss of Caskin2 in vivo promotes increased vascular branching and permeability in mouse and zebrafish models. Caskin2 knockout mice are born in normal Mendelian ratios and appear grossly normal during early adulthood. However, they have consistently elevated systolic and diastolic blood pressure at baseline and significant context-dependent abnormalities in systemic metabolism (e.g., body weight, fat deposition, and glucose homeostasis). Although the precise molecular mechanisms of these effects remain unclear, we have shown that Caskin2 interacts with several proteins known to have important roles in endothelial biology and cardiovascular disease including the serine/threonine phosphatase PP1, the endothelial receptor Tie1, and eNOS, which is a critical regulator of vascular homeostasis. Ongoing work seeks to further characterize the functions of Caskin2 and its mechanisms of action with a focus on how Caskin2-mediated regulation of endothelial phenotype relates to its systemic effects on cardiovascular and metabolic function.</p>

microRNA Regulation of Cellular Immunity

<p>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 na��ve 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.</p><p>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. </p><p>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. </p><p>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.</p><p>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.</p>

Insulin-like growth factor receptor activity in cancer biology & therapy responses

The Insulin-like Growth Factor 1 Receptor (IGF-1R) has an essential function in normal cell growth and in cancer progression. However, anti-IGF-1R therapies have mostly been withdrawn from clinical trials owing to a lack of efficacy and predictive biomarkers. IGF-1R activity and signalling in cancer cells is regulated by its C-terminal tail, and in particular, by a motif that encompasses tyrosines 1250 and 1251 flanked by serines 1248 and 1252 (1248- SFYYS-1252). Mutation of Y1250/1251 greatly reduces IGF-1-promoted cell migration, interaction with the scaffolding protein RACK1 in the context Integrin signalling, and IGF- 1R kinase activity. Here we investigated the phosphorylation of the SFYYS motif and characterise the conditions under which this motif may be phosphorylated under. As phosphorylated residues, the SFYYS motif may also serve to recruit interacting proteins to the IGF-1R. To this end we identified a novel IGF-1R interacting partner which requires phosphorylated residues in the SFYYS motif to interact with the IGF-1R. This interaction was found to be IGF-1-dependent, and required the scaffold protein RACK1. The interaction of this binding protein with the IGF-1R likely functions to promote maximal phosphorylation of Shc and ERK in IGF-1-stimulated cell migration, and may be important for IGF-1 signalling in cancer cells. Lastly, we have investigated possible kinases that may confer resistance or sensitivity to the IGF-1R kinase inhibitor BMS-754807. In this screen we identified ATR as a mediator of resistance and showed that suppression or chemical inhibition of ATR synergised with BMS-754807 to reduce colony formation. This work has contributes to our understanding of IGF-1R kinase regulation and signalling and suggests that administration of anti-IGF-1R drugs with ATR inhibitors may have therapeutic benefit.

Learning to recognize faces incrementally

<p>[EN]Most face recognition systems are based on some form of batch learning. Online face recognition is not only more practical, it is also much more biologically plausible. Typical batch learners aim at minimizing both training error and (a measure of) hypothesis complexity. We show that the same minimization can be done incrementally as long as some form of ���scaffolding��� is applied throughout the learning process. Scaffolding means: make the system learn from samples that are neither too easy nor too difficult at each step. We note that such learning behavior is also biologically plausible. Experiments using large sequences of facial images support the theoretical claims. The proposed method compares well with other, numerical calculus-based online learners.</p>

Bridging discourses in a writing classroom

The aim of this study is to describe and analyse the writing discourse in one classroom and how students learn through studying a topic, i.e. the teaching and learning of written argument. The study takes its stance from a sociocultural perspective and is influenced by discourse analyses, new literacy studies and critical literacy (Fairclough1989; Barton 2007; Janks 2010; Ivani�� 2004). Data from year 6 in Sweden consists of observations, informal conversations, teachers��� planning and students��� written texts, i.e. letters to a newspaper editor. The results are presented in terms of four themes that became apparent during the reading of the data, viz. (1) teaching for learning - deconstruction; (2) dialogue and scaffolding for learning ��� enabling access; (3) reconstruction, feedback and students��� reflections for learning; and (4) writing to learn. The data is analysed and discussed on the basis of four concepts for developing critical literacy, viz. access, deconstruction, reconstruction and domination (cf. Janks 2010:21 ��� 32). The study indicates that explicit teaching of a written argument gives students access to the dominating structure of the genre if they are given the time and tools to reflect and be given feedback from the teacher.