Acoustic and Magnetic Techniques for the Isolation and Analysis of Cells in Microfluidic Platforms

Cancer comprises a collection of diseases, all of which begin with abnormal tissue growth from various stimuli, including (but not limited to): heredity, genetic mutation, exposure to harmful substances, radiation as well as poor dieting and lack of exercise. The early detection of cancer is vital to providing life-saving, therapeutic intervention. However, current methods for detection (e.g., tissue biopsy, endoscopy and medical imaging) often suffer from low patient compliance and an elevated risk of complications in elderly patients. As such, many are looking to “liquid biopsies” for clues into presence and status of cancer due to its minimal invasiveness and ability to provide rich information about the native tumor. In such liquid biopsies, peripheral blood is drawn from patients and is screened for key biomarkers, chiefly circulating tumor cells (CTCs). Capturing, enumerating and analyzing the genetic and metabolomic characteristics of these CTCs may hold the key for guiding doctors to better understand the source of cancer at an earlier stage for more efficacious disease management.

The isolation of CTCs from whole blood, however, remains a significant challenge due to their (i) low abundance, (ii) lack of a universal surface marker and (iii) epithelial-mesenchymal transition that down-regulates common surface markers (e.g., EpCAM), reducing their likelihood of detection via positive selection assays. These factors potentiate the need for an improved cell isolation strategy that can collect CTCs via both positive and negative selection modalities as to avoid the reliance on a single marker, or set of markers, for more accurate enumeration and diagnosis.

The technologies proposed herein offer a unique set of strategies to focus, sort and template cells in three independent microfluidic modules. The first module exploits ultrasonic standing waves and a class of elastomeric particles for the rapid and discriminate sequestration of cells. This type of cell handling holds promise not only in sorting, but also in the isolation of soluble markers from biofluids. The second module contains components to focus (i.e., arrange) cells via forces from acoustic standing waves and separate cells in a high throughput fashion via free-flow magnetophoresis. The third module uses a printed array of micromagnets to capture magnetically labeled cells into well-defined compartments, enabling on-chip staining and single cell analysis. These technologies can operate in standalone formats, or can be adapted to operate with established analytical technologies, such as flow cytometry. A key advantage of these innovations is their ability to process erythrocyte-lysed blood in a rapid (and thus high throughput) fashion. They can process fluids at a variety of concentrations and flow rates, target cells with various immunophenotypes and sort cells via positive (and potentially negative) selection. These technologies are chip-based, fabricated using standard clean room equipment, towards a disposable clinical tool. With further optimization in design and performance, these technologies might aid in the early detection, and potentially treatment, of cancer and various other physical ailments.

Development and Application of Covalent-Labeling Strategies for the Large-Scale Thermodynamic Analysis of Protein Folding and Ligand Binding

Thermodynamic stability measurements on proteins and protein-ligand complexes can offer insights not only into the fundamental properties of protein folding reactions and protein functions, but also into the development of protein-directed therapeutic agents to combat disease. Conventional calorimetric or spectroscopic approaches for measuring protein stability typically require large amounts of purified protein. This requirement has precluded their use in proteomic applications. Stability of Proteins from Rates of Oxidation (SPROX) is a recently developed mass spectrometry-based approach for proteome-wide thermodynamic stability analysis. Since the proteomic coverage of SPROX is fundamentally limited by the detection of methionine-containing peptides, the use of tryptophan-containing peptides was investigated in this dissertation. A new SPROX-like protocol was developed that measured protein folding free energies using the denaturant dependence of the rate at which globally protected tryptophan and methionine residues are modified with dimethyl (2-hydroxyl-5-nitrobenzyl) sulfonium bromide and hydrogen peroxide, respectively. This so-called Hybrid protocol was applied to proteins in yeast and MCF-7 cell lysates and achieved a ~50% increase in proteomic coverage compared to probing only methionine-containing peptides. Subsequently, the Hybrid protocol was successfully utilized to identify and quantify both known and novel protein-ligand interactions in cell lysates. The ligands under study included the well-known Hsp90 inhibitor geldanamycin and the less well-understood omeprazole sulfide that inhibits liver-stage malaria. In addition to protein-small molecule interactions, protein-protein interactions involving Puf6 were investigated using the SPROX technique in comparative thermodynamic analyses performed on wild-type and Puf6-deletion yeast strains. A total of 39 proteins were detected as Puf6 targets and 36 of these targets were previously unknown to interact with Puf6. Finally, to facilitate the SPROX/Hybrid data analysis process and minimize human errors, a Bayesian algorithm was developed for transition midpoint assignment. In summary, the work in this dissertation expanded the scope of SPROX and evaluated the use of SPROX/Hybrid protocols for characterizing protein-ligand interactions in complex biological mixtures.

A Jaworskian analysis of four senior class primary teachers endeavouring to teach mathematics from a constructivist-compatible perspective

A constructivist philosophy underlies the Irish primary mathematics curriculum. As constructivism is a theory of learning its implications for teaching need to be addressed. This study explores the experiences of four senior class primary teachers as they endeavour to teach mathematics from a constructivist-compatible perspective with primary school children in Ireland over a school-year period. Such a perspective implies that children should take ownership of their learning while working in groups on tasks which challenge them at their zone of proximal development. The key question on which the research is based is: to what extent will an exposure to constructivism and its implications for the classroom impact on teaching practices within the senior primary mathematics classroom in both the short and longer term? Although several perspectives on constructivism have evolved (von Glaserfeld (1995), Cobb and Yackel (1996), Ernest (1991,1998)), it is the synthesis of the emergent perspective which becomes pivotal to the Irish primary mathematics curriculum. Tracking the development of four primary teachers in a professional learning initiative involving constructivist-compatible approaches necessitated the use of Borko’s (2004) Phase 1 research methodology to account for the evolution in teachers’ understanding of constructivism. Teachers’ and pupils’ viewpoints were recorded using both audio and video technology. Teachers were interviewed at the beginning and end of the project and also one year on to ascertain how their views had evolved. Pupils were interviewed at the end of the project only. The data were analysed from a Jaworskian perspective i.e. using the categories of her Teaching Triad of management of learning, mathematical challenge and sensitivity to students. Management of learning concerns how the teacher organises her classroom to maximise learning opportunities for pupils. Mathematical challenge is reminiscent of the Vygotskian (1978) construct of the zone of proximal development. Sensitivity to students involves a consciousness on the part of the teacher as to how pupils are progressing with a mathematical task and whether or not to intervene to scaffold their learning. Through this analysis a synthesis of the teachers’ interpretations of constructivist philosophy with concomitant implications for theory, policy and practice emerges. The study identifies strategies for teachers wishing to adopt a constructivist-compatible approach to their work. Like O’Shea (2009) it also highlights the likely difficulties to be experienced by such teachers as they move from utilising teacher-dominated methods of teaching mathematics to ones in which pupils have more ownership over their learning.

What Makes Online Grocers Work? A Case Study Analysis of Factors Contributing to Online Grocery Store Profitability

This research examined the factors contributing to the performance of online grocers prior to, and following, the 2000 dot.com collapse. The primary goals were to assess the relationship between a company’s business model(s) and its performance in the online grocery channel and to determine if there were other company and/or market related factors that could account for company performance. To assess the primary goals, a case based theory building process was utilized. A three-way cross-case analysis comprising Peapod, GroceryWorks, and Tesco examined the common profit components, the structural category (e.g., pure-play, partnership, and hybrid) profit components, and the idiosyncratic profit components related to each specific company. Based on the analysis, it was determined that online grocery store business models could be represented at three distinct, but hierarchically, related levels. The first level was termed the core model and represented the basic profit structure that all online grocers needed in order to conduct operations. The next model level was termed the structural model and represented the profit structure associated with the specific business model configuration (i.e., pure-play, partnership, hybrid). The last model level was termed the augmented model and represented the company’s business model when idiosyncratic profit components were included. In relation to the five company related factors, scalability, rate of expansion, and the automation level were potential candidates for helping to explain online grocer performance. In addition, all the market structure related factors were deemed possible candidates for helping to explain online grocer performance. The study concluded by positing an alternative hypothesis concerning the performance of online grocers. Prior to this study, the prevailing wisdom was that the business models were the primary cause of online grocer performance. However, based on the core model analysis, it was hypothesized that the customer relationship activities (i.e., advertising, promotions, and loyalty program tie-ins) were the real drivers of online grocer performance.

Analysis of Cosmogenic Impurities in Tellurium and Development of Tellurium Loading for the SNO+ Experiment

For the SNO+ neutrinoless double beta decay search, various backgrounds, ranging from impurities present naturally to those produced cosmogenically, must be understood and reduced. Cosmogenic backgrounds are particularly difficult to reduce as they are continually regenerated while exposed to high energy cosmic rays. To reduce these cosmogenics as much as possible the tellurium used for the neutrinoless double beta decay search will be purified underground. An analysis of the purification factors achievable for insoluble cosmogenic impurities found a reduction factor of $>$20.4 at 50\% C.L.. During the purification process the tellurium will come into contact with ultra pure water and nitric acid. These liquids both carry some cosmogenic impurities with them that could be potentially transferred to the tellurium. A conservative limit is set at $<$18 events in the SNO+ region of interest (ROI) per year as a result of contaminants from these liquids. In addition to cosmogenics brought underground, muons can produce radioactive isotopes while the tellurium is stored underground. A study on the rate at which muons produce these backgrounds finds an additional 1 event per year. In order to load the tellurium into the detector, it will be combined with 1,2-butanediol to form an organometallic complex. The complex was found to have minimal effect on the SNO+ acrylic vessel for 154 years.

An analysis of beginning mentors’ critical incidents in English post-compulsory education: navigating stormy waters

This qualitative study explores the barriers and dilemmas faced by beginning and novice mentors in post-compulsory education in the southeast of England. It analyses critical incidents (Tripp, 2012) taken from the everyday practice of mentors who were supporting new teachers and lecturers in the southeast of England. It categorises different types of critical incidents that mentors encountered and describes the strategies and rationales mentors used to support mentees and (indirectly) their learners and colleagues. The study explores ways in which mentors' own values, beliefs and life experiences affected their mentoring practice.  Methodology As part of a specialist master’s-level professional development module, 21 mentors wrote about two critical incidents (Tripp, 2012) taken from their own professional experiences, which aimed to demonstrate their support for their mentee’s range of complex needs. These critical incidents were written up as short case studies, which justified the rationale for their interventions and demonstrated the mentors' own professional development in mentoring. Critical incidents were used as units of analysis and categorised thematically by topic, sector and mentoring strategies used. Findings The research demonstrated the complex nature of decision-making and the potential for professional learning within a mentoring dyad. The study of these critical incidents found that mentors most frequently cited the controversial nature of teaching observations, the mentor’s role in mediating professional relationships, the importance of inculcating professional dispositions in education, and the need to support new teachers so that they can use effective behaviour management strategies. This study contributes to our understanding of the central importance of mentoring for professional growth within teacher education. It identifies common dilemmas that novice mentors face in post-compulsory education, justifies the rationale for their interventions and mentoring strategies, and helps to identify ways in which mentors' professional development needs can be met. It demonstrates that mentoring is complex, non-linear and mediated by mentors’ motivation and values.

Non-Verbal Categories. Analysis of an Galician Oral Narrative

Based on the concept of the triple basic structure of human communication by Poyatos (1994a, 1994b) and on the analytical and theoretical implications that derive from this, the present paper conceives the human communication as an indivisible whole in which verbal communication can not be separated from body behavior. This paper analyzes nonverbal categories used in oral communication. The corpus consists of an oral narration in Galician from which we highlighted certain kinemes (minimum units of body movement with meaning) by using the model proposed by Bouvet (2001), in order to explain the non-verbal categories with examples taken from said recordings.

Proteomic analysis of coronary sinus serum reveals leucine-rich α2-glycoprotein as a novel biomarker of ventricular dysfunction and heart failure

BACKGROUND: Heart failure (HF) prevention strategies require biomarkers that identify disease manifestation. Increases in B-type natriuretic peptide (BNP) correlate with increased risk of cardiovascular events and HF development. We hypothesize that coronary sinus serum from a high BNP hypertensive population reflects an active pathological process and can be used for biomarker exploration. Our aim was to discover differentially expressed disease-associated proteins that identify patients with ventricular dysfunction and HF.

METHODS AND RESULTS: Coronary sinus serum from 11 asymptomatic, hypertensive patients underwent quantitative differential protein expression analysis by 2-dimensional difference gel electrophoresis. Proteins were identified using mass spectrometry and then studied by enzyme-linked immunosorbent assay in sera from 40 asymptomatic, hypertensive patients and 105 patients across the spectrum of ventricular dysfunction (32 asymptomatic left ventricular diastolic dysfunction, 26 diastolic HF, and 47 systolic HF patients). Leucine-rich α2-glycoprotein (LRG) was consistently overexpressed in high BNP serum. LRG levels correlate significantly with BNP in hypertensive, asymptomatic left ventricular diastolic dysfunction, diastolic HF, and systolic HF patient groups (P≤0.05). LRG levels were able to identify HF independent of BNP. LRG correlates with coronary sinus serum levels of tumor necrosis factor-α (P=0.009) and interleukin-6 (P=0.021). LRG is expressed in myocardial tissue and correlates with transforming growth factor-βR1 (P<0.001) and α-smooth muscle actin (P=0.025) expression.

CONCLUSIONS: LRG was identified as a serum biomarker that accurately identifies patients with HF. Multivariable modeling confirmed that LRG is a stronger identifier of HF than BNP and this is independent of age, sex, creatinine, ischemia, β-blocker therapy, and BNP.

An updated comprehensive techno-economic analysis of algae biodiesel

Algae biodiesel is a promising but expensive alternative fuel to petro-diesel. To overcome cost barriers, detailed cost analyses are needed. A decade-old cost analysis by the U.S. National Renewable Energy Laboratory indicated that the costs of algae biodiesel were in the range of $0.53–0.85/L (2012 USD values). However, the cost of land and transesterification were just roughly estimated. In this study, an updated comprehensive techno-economic analysis was conducted with optimized processes and improved cost estimations. Latest process improvement, quotes from vendors, government databases, and other relevant data sources were used to calculate the updated algal biodiesel costs, and the final costs of biodiesel are in the range of $0.42–0.97/L. Additional improvements on cost-effective biodiesel production around the globe to cultivate algae was also recommended. Overall, the calculated costs seem promising, suggesting that a single step biodiesel production process is close to commercial reality.

IMPLEMENTING BACK PAIN TREATMENT PATHWAYS IN CLINICAL PRACTICE: QUALITATATIVE ANALYSIS OF STAKEHOLDER PERCEPTIONS

Background
Evidence-based practice advocates utilising best current research evidence, while reflecting patient preference and clinical expertise in decision making. Successfully incorporating this evidence into practice is a complex process. Based on recommendations of existing guidelines and systematic evidence reviews conducted using the GRADE approach, treatment pathways for common spinal pain disorders were developed.

Aims
The aim of this study was to identify important potential facilitators to the integration of these pathways into routine clinical practice.

Methods
A 22 person stakeholder group consisting of patient representatives, clinicians, researchers and members of relevant clinical interest groups took part in a series of moderated focus groups, followed up with individual, semi-structured interviews. Data were analysed using content analysis.

Results
Participants identified a number of issues which were categorized into broad themes. Common facilitators to implementation included continual education and synthesis of research evidence which is reflective of everyday practice; as well as the use of clear, unambiguous messages in recommendations. Meeting additional training needs in new or extended areas of practice was also recognized as an important factor. Different stakeholders identified specific areas which could be associated with successful uptake. Patients frequently defined early involvement in a shared decision making process as important. Clinicians identified case based examples and information on important prognostic indicators as useful tools to aiding decisions.

Conclusion
A number of potential implementation strategies were identified. Further work will examine the impact of these and other important factors on the integration of evidence-based treatment recommendations into clinical practice.

A comparative study of translation strategies applied in dealing with role languages. : A translation analysis of the video game Final Fantasy XIV

Localisation is the process of taking a product and adapting it to fit the culture in question. This usually involves making it both linguistically and culturally appropriate for the target audience. While there are many areas in video game translations where localisation holds a factor, this study will focus on localisation changes in the personalities of fictional characters between the original Japanese version and the English localised version of the video game Final Fantasy XIV: A Realm Reborn and its expansion Heavensward for PC, PS3 and PS4. With this in mind, specific examples are examined using Satoshi Kinsui's work on yakuwarigo, role language as the main framework for this study. Five non-playable characters were profiled and had each of their dialogues transcribed for a comparative analysis. This included the original Japanese text, the officially localised English text and a translation of the original Japanese text done by myself. Each character were also given a short summary and a reasoned speculation on why these localisation changes might have occurred. The result shows that there were instances where some translations had been deliberately adjusted to ensure that the content did not cause any problematic issues to players overseas. This could be reasoned out that some of the Japanese role languages displayed by characters in this game could potentially cause dispute among the western audience. In conclusion, the study shows that localisation can be a difficult process that not only requires a translator's knowledge of the source and target language, but also display some creativity in writing ability to ensure that players will have a comparable experience without causing a rift in the fanbase.

Analysis of irregular microstrip structures using a full wave MoM scheme

[EN]This article presents the results obtained in the analysis of irregular microstrip structures using a full wave method of moments scheme. The irregular microstrip structures are divided into rectangular subdomains. The EFIE is discretized an solved over the subdomains using a Galerkin type scheme. Base and weight functions are piece wise sinusoidals (PWS) or triangular. Delta gap voltage generators are used as sources]. Green functions are computed using a freely available library developed by our research group. All the calculations are carried out in the so called ”spatial domain” so there is no need of using regular grids during the discretization process.

Ultrassonic Analysis of UO2 Pellets

Ceramic materials have been widely used for various purposes in many different industries due to certain characteristics, such as high melting point and high resistance to corrosion. In the nuclear area, ceramics are of great importance due to the process of fabrication of fuel pellets for nuclear reactors. Generally, high accuracy destructive techniques are used to characterize nuclear materials for fuel fabrication. These techniques usually require costly equipment and facilities, as well as experienced personnel. This paper aims at presenting an analysis methodology for UO2 pellets using a non-destructive ultrasonic technique for porosity measurement. This technique differs from traditional ultrasonic techniques in the sense it uses ultrasonic pulses in frequency domain instead of time domain. Therefore, specific characteristics of the analyzed material are associated with the obtained frequency spectrum. In the present work, four fuel grade UO2 pellets were analyzed and the corresponding results evaluated.

Can the European Union build a bridge over troubled waters? An analysis of the politicised and depoliticised legal approach between the European Union and Cyprus

The Cyprus dispute accurately portrays the evolution of the conflict from ‘warfare to lawfare’ enriched in politics; this research has proven that the Cyprus problem has been and will continue to be one of the most judicialised disputes across the globe. Notwithstanding the ‘normalisation’ of affairs between the two ethno-religious groups on the island since the division in 1974, the Republic of Cyprus’ (RoC) European Union (EU) membership in 2004 failed to catalyse reunification and terminate the legal, political and economic isolation of the Turkish Cypriot community. So the question is; why is it that the powerful legal order of the EU continuously fails to tame the tiny troublesome island of Cyprus? This is a thesis on the interrelationship of the EU legal order and the Cyprus problem. A literal and depoliticised interpretation of EU law has been maintained throughout the EU’s dealings with Cyprus, hence, pre-accession and post-accession. The research has brought to light that this literal interpretation of EU law vis-à-vis Cyprus has in actual fact deepened the division on the island. Pessimists outnumber optimists so far as resolving this problem is concerned, and rightly so if you look back over the last forty years of failed attempts to do just that, a diplomatic combat zone scattered with the bones of numerous mediators. This thesis will discuss how the decisions of the EU institutions, its Member States and specifically of the European Court of Justice, despite conforming to the EU legal order, have managed to disregard the principle of equality on the divided island and thus prevent the promised upgrade of the status of the Turkish Cypriot community since 2004. Indeed, whether a positive or negative reading of the Union’s position towards the Cyprus problem is adopted, the case remains valid for an organisation based on the rule of law to maintain legitimacy, democracy, clarity and equality to the decisions of its institutions. Overall, the aim of this research is to establish a link between the lack of success of the Union to build a bridge over troubled waters and the right of self-determination of the Turkish Cypriot community. The only way left for the EU to help resolve the Cyprus problem is to aim to broker a deal between the two Cypriot communities which will permit the recognition of the Turkish Republic of Northern Cyprus (TRNC) or at least the ‘Taiwanisation’ of Northern Cyprus. Albeit, there are many studies that address the impact of the EU on the conflict or the RoC, which represents the government that has monopolised EU accession, the argument advanced in this thesis is that despite the alleged Europeanisation of the Turkish Cypriot community, they are habitually disregarded because of the EU’s current legal framework and the Union’s lack of conflict transformation strategy vis-à-vis the island. Since the self-declared TRNC is not recognised and EU law is suspended in northern Cyprus in accordance with Protocol No 10 on Cyprus of the Act of Accession 2003, the Turkish-Cypriots represent an idiomatic partner of Brussels but the relations between the two resemble the experience of EU enlargement: the EU’s relevance to the community has been based on the prospects for EU accession (via reunification) and assistance towards preparation for potential EU integration through financial and technical aid. Undeniably, the pre-accession and postaccession strategy of Brussels in Cyprus has worsened the Cyprus problem and hindered the peace process. The time has come for the international community to formally acknowledge the existence of the TRNC.

A Video Analysis of Intra- and Inter-professional Leadership Behaviours Within “The Burns Suite”: Identifying Key Leadership Models

Objective Leadership is particularly important in complex highly interprofessional health care contexts involving a number of staff, some from the same specialty (intraprofessional), and others from different specialties (interprofessional). The authors recently published the concept of “The Burns Suite” (TBS) as a novel simulation tool to deliver interprofessional and teamwork training. It is unclear which leadership behaviors are the most important in an interprofessional burns resuscitation scenario, and whether they can be modeled on to current leadership theory. The purpose of this study was to perform a comprehensive video analysis of leadership behaviors within TBS. Methods A total of 3 burns resuscitation simulations within TBS were recorded. The video analysis was grounded-theory inspired. Using predefined criteria, actions/interactions deemed as leadership behaviors were identified. Using an inductive iterative process, 8 main leadership behaviors were identified. Cohen’s κ coefficient was used to measure inter-rater agreement and calculated as κ = 0.7 (substantial agreement). Each video was watched 4 times, focusing on 1 of the 4 team members per viewing (senior surgeon, senior nurse, trainee surgeon, and trainee nurse). The frequency and types of leadership behavior of each of the 4 team members were recorded. Statistical significance to assess any differences was assessed using analysis of variance, whereby a p < 0.05 was taken to be significant. Leadership behaviors were triangulated with verbal cues and actions from the videos. Results All 3 scenarios were successfully completed. The mean scenario length was 22 minutes. A total of 362 leadership behaviors were recorded from the 12 participants. The most evident leadership behaviors of all team members were adhering to guidelines (which effectively equates to following Advanced Trauma and Life Support/Emergency Management of Severe Burns resuscitation guidelines and hence “maintaining standards”), followed by making decisions. Although in terms of total frequency the senior surgeon engaged in more leadership behaviors compared with the entire team, statistically there was no significant difference between all 4 members within the 8 leadership categories. This analysis highlights that “distributed leadership” was predominant, whereby leadership was “distributed” or “shared” among team members. The leadership behaviors within TBS also seemed to fall in line with the “direction, alignment, and commitment” ontology. Conclusions Effective leadership is essential for successful functioning of work teams and accomplishment of task goals. As the resuscitation of a patient with major burns is a dynamic event, team leaders require flexibility in their leadership behaviors to effectively adapt to changing situations. Understanding leadership behaviors of different team members within an authentic simulation can identify important behaviors required to optimize nontechnical skills in a major resuscitation. Furthermore, attempting to map these behaviors on to leadership models can help further our understanding of leadership theory. Collectively this can aid the development of refined simulation scenarios for team members, and can be extrapolated into other areas of simulation-based team training and interprofessional education.