Development of Microfluidic Instrumentation for Application in the Diagnosis of Rare Anaemias

Globally, the number of children born every year with a rare anaemia exceeds 500,000. The symptoms of rare anaemias range, depending on the mutation, from mild to severe, and in many cases prove to be fatal. The geographical prevalence of rare anaemias is concentrated in developing countries where resources available for diagnosis and treatment are scarce. The gold standard diagnosis of rare anaemia requires a three-tier investigation which is costly and not readily available in the areas most afflicted. As such, there is a need for a low-cost and user friendly method of diagnosis for these diseases. This thesis investigates the diagnostic abilities of a bio-chemical assay that exposes red blood cells to a low pH shock using microfluidic techniques. This involved the development of a novel low-cost microfluidic instrument, which has been named MeCheM, to run Lab-on-a-Chip devices. The experimental techniques and protocols developed are critically reviewed using healthy blood samples as the control. The results from the control population establish baselines for comparison against the diseased samples. Subsequently, the developed methods are investigated for diagnostic capabilities using rare anaemia blood samples. The results from these investigations suggest that there are observable differences for the developed Flow Test in the case of the Thalassaemia and Hereditary Spherocytosis disorders. Similarly, the developed Cell-Surface Adhesion measurements highlighted significant differences among the Sickle Cell samples. Additionally, secondary investigations indicated correlations between the gold standard Red Blood Cell Count and the RBC Count as measured using MeCheM, and Mean Corpuscular Volume and Average Cell Projected Area (pre-acid addition). The development of MeCheM, a novel microfluidic instrument, as a stand-alone device is a key output from this body of work

Adolescent Visual Voices: Discovering Emerging Identities Through Photovoice, Perspective and Narrative

This qualitative multicase study seeks to create dynamic pedagogical space - meaning making capacities encouraging multiple types of participation - where adolescent voices are privileged. Opening pedagogical space sits at the intersection of feminist standpoint theory, critical consciousness and social constructivism. Disturbingly, space supporting the inner lives and voices of students is shrinking in current educational environments, partially due to prescriptive curricula and rigid standards. The rationale for this study emanates from the researcher’s (as co-participant) educational journey and professional experience at the middle school and higher education levels. This study’s purpose explores, “what happens when space is created for middle school students to engage in photovoice participatory action research with narrative self-construction and perspective taking?” The writer’s assumptions comprise thinking around photography stimulating renewal of classroom space for imagining, sharing lived experiences and exploring alternative possibilities. Participants include 15 middle school students across two case studies situated in voluntary after school programs. Participant sites constitute a suburban middle school and an urban University in partnership with community outreach. Qualitative methodology, including a photovoice participatory action hybrid model, informs the two cycle analyses: visual content analysis codes photographs through frequency counts leading to meta-themes while thematic narrative analysis examines discussions and narrative self construction through In Vivo coding leading to meta-theme construction. Framed by three guiding questions, findings are advanced and through reflection and synthesis, the following analytic categories emerge supported by the conceptual framework – pedagogical space reveals strengths; diffuses power; and explores identity. Researcher assumptions are challenged as participants use pedagogical spaces to showcase, “here’s what I am,” rather than, “here’s what I long to be.” Conclusions gleaned from findings include: photographs are multiliteracies opening channels for communication, comprehension and cultural diversity; and middle school students seek power neutral opportunities to explore identity, demonstrate what they know, and engage in topics they care about. Recommendations support classroom habits integrating new literacies, museum components, bi-weekly autobiographical narratives and reflexive memo writing. This research contributes to the fields of adolescent identity, disciplinary literacy, feminist theory, participatory action research, secondary education and visual arts

Model-based scientific discovery--a study in space bioengineering

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Civil and Environmental Engineering, 1992.Includes bibliographical references (leaves 130-138).by Nicolas Groleau.Ph.D

Nuclear Magnetic Resonance Studies of Lithium Metal Anodes

Lithium metal has received a renewed interest as a promising anode material for next-generation, high-energy batteries owing to its high specific capacity (3860 mAh g-1) and low reduction potential (-3.04 V vs. the standard hydrogen electrode). However, lithium metal batteries suffer from low capacity retention, short cycle life and safety problems associated with microstructural and dendritic growth of lithium. In this work, nuclear magnetic resonance (NMR) spectroscopy is used to understand the effect of the solid electrolyte interphase (SEI) on lithium metal deposition. In situ NMR is used to quantify the lithium microstructures formed during plating and allows the current efficiency and porosity of the structures to be estimated. The effect of the fluoroethylene carbonate (FEC) additive is explored along with a range of plating conditions. NMR measurements show that the isotope exchange between a 6Li-enriched lithium metal and a natural abundance electrolyte depends significantly on the electrolyte and the corresponding SEI. A numerical model is developed to describe the processes during isotope exchange and is discussed in the context of the standard model of electrochemical kinetics. The model is used to extract both an exchange current at the open circuit voltage and the SEI formation current as a function of time. In situ NMR methods are then developed to study ‘anode-free’ lithium metal batteries where the lithium is plated directly onto a bare copper current collector from a LiFePO4 cathode. The low cycling stability of lithium metal batteries becomes clear when there is no excess of lithium in the cell. The ‘dead lithium’ and SEI formation can be quantified by NMR and their relative rates of formation are here compared in carbonate and ether-electrolytes. Importantly, the NMR experiments reveal that the dissolution of lithium metal during the periods when the battery is not in use, i.e., when no current is flowing, demonstrating that dissolution of lithium remains a critical issue for lithium metal batteries. Strategies to mitigate lithium corrosion are explored; the work demonstrating that both polymer coatings and the modification of the copper surface chemistry stabilising lithium metal. Overall, this work demonstrates that the NMR approach offers unique insight into the dynamic processes occurring on lithium metal both during electrochemical measurements and at the open circuit voltage

Materials’ Methods: NMR in Battery Research

Improving electrochemical energy storage is one of the major issues of our time. The search for new battery materials together with the drive to improve performance and lower cost of existing and new batteries is not without its challenges. Success in these matters is undoubtedly based on first understanding the underlying chemistries of the materials and the relations between the components involved. A combined application of experimental and theoretical techniques has proven to be a powerful strategy to gain insights into many of the questions that arise from the “how do batteries work and why do they fail” challenge. In this Review, we highlight the application of solid-state nuclear magnetic resonance (NMR) spectroscopy in battery research: a technique that can be extremely powerful in characterizing local structures in battery materials, even in highly disordered systems. An introduction on electrochemical energy storage illustrates the research aims and prospective approaches to reach these. We particularly address “NMR in battery research” by giving a brief introduction to electrochemical techniques and applications as well as background information on both in and ex situ solid-state NMR spectroscopy. We will try to answer the question “Is NMR suitable and how can it help me to solve my problem?” by shortly reviewing some of our recent research on electrodes, microstructure formation, electrolytes and interfaces, in which the application of NMR was helpful. Finally, we share hands-on experience directly from the lab bench to answer the fundamental question “Where and how should I start?” to help guide a researcher’s way through the manifold possible approaches.This project has received funding from the European Union’s Horizon 2020 research and innovation program under the Marie Skłodowska-Curie grant agreement No 655444 (O.P.). K.J.G. thanks the Winston Churchill Foundation of the United States and the Herchel Smith Scholarship for financial support

Computational Molecular Biophysics of Membrane Reactions

Proteins are nanoscale molecules that perform functions essential for biological life. Membranes surrounding cells, for example, contain receptor proteins that mediate communication between the cell and the external milieu, membrane transporters that transport ions and larger compounds across the membranes, and enzymes that catalyze chemical reactions. Likewise, soluble proteins found in interior of the cell include motor proteins that move other proteins around, enzymes that bind to and repair breaks in the DNA, and proteins that help control the cellular clock. Mutations in genes that encode proteins can cause disease, as is the case of cystic fibrosis, a disease that associates with mutation of a chloride channel called the cystic fibrosis transmembrane conductance regulator.1 The essential functions they perform in the cell makes proteins essential drug targets for modern bio-medical applications. An important example here is the programmed death ligand-1 (PD-L1), which is a valuable target for modern immunotherapy.2-4 Predicting how a protein responds to a drug molecule, or using the protein as inspiration for biotechnological applications, require knowledge of how that protein works. As proteins are dynamic entities and protein dynamics are essential for function,5-8 describing the mechanism of action of a protein requires knowledge about the protein motions in fluid environments. Theoretical biophysics provides valuable tools to characterize protein reaction mechanisms and protein motions at the atomic level of detail. This Habilitation Thesis presents research on using theoretical biophysics approaches to decipher how proteins work. The focus of the research is on membrane proteins and reactions that occur at lipid membrane interfaces. The central question I address is the role of dynamic hydrogen (H) bonds in protein function and membrane interactions. The methods used include quantum mechanical (QM) computations of small molecules, combined quantum mechanics/molecular mechanics (QM/MM) of chemical reactions in protein environments, classical mechanical computations of large protein and membrane systems, and bridging numerical simulations to bioinformatics. In my research group we developed algorithms to identify H-bond networks in proteins and membrane environments, and to characterize the dynamics of these networks. To extend the applicability of numerical computations to bio-systems that bind drug-like compounds, we derive parameters for a potential energy function widely used in the field. The main research topics and specific questions addressed are summarized below together with a discussion of the computational approaches used

Use-driven concept formation

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Electrical Engineering and Computer Science, 2010.Cataloged from PDF version of thesis.Includes bibliographical references (p. 161-165).When faced with a complex task, humans often identify domain-specific concepts that make the task more tractable. In this thesis, I investigate the formation of domain-specific concepts of this sort. I propose a set of principles for formulating domain-specific concepts, including a new inductive bias that I call the equivalence class principle. I then use the domain of two-player, perfect-information games to test and refine those principles. I show how the principles can be applied in a semiautomated fashion to identify strategically-important visual concepts, discover highlevel structure in a game's state space, create human-interpretable descriptions of tactics, and uncover both offensive and defensive strategies within five deterministic, perfect-information games that have up to forty-two million states apiece. I introduce a visualization technique for networks that discovers a new strategy for exploiting an opponent's mistakes in lose tic-tac-toe; discovers the optimal defensive strategies in five and six men's morris; discovers the optimal offensive strategies in pong hau k'i, tic-tac-toe, and lose tic-tac-toe; simplifies state spaces by up to two orders of magnitude; and creates a hierarchical depiction of a game's state space that allows the user to explore the space at multiple levels of granularity. I also introduce the equivalence class principle, an inductive bias that identifies concepts by building connections between two representations in the same domain. I demonstrate how this principle can be used to rediscover visual concepts that would help a person learn to play a game, propose a procedure for using such concepts to create succinct, human-interpretable descriptions of offensive and defensive tactics, and show that these tactics can compress important information in the five men's morris state space by two orders of magnitude.by Jennifer M. Roberts.Ph.D

ERK1/2 signalling and protein ubiquitylation in the control of apoptosis

Programmed cell death, or apoptosis, is critical for normal developmental processes that involve cell turnover including embryogenesis and development and function of the immune system. It is preceded by classical changes in cell morphology, driven by biochemical changes including caspase activation. Apoptosis is deregulated in multiple human diseases, with suppression of apoptosis being critical for carcinogenesis. As such, proteins that regulate apoptosis are tightly regulated by cell fate signalling pathways. The ERK1/2 signalling pathway is a key regulator of cell intrinsic apoptosis, in part through regulation of the pro-apoptotic protein ‘BCL2-interacting mediator of cell death’ (BIM). BIM is phosphorylated by ERK1/2 and this serves to drive its K48-linked polyubiquitylation and proteasome-dependent degradation, thereby promoting cell survival. βTrCP, an F-box protein that acts in a larger SCF complex, is one of several E3 ligases that have been proposed to polyubiquitinate BIM. This study demonstrated that of the major isoforms of BIM only BIMEL interacts with βTrCP. ERK1/2-driven phosphorylation of BIMEL is essential for this interaction, leading to BIMEL destabilisation and degradation. As a consequence, tumour cells that are addicted to ERK1/2 signalling undergo BIM-dependent cell death in response to MEK1/2 inhibitors when combined with BH3-mimetics such as ABT263, small molecules that inhibit pro-survival proteins of the apoptotic pathway. The RSK1/2 protein kinases, immediate downstream targets of ERK1/2, have also been implicated in the destabilisation of BIMEL. Specifically phosphorylation of BIMEL by RSK1/2 is proposed to be required for βTrCP binding. This study revealed that whilst the putative RSK1/2 phosphorylation sites in BIMEL may be required for βTrCP binding, inhibition of RSK activity by three distinct RSK inhibitors does not block BIMEL:βTrCP binding or BIMEL turnover. Furthermore, tumour cells that are addicted to ERK1/2 signalling for survival are not addicted to RSK activity, arguing against a role for RSK in the regulation of BIMEL. This suggests that ERK1/2 and an as yet unidentified kinase cooperate to drive BIMEL degradation. Deubiquitylating enzymes (DUBs) remove ubiquitin from target proteins. In the context of BIMEL, DUB activity might oppose E3 ligases and thus cause its accumulation. Until recently the DUB for BIM was unknown however, USP27x has now been suggested. Follow-up validation of the reported interaction between BIMEL and USP27x was challenging but loss of BIMEL polyubiquitination was observed following overexpression of USP27x, suggesting that USP27x may serve as a DUB for BIM. Numerous DUBs control cellular processes that are dysregulated in cancer, including proliferation and apoptosis, making them attractive therapeutic targets. Recent interest in the DUB USP30 has increased as it has been shown to inhibit parkin-mediated mitophagy, with defective mitophagy being linked to Parkinson’s disease. USP30 has also been suggested to play a role in apoptosis and its depletion was shown to sensitise cells to BH3 mimetics. These findings suggest that USP30 depletion or inhibition could provide a means for inducing tumour cell death. Indeed, combining a novel USP30 inhibitor (MTX32), provided by Mission Therapeutics, with the BH3 mimetic, ABT-263, induced apoptotic tumour cell death that required BAX and caspase activation. However, this was not replicated by a more selective USP30 inhibitor (MTX48), suggesting that the observed apoptotic cell death reflected the off-target effects of MTX32 rather than specific inhibition of USP30. Finally, an RNAi screen, targeting 94 DUBs, and 8 sentrin/SUMO-specific proteases (SENPs), in the human genome, was performed to identify DUBs that modulate cell death induced by MEK1/2 or mTOR inhibition. As such, inhibitors of identified ‘hit’ DUBs might be suitable as a combinatorial therapy with MEK1/2 or mTOR inhibitors in the treatment of cancer. The RNAi screen identified several DUBs, that, when knocked down, sensitised HCT116 cells to MEK1/2 inhibitor treatment and enhanced MEK1/2 inhibitor induced cell death in a BAX-dependent fashion. This work is discussed in the context of the role of ERK1/2 signalling as a pro-survival pathway, its specific role in BIM regulation, the potential for co-targeting DUBs and the ERK1/2 pathway to inhibit the growth of ERK1/2 addicted tumour cells and suggestions for future work are outlined.iCase studentship - partially funded by Mission Therpeautic

The Journal of Mine Action Issue 5.1 (2001)

The Journal of Mine Action Issue 5.1 Landmines in Asia and the Pacifi