Development of microfluidic tools for cancer single cell encapsulation and proliferation in microdroplets

The role of microfluidics in liquid biopsy as a more capable solution to address the monitoring of cancer progression in patients is gaining increasing attention. One out of the several difficulties in can-cer monitoring resides with the offset between current cell growth techniques in vitro and the influence of the cellular microenvironment in proliferation. One application of microfluidics consists in the use of microdroplets to replicate the complex dynamic microenvironment that can accurately describe factual 3D models of cancer cell growth. The goal of this thesis was to develop a set of microfluidic-based tools that would enable the encapsulation, proliferation and monitoring of single cancer cells in micro-droplets. For this, a set of microfluidic devices made of PDMS for droplet generation and containment were developed by photo- and soft-lithography techniques, being tested and optimized to ensure single cancer cell encapsulation. After the optimization of the droplet generation parameters in terms of droplet size and long-term stability on-chip, the best performance conditions were selected for cell growth ex-periments. Different densities of MDA-MB-435S cancer cells were combined with various percentages of Matrigel®, an extracellular matrix supplement, to promote cell proliferation. As a result, it was possi-ble to monitor droplets with cancer cells for a range of 1-20 days. A preliminary observation showed signs of cell aggregation, indicating that the tools developed during the thesis have the potential of developing 3D cancer spheroids from cancer single cells

SUBUNIT PEPTIDE-ENCAPSULATING NANOPARTICLE HIV VACCINE TARGETING CYTOTOXIC T LYMPHOCYTE ACTIVATION VIA PROTAC-ENHANCED CROSS PRESENTATION

Development of an effective prophylactic vaccine against HIV remains an ongoing challenge. Protein subunit vaccines have garnered interest recently, due to desirable safety and stability profiles in comparison to whole-pathogen vaccines. However, immunization with subunit vaccines has a proclivity to elicit humoral, rather than cell-mediated, immune responses. For cytotoxic T lymphocyte (CTL) stimulation, antigenic material in the subunit vaccine must 1) be endocytosed by antigen presenting cells, 2) escape from the endosome, 3) undergo degradation by the ubiquitin proteasome pathway, and 4) be loaded onto MHC I for cross-presentation (CP) to CTLs. In this thesis, a novel vaccine is introduced that specifically targets antigens to the proteolysis pathway for amplified CP and augments endosomal escape in a nanoparticulate drug delivery platform. Inspired by proteolysis-targeting chimera (PROTAC) technologies which have been employed for in situ destruction of cancer-related proteins, this project explores the incorporation of a proteolysis targeting moiety (ProTM) in vaccination. We hypothesize that by conjugating an E3 ubiquitin ligase ligand to synthetic long peptide (SLP) antigens, SLPs can be targeted for proteasomal processing and CP, ultimately resulting in improved CTL activation. Organic synthesis and click-chemistry methods were utilized to covalently modify SLPs with a ProTM. Poly(lactic-co-glycolic acid) nanoparticles that co-encapsulated the modified SLPs and the endosomal escape-promoting molecule, chloroquine, were formulated via single and double emulsion solvent evaporation. Size and loading efficiencies of nanoparticles were characterized using dynamic light scattering and absorbance. In vitro experiments demonstrated uptake of the ~200 nm particles by dendritic cells; quantitative colocalization analysis of fluorescently labeled SLPs and lysosomes was performed to delineate endosomal escape. With this strategy, statistically significant differences were not observed between chloroquine-containing and lacking nanoparticles. mRuby-labeled Galectin-8 cell lines were engineered for future experimental elucidation of endosomal escape. In vivo experiments tested the vaccine in a prime-boost vaccination schedule, showing the highest relative antigen-specific CTL activation in the group receiving the complete formulation. The ProTM-SLP subunit vaccine technology described here can feasibly be delivered in a biodegradable, polymeric nanoparticle formulation and shows strong potential to improve cellular immune responses that are clinically relevant for vaccination against HIV and other pathogens

Dynamically fighting bugs : prevention, detection and elimination

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Electrical Engineering and Computer Science, 2009.Cataloged from PDF version of thesis.Includes bibliographical references (p. 147-160).This dissertation presents three test-generation techniques that are used to improve software quality. Each of our techniques targets bugs that are found by different stake-holders: developers, testers, and maintainers. We implemented and evaluated our techniques on real code. We present the design of each tool and conduct experimental evaluation of the tools with available alternatives. Developers need to prevent regression errors when they create new functionality. This dissertation presents a technique that helps developers prevent regression errors in object-oriented programs by automatically generating unit-level regression tests. Our technique generates regressions tests by using models created dynamically from example executions. In our evaluation, our technique created effective regression tests, and achieved good coverage even for programs with constrained APIs. Testers need to detect bugs in programs. This dissertation presents a technique that helps testers detect and localize bugs in web applications. Our technique automatically creates tests that expose failures by combining dynamic test generation with explicit state model checking. In our evaluation, our technique discovered hundreds of faults in real applications. Maintainers have to reproduce failing executions in order to eliminate bugs found in deployed programs. This dissertation presents a technique that helps maintainers eliminate bugs by generating tests that reproduce failing executions. Our technique automatically generates tests that reproduce the failed executions by monitoring methods and storing optimized states of method arguments.(cont.) In our evaluation, our technique reproduced failures with low overhead in real programs Analyses need to avoid unnecessary computations in order to scale. This dissertation presents a technique that helps our other techniques to scale by inferring the mutability classification of arguments. Our technique classifies mutability by combining both static analyses and a novel dynamic mutability analysis. In our evaluation, our technique efficiently and correctly classified most of the arguments for programs with more than hundred thousand lines of code.by Shay Artzi.Ph.D

Towards Populating Generalizable Engineering Design Knowledge

Aiming to populate generalizable engineering design knowledge, we propose a method to extract facts of the form head entity :: relationship :: tail entity from sentences found in patent documents. These facts could be combined within and across patent documents to form knowledge graphs that serve as schemes for representing as well as storing design knowledge. Existing methods in engineering design literature often utilise a set of predefined relationships to populate triples that are statistical approximations rather than facts. In our method, we train a tagger to identify both entities and relationships from a sentence. Given a pair of entities thus identified, we train another tagger to identify the relationship tokens that specifically denote the relationship between the pair. For training these taggers, we manually construct a dataset of 44,227 sentences and corresponding facts. We also compare the performance of the method against typically recommended approaches, wherein, we predict the edges among tokens by pairing the tokens independently and as part of a graph. We apply our method to sentences found in patents related to fan systems and build a domain knowledge base. Upon providing an overview of the knowledge base, we search for solutions relevant to some key issues prevailing in fan systems. We organize the responses into knowledge graphs and hold a comparative discussion against the opinions from ChatGPT

Contemporary arts practice: Disequilibrium/Limbo/Resistance

This exegesis proposes that conflict and tension are transformative factors in contemporary arts practice, from making through to methodologies; viewing the dialogic nature of art-making and the artist-in-the-world, linked to, yet separate from, surrounding entities. It is the culmination of conceptual research and creative production … the exegetical and the artwork … that together comprise an original contribution to new knowledge, with broad implications - and specific applications - significant to both practice and practitioner

An investigation into mesenchymal stromal cells’ behaviour in 3D environment of PNIPAM-based hydrogel

Mesenchymal stromal cells (MSCs) are multipotent cells, known for the ability to differentiate into cells of bone, fat and cartilage. MSCs are commonly sourced from the bone marrow environment, where these cells reside in a 3-dimensional (3D) environment and are exposed to components of the extracellular matrix (ECM), other cells types, biochemical and mechanical stimuli. Conventional monolayer culture cannot replicate the complexity of the in vivo bone marrow environment. Therefore, a more representative MSC culture environment is required. The aim of this project was to develop a highly tunable synthetic hydrogel, on the basis of poly(N-isopropylacrylamide) (PNIPAM), to allow temperature-driven encapsulation and subsequent study of MSC behaviour in three dimensional (3D) environment. The highly branched (HB) architecture of PNIPAM polymer was obtained by means of living radical polymerisation. Further polymer functionalisation with tri-arginine peptide sequence (RRR) has stabilised hydrogel structure and reduced solvent expulsion (syneresis). Rheological studies have revealed overall resistance to deformation (G*; complex modulus) of 5wt% HB PNIPAM+RRR to be equal to 542.3 Pa at 37˚C. MSC single cell suspensions were successfully encapsulated in HB PNIPAM+RRR 3D droplet hydrogels, demonstrating rounded morphology, absence of proliferation and stable cell viability. Differentiation potential studies of the cell-loaded hydrogels, cultured in osteogenic or adipogenic media, demonstrated osteo-conductive, osteo-inductive and adipo-inhibitive responses. In summary, HB PNIPAM+RRR is a novel chemical entity with a thermo-responsive nature, which forms a porous and hydrated scaffold with osteo-inductive properties for MSC encapsulation at physiologically relevant temperature. HB PNIPAM is a highly functional and amenable hydrogel platform for assessment of MSC behaviour and guidance of differentiation in 3D environment

Implementing statically typed object-oriented programming languages

A paraîtreInternational audienceObject-oriented programming languages represent an original implementation issue due to the mechanism known as late binding, aka message sending. The underlying principle is that the address of the actually called procedure is not statically determined, at compile-time, but depends on the dynamic type of a distinguished parameter known as the receiver. In statically typed languages, the point is that the receiver's dynamic type may be a subtype of its static type. A similar issue arises with attributes, because their position in the object layout may depends on the object's dynamic type. Furthermore, subtyping introduces another original feature, i.e. subtype checks. All three mechanisms need specific implementations, data structures and algorithms. In statically typed languages, late binding is generally implemented with tables, called virtual function tables in C++ jargon. These tables reduce method calls to pointers to functions, through a small fixed number of extra indirections. It follows that object-oriented programming yields some overhead, as compared to usual procedural languages. The different techniques and their resulting overhead depend on several parameters. Firstly, inheritance and subtyping may be single or multiple and a mixing is even possible, as in JAVA, which presents single inheritance for classes and multiple subtyping for interfaces. Multiple inheritance is a well known complication. Secondly, the production of executable programs may involve various schemes, from global compilation frameworks, where the whole program is known at compile time, to separate compilation and dynamic loading, where each program unit---usually a class in an object-oriented context---is compiled and loaded independently of any usage. Global compilation is well known to facilitate optimization. In this paper, we review the various implementation schemes available in the context of static typing and in the three cases of single inheritance, multiple inheritance, and single inheritance but with multiple subtyping, e.g. JAVA. The survey focuses on separate compilation and dynamic loading, as it is the most commonly used framework and the most demanding. However, many works have been recently undertaken in the global compilation framework, mostly for dynamically typed languages but also applied to the EIFFEL language in the SMARTEIFFEL compiler. Hence, we examine global techniques and how they can improve implementation efficiency. Finally, a mixed framework is considered, where separate compilation is followed by a global step, similar to linking, which uses global techniques, as well for implementation, with coloring, as for optimization, with type analysis. An application to dynamic loading is sketched

The Bookish Turn: Assessing the Impact of the Book-Roll on Authorial Self- Representation in Early Hellenistic Poetry

My thesis takes its start from the oft-used description of Hellenistic poetry as ‘bookish’, but looks beyond the connotations of this label as denoting a milieu which was self-consciously intellectual, and instead considers the more fundamental ramifications of the designation: that Hellenistic poetry was bookish in its form, as much as in outlook. To consider the implications of this, I focus upon a period, and a significant poetic topos, wherein the effects of the book-roll can be most keenly discerned, assessing the impact of the medium upon authorial self- representations - particularly in the construction of authorial personae - undertaken in early Hellenistic poetry (c.323-246 BC). In Part I of the thesis, I assess the evolution of authorial self-representation in epigram, charting developments from the inscribed form of the genre through to the book-epigram collections of the Hellenistic period: I argue that the author acquired a newfound prominence in this medial transition, asserting their presence as a voice within the text as opposed to a figure situated strictly in antecedence to it. I demonstrate this through analyses of Posidippus, Callimachus, Nossis, Asclepiades, and the epigrams ascribed to Erinna, and suggest that we repeatedly observe authors undertaking composite processes of self-representation, as a direct result of the composite context of the book-roll. In Part II of the thesis, I examine the Mimiambs of Herodas. Through the analysis of Mimiamb 8 (in which Herodas constructs an authorial persona, and defines his poetic programme) in conjunction with an appraisal of the metapoetic dimension of the other Mimiambs, I assess the manner in which Herodas undertakes a complex, intertextual process of self-representation. Arguing that the author reflects upon the generic and medial innovations of his poetic practice across his corpus, I demonstrate that this process of reflection complements Herodas’ overt authorial self-representation in Mimiamb 8. In summary, I argue that the impact of the book-roll on authorial self-representation was wide- ranging, but that the most significant consequence of the medium was the evolution of authorial self-representation as a composite, roll-spanning activity