VictorJuiz/thesis huggingface dataset:
Evaluation of Photophysical Methods for Photodynamic Therapy Dosimetry
Keywords: Singlet Oxygen
Luminescence
Photodynamic Therapy
Dosimetry

In photodynamic therapy (PDT), the combination of light, photosensitizer and molecular oxygen generates reactive oxygen species, including singlet oxygen (1O2), which is regarded as the primary cytotoxin and effector with most clinical photosensitizers. PDT has gained some acceptance for the treatment of cancer and other conditions. However, its clinical utility and effectiveness has been limited by variability in treatment response and failure to integrate adequate treatment planning and dosimetry. Direct PDT dosimetry through the detection of ultra-weak near-infrared 1O2 luminescence emission at 1270 nm (SOL) collapses the complexity of PDT into a single parameter, the 1O2 concentration. Prior to the present studies, it was shown that SOL was well correlated with PDT response in vitro and in vivo under controlled experimental conditions. However, SOL detection is technically challenging because of the very low radiative probability of 1O2 (~ 10-8 in biological environments), dynamic background signals and limited sensitivity of suitable photodetectors in this wavelength region. A technologically simpler and less costly PDT dosimetry approach is to use photosensitizer photobleaching to estimate the 1O2 dose. The first objective in this thesis was to characterize the dynamics of SOL measurements, in particular the influence of oxygen depletion, in order to improve the quantification of SOL and its use as an accurate PDT dose metric. Subsequently, direct comparison of SOL and photobleaching-based dosimetry during in vitro PDT treatment with meso-tetra(hydroxyphenyl)chlorin (mTHPC) showed that SOL dosimetry is robust but that photobleaching-based dosimetry can fail under hypoxic conditions. However, the latter can be salvaged through the identification of a previously unreported 613 nm emission from mTHPC that indicates hypoxia. These studies were carried forward into an in vivo dorsal skin-fold window chamber tumor model, which showed promising initial correlation between 1O2 dose and tumor response. This work also identified SOL detection limitations and opportunities for further development. Additionally, SOL measurements were used as a ‘gold standard’ to evaluate novel activatable PDT beacons and a novel “PDT biodosimeter” based on STAT3 cross-linking. Future work includes further tumor dose-response studies, characterization of novel photosensitizing agents, improvement on signal detection and processing, and studies in normal human skin.
Aerostructural Shape and Topology Optimization of Aircraft Wings
Keywords: Multidisciplinary Design Optimization
Topology Optimization

A series of novel algorithms for performing aerostructural shape and topology optimization are introduced and applied to the design of aircraft wings. An isoparametric level set method is developed for performing topology optimization of wings and other non-rectangular structures that must be modeled using a non-uniform, body-fitted mesh. The shape sensitivities are mapped to computational space using the transformation defined by the Jacobian of the isoparametric finite elements. The mapped sensitivities are then passed to the Hamilton-Jacobi equation, which is solved on a uniform Cartesian grid. The method is derived for several objective functions including mass, compliance, and global von Mises stress. The results are compared with SIMP results for several two-dimensional benchmark problems. The method is also demonstrated on a three-dimensional wingbox structure subject to fixed loading. It is shown that the isoparametric level set method is competitive with the SIMP method in terms of the final objective value as well as computation time. In a separate problem, the SIMP formulation is used to optimize the structural topology of a wingbox as part of a larger MDO framework. Here, topology optimization is combined with aerodynamic shape optimization, using a monolithic MDO architecture that includes aerostructural coupling. The aerodynamic loads are modeled using a threedimensional panel method, and the structural analysis makes use of linear, isoparametric, hexahedral elements. The aerodynamic shape is parameterized via a set of twist variables representing the jig twist angle at equally spaced locations along the span of the wing. The sensitivities are determined analytically using a coupled adjoint method. The wing is optimized for minimum drag subject to a compliance constraint taken from a 2g maneuver condition. The results from the MDO algorithm are compared with those of a sequential optimization procedure in order to quantify the benefits of the MDO approach. While the sequentially optimized wing exhibits a nearly-elliptical lift distribution, the MDO design seeks to push a greater portion of the load toward the root, thus reducing the structural deflection, and allowing for a lighter structure. By exploiting this trade-off, the MDO design achieves a 42% lower drag than the sequential result.
Functional Tissue Engineering of Myocardium Through Cell Tri-culture
Keywords: Tissue Engineering
Cardiac Tissue Engineering
Biomedical Engineering
Vascularization
Tri-culture
Microfabrication
Cell Tracking
VEGF
Connexin-43
Pre-culture
Milica Radisic
Rohin Iyer
Bioreactor
Microbioreactor
Soft Lithography
Cardiomyocyte
Fibroblast
Endothelial
Sequential Pre-culture
Pericyte
Cords
Endothelial Cord

Cardiac tissue engineering promises to create therapeutic tissue replacements for repair of diseased native myocardium. The main goals of this thesis were four-fold: 1) to evaluate cardiac tissues engineered using multiple cell types including endothelial cells (EC), fibroblasts (FB), and cardiomyocytes (CM); 2) to spatiotemporally track cells in organoids and optimize their seeding percentages for improved function; 3) to enhance vascular cord formation through sequential versus simultaneous seeding of ECs and FBs; and 4) to perform mechanistic studies to elucidate the role of soluble factors in cell-cell communication. Microscale templates fabricated from photocrosslinkable poly(ethylene glycol) diacrylate (PEG-DA) were used for all studies for rapid screening. When ECs and FBs were precultured for two days prior to seeding enriched CMs, cells self-assembled into three-dimensional, beating organoids, compared to simultaneously tricultured EC/ FB / CM which formed non-contractile clusters. Fluorescent dyes were used to label and track each cell type for up to 4 days, demonstrating an even distribution of cells within precultured organoids versus EC clustering in simultaneous triculture. When ECs were seeded first, followed by FBs 24 hours later and CMs 48 hours later, vascular-like cords formed that persisted with time in a seeding density-dependent manner. Vascular endothelial growth factor (VEGF) signaling was quantified, showing higher endogenous VEGF secretion rates in sequential preculture (16.6 ng/mL/hr) compared to undetectable VEGF secretion in simultaneous triculture. Blocking of endogenous VEGF signaling through addition of VEGF antibody / VEGFR2 inhibitor resulted in a significant decrease in mRNA and protein expression of the key cardiac gap junctional marker connexin-43. These findings provide a foundation for future work into the mechanisms governing functional cardiac tissue engineering performance and may aid in the development of novel therapies for heart failure based on growth factor signaling and engineering of vascularized, clinically relevant cardiac tissue patches.
Hunting Specialisation and the Broad Spectrum Revolution in the Early Epipalaeolithic: Gazelle Exploitation at Urkan e-Rubb IIa, Jordan Valley
Keywords: zooarchaeology
taphonomy
Epipalaeolithic
Kebaran
broad spectrum
specialisation
gazelle
subsistence

This present research explores the impact of Flannery’s (1969) Broad Spectrum Revolution model within the context of the Levantine Epipalaeolithic, specifically the early (Kebaran) Epipalaeolithic. This model attempts to explain changes in subsistence behaviour associated with the end of the Pleistocene leading up to the development of agriculture. The Jordan valley represents an ideal model for Flannery’s marginal habitat because of the range of different flora and fauna required more intensive resource exploitation strategies. The discussion here focuses on the zooarchaeological and taphonomic analysis of two faunal assemblages (representing two sub-horizons) from Urkan e-Rubb IIa, a Kebaran site located in the lower Jordan Valley, and excavated by E. Hovers (Institute of Archaeology, Hebrew University Jerusalem), in 1986 and 1988. This site shows evidence of specialised gazelle hunting and processing. Initial interpretations of the site argued for a multi-purpose residential base camp, supported by lithic tool and shell bead manufacturing. Recent discussions of Epipalaeolithic diet breadth have concentrated on the end of the Pleistocene (Munro 2001, 2003, 2004, 2009; Stiner 2001; Stiner & Munro 2002; Stiner et al. 1999, 2000; Stutz et al. 2009) and have been directed towards explaining changing subsistence patterns towards the end of the Epipalaeolithic (i.e. the Natufian), where an increased use of wild cereals and small game has been well documented. The view that it is not until the Natufian that broad spectrum resource strategies were used is questioned here. Major questions that are addressed with this research include: What do the faunal assemblages from Urkan e-Rubb IIa tell us about Kebaran subisistence behaviour? Do broad spectrum models help to shed light on subsistence adaptations at the end of the Last Glacial Maximum? What can recent analyses of Jordan Valley faunal assemblages contribute to the discussion? These are addressed through a detailed zooarchaeological and taphonomic analysis of the Urkan e-Rubb IIa fauana, followed by synchronic and diachronic analyses of Levantine Epipalaeolithic assemblages, through a combination of taxonomy-based diversity indices and prey-ranking indices.
A Metagenome-based Examination of Dechlorinating Enrichment Cultures: Dehalococcoides and the Role of the Non-dechlorinating Microorganisms.
Keywords: metagenomics
bioremediation

Bioremediation of chlorinated solvents to a non-toxic end product can be achieved with Dehalococcoides sp., through reductive dehalogenation of the chlorinated organics. Dehalococcoides sp. are typically maintained in enrichment cultures containing multiple microorganisms, which often exhibit better growth and dechlorination rates than Dehalococcoides isolates. This thesis examines the nature of the relationships between the Dehalococcoides and the non-dechlorinating organisms in enrichment cultures. Comparative metagenomics revealed differences and similarities in taxonomy and functional gene complements between three Dehalococcoides-containing enrichment cultures. This allowed identification of pivotal supporting organisms involved in maintaining dechlorination activity through provision of nutrients and other factors to the Dehalococcoides. A Dehalococcoides pan-genus microarray was designed using available sequenced genomes as well as a draft genome generated from an in-house metagenome sequence. The array leverages homolog clustering during probe design to improve detection of the Dehalococcoides genus, including strains not utilized in the array design. A phylogenetic examination of the reductive dehalogenase gene family showed that organism and gene phylogenies are not linked, indicating vertical inheritance of reductive dehalogenases is not a primary mechanism of acquisition. Design of a universal PCR primer suite targeting a curated database of reductive dehalogenase homologous genes was used to characterize the reductive dehalogenase complement of four environmental sites and two enrichment cultures. Using an enrichment culture containing three phylogenetically distinct dechlorinating organisms, the interactions of niche-specific organisms were examined through single-cell genome sequencing. From the partial genome sequences, novel reductive dehalogenase genes were identified, as well as evidence of lateral gene transfer between the three dechlorinating organisms. This research primarily utilizes genomic and metagenomic datasets, which serve as metabolic blueprints for prediction of organisms’ functions. The results presented in this thesis advocate in favour of phylogenetic diversity within enrichment cultures to maintain functional redundancy, leading to more robust cultures for bioremediation efforts.
Pancreatic Alpha-cell Characterization in Healthy and Type 1 Diabetic Mice Employing Organotypic Tissue Slice Preparations
Keywords: Diabetes
Electrophysiology

Pancreatic alpha- and beta-cells play vital roles in maintaining glucose homeostasis. While much work has investigated beta-cell biology, alpha-cell research has been scarce. This is due to limitations in conventional methods of alpha-cell preparation, which expose alpha-cells on the islet mantle to enzymatic and mechanical injury inherent in the preparation. I have employed the pancreas tissue slice preparation, which surmounts these limitations. Pancreas slices can be prepared efficiently, and islet cells examined in situ without requiring culture conditions. Alpha-cells are preserved in their native cellular environment not only in health, but more remarkably, also in disease (type 1 diabetes; T1D) states, which was not previously feasible. In the first part of my study, I deployed this preparation to assess normal mouse alpha-cell physiology. Alpha-cells exhibited well-described features of INa, IKATP, small cell size, low resting membrane conductance, and inducible low and high voltage-activated ICa, the latter correlating with exocytosis determined by capacitance measurements. In contrast to previous reports, our large sampling of alpha-cells revealed a wide-range data distribution of several ion channel parameters. My findings explain the apparent inconsistency of previous reports wherein alpha-cell ion channel properties appeared skewed within narrow portions of this wide distribution, likely caused by different preparations. In the second part of my thesis, I assessed alpha-cell perturbation in streptozotocin-induced T1D in the GluCre-ROSA26EYFP (GYY) mouse. In this T1D model, alpha-cells exhibited more glucagon content per cell, which can be exocytosed in greater quantity upon serial depolarization. Membrane electrical properties revealed larger Na+ current and reduced KV-transient current, which contributed to the apparent increased amplitude and firing frequency of action potentials in membrane electrical recording. These electrical events likely prime alpha-cells to release more glucagon, culminating in larger in vivo glucagon secretory responses to low glucose stimulation in this T1D model. We are now well-positioned to employ this in situ model of pancreas slice preparation to address many other apparently unanswerable questions in alpha-cells in normal and pathophysiologic states, such as diabetes.
Generation and Verification of Plans with Loops
Keywords: planning
knowledge representation

This thesis studies planning problems whose solution plans are program-like structures that contain branches and loops. Such problems are a generalization of classical and conditional planning, and usually involve infinitely many cases to be handled by a single plan. This form of planning is useful in a number of applications, but meanwhile challenging to analyze and solve. As a result, it is drawing increasing interest in the AI community. In this thesis, I will give a formal definition of planning with loops in the situation calculus framework, and propose a corresponding plan representation in the form of finite-state automata. It turns out that this definition is more general than a previous formalization that uses restricted programming structures for plans. For the verification of plans with loops, we study a property of planning problems called finite verifiability. Such problems have the property that for any candidate plan, only a finite number of cases need to be checked in order to conclude whether the plan is correct for all the infinitely many cases. I will identify several forms of finitely-verifiable classes of planning problems, including the so-called one-dimensional problems where an unknown and unbounded number of objects need independent processing. I will also show that this property is not universal, in that finite verifiability of less restricted problems would mean a solution to the Halting problem or an unresolved mathematical conjecture. For the generation of plans with loops, I will present a novel nondeterministic algorithm which essentially searches in the space of the AND/OR execution trees of an incremental partial plan on a finite set of example instances of the planning problem. Two different implementations of the algorithm are explored for search efficiency, namely, heuristic search and randomized search with restarts. In both cases, I will show that the resulting planner generates compact plans for a dozen benchmark problems, some of which are not solved by other existing approaches, to the best of our knowledge. Finally, I will present generalizations and applications of the framework proposed in this thesis that enables the analysis and solution of related planning problems recently proposed in the literature, namely, controller synthesis, service composition and planning programs. Notably, the latter two require possiblynon-terminating execution in a dynamic environment to provide services to coming requests. I will show a generic definition and planner whose instantiation accommodates and solves all the three example applications. Interestingly, the instantiations are competitive with, and sometimes even outperform, the original tailored approaches proposed in the literature.
Regulators of Hedgehog Signaling in Chondrocytes: Sufu, Kif7, and Primary Cilium
Keywords: Hedgehog signaling
chondrogenesis

The Hedgehog (Hh) signaling pathway has received attention regarding its important role in embryonic development, however the mechanism by which pathway regulators, such as Suppressor of fused (Sufu), Kinesin family member 7 (Kif7), and primary cilium, mediate Hh signaling transduction is not entirely understood. The work presented here examines the roles of Sufu and Kif7 in regulating Hh signaling in growth plate chondrocytes, as well as how they mediate parathyroid hormone-like hormone (Pthlh) signaling during chondrocyte development. I show here that Sufu and Kif7 are essential regulators of Indian hedgehog (Ihh) signaling. While Sufu negatively regulates Gli transcription factors, Kif7 functions both positively and negatively in chondrocytes. Kif7 plays a role in Sufu protein degradation and the exclusion of Sufu-Gli complexes from the primary cilium. Importantly, halving the dosage of Sufu restores normal Hh pathway activity and chondrocyte development in Kif7-null mice, demonstrating that the positive role of Kif7 is to restrict the inhibitory function of Sufu. Furthermore, Kif7 exerts inhibitory function on Gli transcriptional activity in chondrocytes when Sufu function is absent. Therefore, Kif7 regulates the activity of Gli transcription factors through both Sufu-dependent and Sufu-independent mechanisms. I show that Sufu is crucial for mediating the negative effect of Pthlh on Gli transcriptional activity and chondrocyte hypertrophic differentiation, whereas Kif7 and primary cilium are dispensable in this process. Although primary cilium is required for Hh ligand-mediated activation of Gli transcription, Pthlh negatively controls Gli transcriptional activity in a cilia-independent manner. The results of this work provide insight into how Hh signaling is regulated by Sufu and Kif7 in the context of primary cilium, but also suggest Sufu serves as an important link between Ihh and Pthlh signaling during growth plate chondrocyte development.
The Acute Regulation of Intestinal Chylomicron Secretion by Glucagon-like Peptides
Keywords: apolipoprotein B
insulin resistance
postprandial dyslipidemia

Postprandial overproduction of apolipoprotein B48 (apoB48)-containing lipoproteins has been observed in states of insulin resistance and is important to the sequelae of cardiovascular disease, but little is understood about factors that regulate their secretion. The glucagon-like peptides (GLPs) are released from ileal enteroendocrine L-cells following lipid ingestion. I hypothesized that the GLPs could acutely affect the production of apoB48-containing triglyceride (TG)-rich lipoproteins (TRL) in the small intestine. Using the Syrian golden hamster, I first characterized the gross effects of the GLPs on TRL secretion in response to an oral fat load and then continued to dissect the mechanisms of these changes using primary intestinal cell cultures and a variety of knockout mouse models. An exogenous GLP-1 receptor (GLP-1R) agonist was found to acutely inhibit chylomicron secretion in both hamsters and mouse models, and extending the bioactivity of endogenously-secreted GLP-1 with a dipeptidyl peptidase-4 inhibitor had suppressive effects in insulin-resistant fructose-fed hamsters. The insulinotropic and delayed gastric emptying functions do not completely account for the hypolipidemic effect of GLP-1R agonism, and the effect of the GLP-1R agonist exendin-4 could be seen directly in the apoB48 secretion of primary enterocytes. In contrast, the sister peptide GLP-2 was a potent acute stimulator of chylomicron secretion in hamsters and mice. The hyperlipidemic effect of GLP-2 could be attributed to an increased rate of luminal FA uptake mediated by the posttranslational modification of the FA transporter CD36, and CD36-deficient mice were found to be refractory to the stimulatory effects of GLP-2. The activity of nitric oxide synthase was also found to be essential to the hyperlipidemic action of GLP-2. I identified a set of intercellular communications that could contribute in mediating the action of GLP-2, in which GLP-2 secreted from the enteroendocrine L-cell stimulates intestinal subepithelial myofibroblasts to release vascular endothelial growth factor, which directly activated the enterocyte to secrete apoB48. In summary, this thesis demonstrates that two co-secreted postprandial hormones have considerable but completely opposite influences on chylomicron production. Changing the balance of the GLPs’ actions in vivo could provide a therapeutic strategy to combat postprandial dyslipidemia.