VictorJuiz/thesis huggingface dataset:
Development and Intratumoral Distribution of Block Copolymer Micelles as Nanomedicines for the Targeted Delivery of Chemotherapy to Solid Tumors
Keywords: cancer
nanoparticle
nanomedicine
block copolymer micelle
chemotherapy
biomaterials
tumor
tumor penetration
tumor distribution
docetaxel
Taxotere
targeted delivery

Recent advancements in pharmaceutical technology based on principles of nanotechnology, polymer chemistry, and biomedical engineering have resulted in the creation of novel drug delivery systems with the potential to revolutionize current strategies in cancer chemotherapy. In oncology, realization of significant improvements in therapeutic efficacy requires minimization of drug exposure to healthy tissues and concentration of the drug within the tumor. As such, encapsulation of chemotherapeutic agents inside nanoparticles capable of enhancing tumor-targeted drug delivery is a particularly promising innovation. Yet, initial investigations into the intratumoral fate of nanomedicines have suggested that they may be heterogeneously distributed and achieve limited access to cancer cells located distant from the tumor vasculature. As such, uncovering the determinants of nanoparticle transport at the intratumoral level is critical to the development of optimized delivery vehicles capable of fully exploiting the therapeutic potential of nanomedicines. In this work, the chemotherapeutic agent, docetaxel (DTX), was incorporated into nano-sized, biocompatible PEG-b-PCL block copolymer micelles (BCMs). Encapsulation of DTX in micelles via chemical conjugation or physical entrapment resulted in a dramatic increase in drug solubility and customizable drug release rate. The use of multicellular tumor spheroids (MCTS) was established as a viable platform for assessing the efficacy and tumor tissue penetration of nanomedicines in vitro. A series of complementary assays was validated for analysis of DTX-loaded micelle (BCM+DTX) toxicity in monolayer and spheroid cultures relative to Taxotere®. Cells cultured as spheroids were less responsive to treatment relative to monolayer cultures due to mechanisms of drug resistance associated with structural and microenvironmental properties of the 3-D tissue. Computational, image-based methodologies were used to assess the spatial and temporal penetration of BCMs in spheroids and corresponding human tumor xenografts. Using this approach, the tumor penetration of micelles was found to be nanoparticle-size-, tumor tissue type- and time- dependent. Furthermore, spheroids were found to be a valuable platform for the prediction of trends in nanoparticle transport in vivo. Overall, the results reported herein serve to demonstrate important determinants of nanoparticle intratumoral transport and to establish computational in vitro and in vivo methodologies for the rational design and optimization of nanomedicines.
Subwavelength Imaging using Scanning Near-field Antenna Arrays
Keywords: subwavelength
imaging
near field
antenna array
focusing

This thesis examines a series of near-field antenna arrays used to perform subwavelength focusing and subwavelength imaging outside the extreme near field. For this purpose, slot and dipole arrays have been designed to produce a subwavelength focal spot at a distance of a quarter wavelength from the array. The dipole arrays are then used as scanning probes to produce images with subwavelength resolution based on perturbations in the scattered field. Unlike negative-refractive-index metamaterial superlenses, the imaging resolution is not affected by losses in the array. Furthermore, the arrays are simple to fabricate and are frequency scalable up to Terahertz frequencies and beyond. A near-field analogue to classic antenna-array theory called ``shifted beam theory'' is presented as a design tool. Based on the linear independence of element field patterns in the near field, this theory is very intuitive and provides a simplified way to calculate the element current weights necessary to generate a given target near-field pattern. Two-dimensional near-field subwavelength focusing is demonstrated using a slotted transmission-screen, or ``meta-screen'', under plane-wave incidence. At a distance of a quarter wavelength, the transverse electric field was measured in experiment to have a full-width half-maximum beamwidth of 0.40 by 0.27 wavelengths. This is compared to a single slot transmission-screen which had a beamwidth of 0.60 by 0.58 wavelengths. Broadside and end-fire dipole arrays are used to perform subwavelength imaging in one and two dimensions, respectively. The experimental minimum resolvable separation between two objects at a quarter-wavelength distance was 0.26 wavelengths using the end-fire array probe, as compared to 0.43 wavelengths for a single monopole probe. For an experiment using eight objects scattered over a one-square-wavelength area, however, the array probe imaging resolution remained around 0.25 wavelengths while the baseline monopole probe was no longer able to resolve any of the objects. Experiments were also conducted using objects buried behind a dielectric barrier as well as objects immersed within a dielectric. These results were consistent with the resolution improvements observed in the free-space resolution experiments.
Genome-environment Interactions in Type 1 Diabetes
Keywords: Immunology
Genetics
Autoimmunity
Type 1 Diabetes

This project aims to integrate knowledge of genetic susceptibility, immune cell function, and environmental modifiers in determining risk for type 1 diabetes (T1D) in the non-obese diabetic (NOD) mouse model. Similar to human T1D, disease risk in the NOD mouse is polygenic and has been mapped to multiple Idd loci. We have fine-mapped the Idd4.1 locus and identified Nlrp1b as its candidate gene. We report an alternatively spliced isoform in the diabetes-resistant Nrlp1b allele, resulting in a truncated NLRP1b protein that is unable to activate release of IL-1β. In another aspect of this project, we have characterized the critical contribution to T1D pathogenesis by γδ T cells. We report that CD27- γδT cells infiltrate islets of pre-diabetic NOD mice. Adoptive transfer of T1D to lymphocyte-deficient NOD.SCID recipients was potentiated when CD27- γδ T cells were transferred, compared to transfer of αβ T cells alone. Antibody-mediated blockade of IL-17 prevented T1D transfer in this setting. Moreover, introgression of genetic Tcrd deficiency onto the NOD background provided robust T1D protection. Finally, we report novel relationships between the gut microbiome, host sex hormones and metabolism, and T1D pathogenesis in the NOD mouse. Using germ-free, specific pathogen free, and microbiome-transplanted NOD mice, we show that colonization by commensal microbes elevated serum testosterone and protected NOD males from T1D. Transfer of gut microbiota from adult males to immature females altered the recipients’ microbiota, resulting in elevated testosterone and metabolomic changes, reduced islet inflammation and autoantibody production, and endowed robust T1D protection. Collectively, the data presented in this thesis describe a novel genetic lesion involved in T1D risk and its immunological consequences, demonstrate a potent role for IL-17-producing γδ T cells in NOD mouse model, and uncover a novel relationship between the gut microbiome, host hormonal and metabolic phenotypes, and autoimmunity risk.
The Molecular Pathogenesis of Noonan Syndrome-Associated RAF1 Mutations
Keywords: Noonan syndrome
signal transduction
RAS/ERK pathway
mouse model

Noonan syndrome (NS) is one of several autosomal dominant “RASopathies” caused by mutations in components of the RAS-RAF-MEK-ERK MAPK pathway. Germ line mutations in RAF1 (encoding the serine-threonine kinase for MEK1/2) account for ~3-5% of NS, and unlike other NS alleles, RAF1 mutations that confer increased kinase activity are highly associated with hypertrophic cardiomyopathy (HCM). Notably, some NS-associated RAF1 mutations show normal or decreased kinase activity. To explore the pathogenesis of such mutations, I generated “knock-in” mice that express kinase-activating (L613V) or -impaired (D486N) Raf1 mutants, respectively. Knock-in mice expressing the kinase-activated allele Raf1L613V developed typical NS features (short stature, facial dysmorphia, haematological abnormalities), as well as HCM. As expected, agonist-evoked Mek/Erk activation was enhanced in multiple cell types expressing Raf1L613V. Moreover, postnatal Mek inhibition normalized the growth, facial, and cardiac defects in L613V/+ mice, showing that enhanced Mek/Erk activation by Raf1 mutant is critical for evoking NS phenotypes. D486N/+ female mice exhibited a mild growth defect. Male and female D486N/D486N mice developed concentric cardiac hypertrophy and incompletely penetrant, but severe, growth defects. Remarkably, Mek/Erk activation was enhanced in Raf1D486N-expressing cells compared with controls. In both mouse and human cells, RAF1D486N, as well as other kinase-impaired RAF1 mutants, show increased heterodimerization with BRAF, which is necessary and sufficient to promote increased MEK/ERK activation. Furthermore, kinase-activating RAF1 mutants also require heterodimerization to enhance MEK/ERK activation. Our results suggest that increased heterodimerization ability is the common pathogenic mechanism for NS-associated RAF1 mutations.
An Efficient, Extensible, Hardware-aware Indexing Kernel
Keywords: Publish/Subscribe
Event Processing
Complex-Event Processing
Boolean Expression Indexing
Indexing Kernel
Data Structure
Data Indexing
Query Indexing
Storage Hierarchy
Index Maintenance
Compressed Matching
Delta Compression
Event Matching
Content-based Publish/Subscribe
FPGA
SSD
GPU
Multicore

Modern hardware has the potential to play a central role in scalable data management systems. A realization of this potential arises in the context of indexing queries, a recurring theme in real-time data analytics, targeted advertising, algorithmic trading, and data-centric workflows, and of indexing data, a challenge in multi-version analytical query processing. To enhance query and data indexing, in this thesis, we present an efficient, extensible, and hardware-aware indexing kernel. This indexing kernel rests upon novel data structures and (parallel) algorithms that utilize the capabilities offered by modern hardware, especially abundance of main memory, multi-core architectures, hardware accelerators, and solid state drives. This thesis focuses on presenting our query indexing techniques to cope with processing queries in data-intensive applications that are susceptible to ever increasing data volume and velocity. At the core of our query indexing kernel lies the BE-Tree family of memory-resident indexing structures that scales by overcoming the curse of dimensionality through a novel two-phase space-cutting technique, an effective Top-k processing, and adaptive parallel algorithms to operate directly on compressed data (that exploits the multi-core architecture). Furthermore, we achieve line-rate processing by harnessing the unprecedented degrees of parallelism and pipelining only available through low-level logic design using FPGAs. Finally, we present a comprehensive evaluation that establishes the superiority of BE-Tree in comparison with state-of-the-art algorithms. In this thesis, we further expand the scope of our indexing kernel and describe how to accelerate analytical queries on (multi-version) databases by enabling indexes on the most recent data. Our goal is to reduce the overhead of index maintenance, so that indexes can be used effectively for analytical queries without being a heavy burden on transaction throughput. To achieve this end, we re-design the data structures in the storage hierarchy to employ an extra level of indirection over solid state drives. This indirection layer dramatically reduces the amount of magnetic disk I/Os that is needed for updating indexes and localizes the index maintenance. As a result, by rethinking how data is indexed, we eliminate the dilemma between update vs. query performance and reduce index maintenance and query processing cost substantially.
Single Proteins under the Microscope: Conformations, Dynamics and Medicinal Therapies
Keywords: Single Molecule Fluorescence Spectroscopy
Intrinsically Disordered Protein
New Cancer Therapies
Protein Membrane Anchorage

We applied single-molecule fluorescence (SMF) methods to probe the properties of individual fluorescent probes, and to characterize the proteins of interest to which these probes were attached. One remarkable advantage of SMF spectroscopy is the ability to investigate heterogeneous subpopulations of the ensemble, which are buried in ensemble averaging in other measurements. Other advantages include the ability to probe the entire dynamic sequences of a single molecule transitioning between different conformational states. For the purpose of having an extended observation of single molecules, while maintaining the native nanoscale surroundings, we developed an improved vesicle preparation method for encapsulating scarce biological samples. SMF investigations revealed that molecules trapped in vesicles exhibit nearly ideal single-emitter behavior, which therefore recommends the vesicle encapsulation for reproducible and reliable SMF studies. Hyperactive Signal-Transducer-and-Activator-of-Transcription 3 (STAT3) protein contributes significantly to human cancers, such as leukemia and lymphoma. We have proposed a novel therapeutic strategy by designing a cholesterol-based protein membrane anchor (PMA), to tether STAT3 to the cell membrane and thus inhibit unwanted transcription at the cell nucleus. We designed in vitro proof-of-concept experiments by encapsulating STAT3 and PMAs in phospholipid vesicles. The efficiency and the stability of STAT3 anchoring in the lipid membrane were interrogated via quantitative fluorescence imaging and multiparameter SMF spectroscopy. Our in vitro data paved the way for the in vivo demonstration of STAT3 inhibition in live cells, thus demonstrating that PMA-induced protein localization is a conceptually viable therapeutic strategy. The recent discovery of intrinsically disordered proteins (IDPs) highlights important exceptions to the traditional structure-function paradigm. SMF methods are very suited for probing the properties of such highly heterogeneous systems. We studied in detail the effects of electrostatics on the conformational disorder of an IDP protein, Sic1 from yeast, and found that the electrostatic repulsion is a major factor controlling the dimensions of Sic1. Based on our data we also conclude that a rod-like shape seems a better candidate than a random Gaussian chain to describe and predict the behavior of Sic1.
In Vitro Modeling of Pancreatic Duct Cell Carcinogenesis
Keywords: KRAS
Smad4
LCN2
NGAL
pancreatic cancer
HPDE

Pancreatic adenocarcinoma (PDAC) putatively arises from the pancreatic duct, thus usage of the normal human pancreatic duct epithelial (HPDE) cell line is an ideal model to examine the successive accumulation of genetic alterations involved in carcinogenesis. KRAS mutations have been reported in 90% of PDACs. Oncogenic KRAS elicits activation of downstream pathways involved in survival, motility, and cell cycle progression. KRASG12V introduction in the HPDE cell line upregulates Lipocalin-2 (LCN2) expression. LCN2 has been identified in numerous carcinomas and is associated with survival, tumorigenicity, and invasion. In this work, LCN2 was found to be commonly expressed in high grade pancreatic duct neoplastic precursor lesions and PDAC illustrating its potential as a biomarker. Moreover, in vitro and in vivo studies demonstrate that high LCN2 expression promotes gemcitabine resistance, MMP-9 activity, angiogenesis, and tumorigenicity. Loss of Smad4 function is found in 55% of PDAC cases. Smad4 is a critical component in the TGF-β signaling which mediates the transcription of genes involved in processes such as cell cycle arrest, apoptosis, and invasion. This work examined the consequences of KRASG12V expression and Smad4 loss in the HPDE model. Cellular invasion was promoted by KRASG12V expression or knocking down Smad4 by 80% in the HPDE model. A TGF-β resistant HPDE cell line, TβR, was shown to lack Smad4 expression due to deletion, promoter methylation, and nonsense mutation. KRASG12V expression in the TβR model (TβR KRAS) promoted neoplastic transformation and tumour formation in immunodeficient mice with complete penetrance. Smad4 expression in the TβR KRAS cell line reinstated TGF-β signaling, delayed tumour formation, and decreased metastatic spread. This study provides evidence that Smad4 acts as a restriction point in the transformation of HPDE cells. Overall, this work examines the contribution of genes involved in transformation, and identifies a potential therapeutic and diagnostic biomarker in PDAC.
Economic Evaluation of Strategies to Prevent and Treat Febrile Neutropenia in Lymphoma Patients
Keywords: lymphoma
cost-effectiveness analysis

This thesis employed methods used in health care decision making to evaluate strategies for prevention and treatment of febrile neutropenia (FN) in non-Hodgkin lymphoma (NHL) patients. The objectives of this thesis were to quantify the cost-effectiveness of filgrastim and pegfilgrastim as primary prophylaxis against FN in NHL patients, to develop an algorithm for converting health-related quality of life data collected in non-Hodgkin lymphoma patients into preference-based health utility values, and to evaluate NHL patients&rsquo; preferences for outpatient treatment of FN. The cost-effectiveness analysis demonstrated that neither filgrastim, nor pegfilgrastim are cost-effective, with respective incremental cost-effectiveness ratios [95% confidence interval] of $4,599,000/QALY [$597,045, dominated] and $6,272,000/QALY [$730,692, dominated], well above the normally accepted threshold of $50,000/QALY. The algorithm for deriving health utility values was based on a regression model that used health utility values obtained from the EQ-5D instrument as the outcome variable and the four subscales of the Functional Assessment of Cancer Therapy &ndash; General (FACT-G) questionnaire as the predictor variables. The model final model included three of the FACT-G subscales, and had an R-squared value of 0.502 and a mean squared error of 0.013. A discrete choice experiment was used to examine patients&rsquo; preferences for out patient treatment of FN, and demonstrated that out-of-pocket costs, unpaid caregiver time required daily, and probability of return to hospital are all significant attributes when considering outpatient therapy for FN. Adjusted odds ratios [95% confidence intervals] of accepting outpatient treatment for FN were 0.84 [0.75 to 0.95] for each $10 increase in out-of-pocket cost; 0.82 [0.68 to 0.99] for each 1 hour increase in daily unpaid caregiver time; and 0.53 [0.50 to 0.57] for each 5% increase in probability of return to hospital. These results provide important information for clinicians and health care decision makers involved in implementing programs for NHL patients with FN.
Assessment of the Potential Health Risks of the Folic Acid Fortification Program on Acute Lymphoblastic Leukemia and Colorectal Cancer
Keywords: Folic acid
Folate
Meta-analysis
Epidemiology
Systematic review
Colorectal cancer
Folate Fortification
Colorectal adenoma recurrence
Acute Lymphoblastic Leukemia
Perception of risk
Methotrexate

Neural tube defects (NTD) result from the failure of the neural tube to close properly very early in gestation. A child born with an NTD may experience an early death or life-long disability. In the 1990s, the critical role of folic acid in the prevention of NTDs was confirmed and as a strategy to increase blood folate concentrations of women of childbearing age, folic acid fortification programs were mandated in Canada and the US. However, this change impacted the entire population not just women of childbearing age and not everyone may benefit from the increased folate intake. The objective of this research was to investigate the impact of higher intakes of folates on the mortality rates of children with acute lymphoblastic leukemia (ALL) and the risk of colorectal cancer (CRC) in adult populations. To address the impact in children with ALL, a comparison of the mortality rates between the pre- and post-fortification time periods in Ontario was performed using data from the Pediatric Oncology Group of Ontario. A second comparison between the mortality rates in these children in non-folic acid fortifying countries and the US was also completed. These analyses suggest that folic acid fortification is not negatively impacting mortality. With respect to CRC, one systematic review and two meta-analyses were conducted investigating folate intake and the risk of CRC or adenoma recurrence. The first analysis, in observational studies, compared high versus low folate intake and the risk of CRC. The second examined folate intake within the various polymorphisms of the methylene tetrahydrofolate reductase enzyme. The final study examined the impact of supplementation of 1 milligram or more per day of folic acid and the risk of colorectal adenoma recurrence in those adults with a history of colorectal adenomas. The findings from the completed observational studies suggest that there is an associated risk reduction in colorectal cancer from the intake of higher levels of folates. The investigations into the impact of the folic acid fortification program suggest that the program is not associated with having a negative impact on mortality of children with ALL or on the risk of colorectal cancer.
Global Proteomic Assessment of Classical Protein-tyrosine Phosphatases
Keywords: Protein-tyrosine phosphatases
Oxidation
Mass spectrometry
Reactive oxygen species
SHP2
AP-MS

Tyrosyl phosphorylation plays an important role in many fundamental cellular processes, including cell growth, differentiation and proliferation. The levels of phosphotyrosine (pY) are regulated by the opposing actions of protein-tyrosine kinases (PTKs) and protein-tyrosine phosphatases (PTPs). A limitation to understanding the roles of PTPs in physiological and pathological cell signaling has been the absence of global proteomic approaches that enable the systematic and comprehensive analysis of PTP expression, regulation and function. This dissertation describes the development and application of novel proteomic methodologies that permit the global analysis of PTP expression (qPTPome), regulation (by oxidation and nitrosylation; q-oxPTPome) and substrates/binding proteins. These methods provide a workflow to begin assessing PTP function at a systems level, rather than its current targeted format. Application of these techniques will provide invaluable information to begin bridging the gap in our understanding of PTP and PTK function in normal and malignant cell signaling.