Thesis (Ph. D.)--Harvard-MIT Division of Health Sciences and Technology, 2011. / Cataloged from PDF version of thesis. / Includes bibliographical references (p. 114-133). / Bone marrow mesenchymal stem cells (MSCs) may soon become the first FDA-approved stem cell therapy for autoimmune and inflammatory disease. Our lab originally hypothesized that much of the therapeutic activity of MSCs may be attributed to molecules secreted by these cells. This thesis will test this hypothesis, with an emphasis on translational steps towards clinical product development, including the identification of novel proteins secreted by MSCs. The first part of the thesis consists of studies we performed to test whether MSC conditioned medium (MSC-CM) can treat rats undergoing cisplatin-induced acute kidney injury (AKI). When AKI rats were treated with MSC-CM, we observed a survival benefit and significant protection of renal function compared to controls. The second part of the thesis will describe the development of a device designed for sustained delivery of MSC secreted factors to dialysis-dependent AKI subjects. We tested these devices for cell function, stability and viability when subjected to conditions that model future clinical operation. Finally, inspired by the therapeutic capacity of MSC secreted factors, this thesis will conclude with the introduction of a new method that we developed to uncover novel anti-inflammatory proteins from MSCs. This method revealed four previously unidentified cytokine modulators, two of which we found significantly promote IL-1 0 and suppress TNF-a in mice challenged with endotoxin. When leveraged as novel therapeutics for lethal endotoxemic shock, these two most potent modulators protected mice and provided for a significant survival benefit compared to vehicle controls. Together, these results demonstrate the power of MSC secreted factors in the context of inflammatory disease, and propose new tactics for elucidating potent secreted products from cells. / by Jack Miles Milwid. / Ph.D.

Thesis (S.M.)--Harvard-MIT Program in Health Sciences and Technology, 2012. / Cataloged from PDF version of thesis. / Includes bibliographical references (p. 80-87). / BACKGROUND: There is a high prevalence of disease marketing actions in the United States that are targeted towards patients with chronic illness. However, no study has assessed the direct effects of these marketing actions on patient coping attitudes and behaviors. OBJECTIVES: This study aims to investigate whether the mere presence of disease marketing impacts patient coping and if so, how do they affect patients' coping attitudes and behaviors. METHODS: We conducted a controlled experiment using online questionnaires to assess the disease perceptions, coping decisions and disease disclosure behaviors of 108 subjects. The subjects were divided into two groups where the experimental group (N = 55) was shown marketing actions associated with a fictitious disease called Karlsen's Disease while the control group (N = 53) was not shown any marketing actions. The subjects were then asked a series of questions related to health-related coping behaviors and non-health related social behaviors. T-tests and chi-square analyses were used to analyze the behavioral differences between the experimental (high-marketing) and control (no-marketing) groups. RESULTS: Subjects in the high-marketing group were overall significantly more willing to draft a will than subjects in the no-marketing group (t(106) = 2.64, p = 0.01); High-marketing group subjects were overall significantly more likely to wear a medical ID bracelet than no-marketing group subjects (c²(1, N = 108) = 3.71, p = 0.05); Among subjects who were willing to request a menu accommodation at a dinner party, those who were in the high-marketing group were significantly more likely to disclose their disease to the party host (c²(1, N = 90) = 4.65, p = 0.03); Subjects in the high-marketing group were also significantly more likely to anticipate greater understanding from the party host towards their menu accommodation request. When controlled for gender, women in the high-marketing group were more likely to join a patient support group (t(61) = 1.75, p = 0.09), and less likely to ask family and friends to shave their heads in show of solidarity (t(18) = -1.97, p = 0.07) than women in the no-marketing group; Men in the high-marketing group were more likely than men in the no-marketing group to disclose their health condition to the dinner party host (c²(1, N = 47) = 3.61, p = 0.06). Finally, among subjects with at least a 4-year college degree, those in the high-marketing group were more willing than those in the no-marketing group to wear a face mask to protect themselves from airborne pathogens in crowded public places (t(61) = 1.79, p = 0.08). CONCLUSIONS: Based on our results, the presence of disease marketing is anticipated to have a general positive impact on patient coping attitudes and behaviors. Chronically ill patients exposed to disease marketing actions are expected to anticipate less stigma from others, have increased willingness to disclose their illness and adopt health seeking behaviors. Disease marketing is also expected to have differential impact on patients based on their gender and level of education. Follow-up studies using real patients with chronic illness should be carried out to confirm the findings from this study. / by Hew Mun Lau. / S.M.

Thesis (S.M.)--Harvard-MIT Division of Health Sciences and Technology, 2004. / Includes bibliographical references (p. 40-43). / Background: Local adaptation of guidelines may increase compliance with guidelines that have been developed at a national level and are often not used in practice because of contextual factors. We have developed a representation scheme known as HieroGLIF, that facilitates a two-step approach to development and implementation of guidelines. In the first step, professional medical societies create a setting-independent guideline. In the second step, the guideline is adapted to local settings. The scheme represents the setting-independent guideline knowledge in a hierarchical structure for which we use axiomatic design theory to guide a top-down design. This representation extends the Guideline Interchange Format (GLIF), a frame-based ontology that has been previously developed for representation of guidelines. Methods: We encoded conventional national guidelines in the setting-independent format which were adapted to local clinical settings of their practice by primary care physicians (PCPs). We conducted a qualitative analysis of the type of changes that were made by the local adaptors. For each of the guidelines two patients' scenarios were created for which two sets of guideline recommendations were generated, one from the adapted hierarchical guideline and one from the conventional, non-hierarchical guideline. We evaluated in a randomized controlled trial the potential impact of the local adaptation methodology on adherence to guidelines. For each recommendation, 70 PCPs responded to a questionnaire that inquired if PCPs would follow the recommendations and their ratings for several relevant attributes. We also analyzed the data to look for attributes of recommendations that are important for their acceptance by PCPs. Results: In 8 out of / (cont.) recommendations the changes made were significant. The most common types of changes were additions of practical information (6/19) and deletions of unnecessary information (6/19). The PCPs significantly preferred the adapted version for one recommendation in which addition of practical information and deletions of unnecessary information were done. The response for changes in clinical content in another recommendation resulted in rejection of the adapted version. The most important attributes for acceptance of recommendations found were agreement with the clinical content and then in descending order--fitness to the practice environment, availability of practical information and succinctness. Conclusions: We conclude that the potential role of local adaptation is mainly for adding relevant practical information and deletions of unnecessary information. This combination of changes led to significant preference of adapted guidelines' version. On the contrary, changes to clinical content led to rejection of an adapted version recommendation and should be done cautiously. In addition, efforts should be made to make clinical recommendations fit the practice environment, contain practical information and be succinct. We consider the real impact of local adaptation in this study lower that expected. We also believe that the study sharpens the issues of the optimal level of adaptation in specific and the real impact of local adaptation in general. / by Yaron Denekamp. / S.M.

Thesis (Ph. D. in Biomedical Engineering)--Harvard-MIT Division of Health Sciences and Technology, 2009. / Cataloged from PDF version of thesis. / Includes bibliographical references. / In the continuing search for effective treatments for cancer, the emerging model is the combination of traditional chemotherapy with anti-angiogenesis agents that inhibit blood vessel growth. However, the implementation of this strategy has faced two major obstacles. First, the long-term shutdown of tumor blood vessels by the anti-angiogenesis agent can prevent the tumor from receiving a therapeutic concentration of the chemotherapy agent. Second, inhibiting blood supply drives the formation of intra-tumoral hypoxia, or a lack of oxygen, which has been correlated with increased tumor invasiveness and resistance to chemotherapy. In this thesis we report the disease-driven engineering of a drug delivery system, a 'nanocell', which overcomes these barriers unique to solid tumors. The nanocell comprises a nuclear nanoparticle encapsulated within a lipid membrane and is preferentially taken up by the tumor. The nanocell delivers a temporal release of two drugs within the tumor core: the outer lipid envelope first releases an anti-angiogenesis agent, causing a vascular shutdown; the inner nanoparticle, which is trapped inside the tumor, then releases a chemotherapy agent. This focal release within the tumor targets cells most at risk for hypoxia and results in improved therapeutic index with reduced toxicity. The technology can be extended to additional agents, so as to target multiple signaling pathways or distinct tumor compartments, enabling the model of an 'integrative' approach in cancer therapy. / (cont.) In the second part of the thesis we report new tools for the optimization of nanocell formulations. We present a new, three-dimensional, voxel-based computational model for Monte Carlo simulations of nanoparticle delivery systems that enables direct investigation of the entire vehicle during particle degradation and drug release. Use of this model in combination with emerging mechanistic understandings of nanoparticle drug release will facilitate optimization of nanocell combination therapy release profiles. We additionally report the generation and characterization of a set of carbohydrate-based chemotherapeutic agents that have the potential for use in nanocells as reduced toxicity alternatives to traditional chemotherapy agents. / by David A. Eavarone. / Ph.D.in Biomedical Engineering

Thesis: Ph. D., Harvard-MIT Program in Health Sciences and Technology, 2014. / Cataloged from PDF version of thesis. / Includes bibliographical references. / This work investigates the fundamental processes used by mammalian cells and organisms to make decisions during embryonic development. Current technologies that evaluate biological phenomenon often force a compromise between quantification of gene expression via bulk assays and qualitative imaging of cell and tissue heterogeneity. There are few options that allow for quantitative, high-resolution, single-cell analysis that is robust but not associated with a high degree of technical difficulty or obscured by amplification. Here, we address these issues using two model systems, the developing mammalian inner ear and single mouse embryonic stem cells (mESCs) during the process of X inactivation, to demonstrate our ability to perform single-cell, single-molecule assays that reveal both highly quantitative and spatial information. Accordingly, we adapted a high resolution, single-molecule RNA fluorescent in situ hybridization technique (smFISH) to study gene expression in the inner ear and perform allele-specific detection of the X chromosome in mESCs. We used previously-published smFISH procedures as our initial template for investigating biological signaling phenomena in these two systems. To study gene expression in the mouse inner ear, we developed a modified smFISH strategy to investigate mRNA transcript expression patterns in the cochlea during auditory hair cell development. The mammalian cochlea, a highly specialized and complex organ, beautifully demonstrates both the depth and breadth of the smFISH technique. To assay signaling behavior and topological changes of the X chromosome prior to X inactivation, we incorporated a novel allele-specific modification into the smFISH technique. We investigate the allele-specific expression patterns of eight genes that tile the X chromosome, which were chosen for their varied putative roles before, during and after X chromosome inactivation. Taken together, these two systems recapitulate the strength of the smFISH technique and its adaptations. The goals of this thesis were twofold: (1) expand the smFISH technique to work in specialized mammalian systems such as the cochlea and (2) demonstrate allele-specific DNA topological changes and expression patterns in mESCs. Elucidating high-resolution, single-molecule quantifiable imaging methods for application to complex tissues or allele-specific probing will have profound impacts on future investigations and promote a deeper comprehension of these systems. / by Annalisa M. Pawlosky. / Ph. D.

Thesis: Ph. D. in Medical Engineering and Medical Physics, Harvard-MIT Program in Health Sciences and Technology, 2018. / Cataloged from PDF version of thesis. / Includes bibliographical references. / Complex hierarchical structures are a hallmark of life. Within multicellular organisms, the building blocks of these structures are cells; within cells, they are genes. The interdependence of these building blocks is difficult to measure but is integral to the biological processes of health and disease, which emerge from the dynamism of thousands of interacting genes. This cooperativity manifests in particular mutations which accumulate over the course of cancer progression, gender-specific medical conditions, and transcription factor cocktails used to reprogram differentiated cells into stem cells. However, it is experimentally intractable to test the significance of perturbing every unique combination of genes. Instead, we explore gross features of this interaction space to determine how prevalent these synergies are. We take a top-down approach, creating new methods to measure the effects of removing genes from the full set. In the first, we develop a method to measure the transcriptional response to genetic perturbations across hundreds of thousands of cells revealing opposing classes of transcription factors regulating the immune response of dendritic cells. In the second, we create a method to measure how millions of combinations of genetic perturbations impact the growth rate of cancer cell lines. / by Atray Dixit. / Ph. D. in Medical Engineering and Medical Physics

Thesis: Ph. D., Harvard-MIT Program in Health Sciences and Technology, 2015. / Cataloged from PDF version of thesis. / Includes bibliographical references (pages 243-263). / Two parallel research efforts were pursued. First, we conducted a systematic exploration of how the genomic landscape of cancer shapes and is shaped by anti-tumor immunity. Using large-scale genomic data sets of solid tissue tumor biopsies, we quantified the cytolytic activity of the local immune infiltrate and identified associated properties across 18 tumor types. The number of predicted MHC Class I-associated neoantigens was correlated with cytolytic activity and was lower than expected in colorectal and other tumors, suggesting immune-mediated elimination. We identified recurrently mutated genes that showed positive association with cytolytic activity, including beta-2- microglobulin (B2M), HLA-A, -B and -C and Caspase 8 (CASP8), highlighting loss of antigen presentation and blockade of extrinsic apoptosis as key strategies of resistance to cytolytic activity. Genetic amplifications were also associated with high cytolytic activity, including immunosuppressive factors such as PDL1/2 and ALOX12B/15B. Our genetic findings thus provide evidence for immunoediting in tumors and uncover mechanisms of tumor-intrinsic resistance to cytolytic activity. Second, we combined measurements of protein production and degradation and mRNA dynamics so as to build a quantitative genomic model of the differential regulation of gene expression in lipopolysaccharide-stimulated mouse dendritic cells. Changes in mRNA abundance play a dominant role in determining most dynamic fold changes in protein levels. Conversely, the preexisting proteome of proteins performing basic cellular functions is remodeled primarily through changes in protein production or degradation, accounting for more than half of the absolute change in protein molecules in the cell. Thus, the proteome is regulated by transcriptional induction for newly activated cellular functions and by protein lifecycle changes for remodeling of preexisting functions. / by Michael Steven Rooney. / Ph. D.

Thesis: Ph. D., Harvard-MIT Program in Health Sciences and Technology, 2015. / Page 283 blank. Cataloged from PDF version of thesis. / Includes bibliographical references. / RNA interference (RNAi) is a powerful technology that provides a means to alter the expression of a specific protein based on a targeted RNA sequence. This is done by taking advantage of existing cellular machinery present within all eukaryotic cells which use short double-stranded RNA sequences as guides for RNA induced silencing. The potential for RNAi in medicine is enormous, providing a new approach to treat the complex biological dysregulation underlying many diseases. This promise of a new branch of therapeutics however has been mired with difficulties. RNA is quickly degraded by nucleases that are prevalent in the blood and throughout the body, it is highly immunogenic, and systemic delivery is complicated by high clearance rates. As such, developing formulations for the effective delivery of short RNAs presents significant hurdles. Local delivery can limit numerous unwanted systemic side effects of therapies and it maintains the highest therapeutic index possible in a targeted area before clearance. As such, the local delivery of siRNA may hold just as much potential as systemic delivery with significantly reduced complications. Layer-by-layer (LbL) assembly is a robust method that has been successfully demonstrated for the localized and sustained delivery of many biologic therapeutics and biomolecules. Developing an LbL film capable of delivering siRNA locally would offer a powerful new approach to the treatment of local disorders. This approach could be combined with existing medical devices to improve patient outcomes by directly addressing pathologic dysregulation in the area of interest. One field where the local treatment of dysregulation could be of particular interest is that of wound healing. Wound healing is a complex and highly synchronized process of multiple biological pathways, consisting of an assortment of cytokines, growth factors, and varied cell types which evolves over time. The development of a drug delivery system that can locally modify cell behavior on the basic level of gene transcription would be a powerful tool to alter the dynamics of wound healing. There are many known complications of wound healing, ranging from chronic ulcerative wounds to hypertrophic contractile scars, which dramatically affect the lives of tens of millions of patients every year. Through RNA interference, one could specifically target the key mediators of these complications, providing a means to more effectively regulate the wound healing process in vivo. The capability to deliver siRNA locally to address these complications is a significant advance in the current state of wound treatment. As such, this work presents an opportunity to substantially improve the current standard of treatment for patients and their wound healing outcomes. Herein, we present the design and preclinical evaluation of a number of strategies to develop ultra-thin polymer coatings for the controlled delivery of RNA both in vitro and in vivo. We used Layer-by-Layer assembly to create siRNA containing polymer-based films that can sustain the release of complexed siRNA over physiologically relevant timescales for local delivery into tissues. We describe the development of the first high-throughput approach for LbL assembly and screening and its use to identify lead candidate film architectures for RNA delivery. We then apply these findings to treat dysregulation in two distinct animal models; a chronic diabetic mouse wound model and a third-degree bum model in rats, targeting three different genes of interest independently. These coatings were demonstrated to effectively coat a number of medically relevant substrates including bandages, sutures, surgical staples, nanoparticles, microparticles, and microneedles. This body of work provides insight into how siRNA can be incorporated into thin film assemblies and the design criteria to achieve successful gene knockdown in vitro and in vivo. / by Steven Andrew Castleberry. / Ph. D.

No description available.

Thesis: Ph. D., Harvard-MIT Program in Health Sciences and Technology, 2019 / Cataloged from student-submitted PDF version of thesis. / Includes bibliographical references. / As profiling the molecular states of cellular subpopulations has become increasingly important to understand complex systems in biology and medicine, considerable efforts are being made to develop multiplexed techniques. While current fluorescent probes play indispensable roles, their broad emission spectra (about 30-100 nm) limit multiplexing capability. Recently, optical probes emitting narrowband laser spectra (about 0.1-1 nm), called 'laser particles', has drawn attention. Semiconductor microdisk lasers fabricated by top-down lithography have shown potential for massive multiplexing of thousands to millions of samples. In the thesis, I investigated lead halide perovskites (LHP) as a novel material for scalable production of laser particles in a lab flask. I discovered a sonochemical method to produce a large quantity (10 billions/L) of high-quality LHP micro- and sub-micron particles in a polar solvent within minutes. This method enabled me to coat the surface of individual CsPbBr3 laser particles using poly-catecholamine and thereby to improve optical properties and material stability against moisture. With CsPbBr3 microparticles coated with nano-scatterers, I realized disordered lasing based on Anderson localization. In addition, by incorporating plasmonic materials, I demonstrated plasmonic-lasing particles as small as 580 nm. This work paves the way for highly multiplexable laser particles for biomedical applications. / by Sangyeon Cho. / Ph. D. / Ph.D. Harvard-MIT Program in Health Sciences and Technology