Thesis: Ph. D. in Materials Science and Medical Engineering, Harvard-MIT Program in Health Sciences and Technology, February 2010. / Cataloged from PDF version of thesis. / Includes bibliographical references (pages 135-139). / Endothelial cells (EC) are ubiquitous - as vascular epithelial cells they line the inner surface of all vessels and are the contact surface with flowing blood. Macrovascular EC are the first line barrier between flowing blood and mural structures. The microvasculature includes EC-lined vessels that contact virtually every cell in the body. These EC are potent bioregulatory cells, modulating thrombosis, inflammation and control over mural smooth muscle cells and vascular health. The biochemical roles of EC can be retained when cells are embedded within three-dimensional matrices without recapitulation of the full vessel architecture. Within these matrices, surface and structural properties impose a set of forces on the embedded EC. Indeed, substrata pore size and modulus have profound effects on phenotype and function of a range of cell types. In the first part of this work, we examined the effect of pore size, matrix relative density and modulus on matrix-embedded EC growth and secretion and found a greater biological dependence on modulus than pore size or density. In the second part of this work, we examined the effect of isolated changes in modulus on BC growth, secretion of growth regulators, and modulation of smooth muscle cell growth. EC growth is maximal at intermediate moduli over a range from 50 Pa- 1500 Pa. Secretion of heparan sulfate proteoglycans (HSPGs), which inhibit smooth muscle cell growth, is maximal at low moduli and flat at high moduli. Secretion of growth factors such as FGF2 and PDGF-BB were also modulus responsive. Inhibition of smooth muscle cell growth rose as modulus decreased from 510 Pa to 50 Pa and was the result of a balance between increased HSPG secretion and reduced secretion of vasoactive growth factors. Changes in endothelial function correlated with extracellular matrix gene and integrin aP 3 and c41 expression. Changes in the forces experienced by the cell - a change in substrate modulus - cause the cell to alter its ECM and integrin expression in an effort to return the force balance to normal, leading to downstream effects on cell function. While growth stimulatory function largely conserved, growth inhibitory function was altered to a much larger degree. In the final part of this work, we examined the effect of scaffold modulus on EC response to inflammatory stimuli, and attempted to correlate it to changes in smooth muscle cell regulation and integrin expression. While cytokine secretion was independent of modulus, surface expression of ICAM- 1 and VCAM-1, and induction of CD4' T cell proliferation followed a similar pattern to smooth muscle cell inhibition, suggesting that similar mechanisms may be involved in their regulation by substrate modulus. Alteration of scaffold modulus has a profound impact on EC function including growth regulation and inflammatory response. The model offered in this thesis - wherein EC attempt to neutralize changes in environmental force balance by altering ECM and integrin expression, leading to changes in downstream function - offers insight into how environmental changes effect functional changes in ECs. / by Sylaja Murikipudi. / Ph. D. in Materials Science and Medical Engineering

Thesis (Ph. D.)--Harvard-MIT Division of Health Sciences and Technology, 2008. / This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections. / Includes bibliographical references (p. 221-232). / Cells are continuously sensing and processing information from their environments and responding to it in sensible ways. The communication networks on which such information is handled often consist of systems of chemical reactions, such as signaling pathways or metabolic networks. This thesis studies the dynamics of systems of chemical reactions in the context of biological cells. The first part of this thesis analyzes the osmo-regulation network in yeast, responsible for the regulation of internal osmolarity. We measure the system's step response in single cells, and find that the steady state is independent of the input, a property termed perfect adaptation that relies on integral feedback control. We then consider the signaling cycle, a pattern of chemical reactions that is often present in signaling pathways, in which a protein can be either active (e.g., phosphorylated) or inactive (e.g., unphosphorylated). We identify new regimes of static and dynamic operation, and find that these cycles can be tuned to transmit or digitize time-varying signals, while filtering input noise. The second part of this thesis considers systems of chemical reactions where stochastic effects are relevant, and simplifies the standard models. We develop an approximate model for the time-evolution of the average concentrations and their variances and covariances in systems with and without spatial gradients. We also describe a framework to identify and derive approximate models for variables that evolve at different time scales in systems without spatial gradients. These tools can help study the impact of stochastic and spatial effects on system behavior. / by Carlos Alberto Gómez Uribe. / Ph.D.

Thesis (Ph.D.)--Harvard--Massachusetts Institute of Technology Division of Health Sciences and Technology, 1998. / Includes bibliographical references (leaves 219-232). / by Gerald C. Koenig. / Ph.D.

Thesis: Ph. D., Harvard-MIT Program in Health Sciences and Technology, 2016. / Cataloged from PDF version of thesis. / Includes bibliographical references (pages 133-138). / Nanoparticle (NP)-mediated drug delivery typically relies on cargo release to occur passively or in response to environmental stimuli. In the first part of this work, we present a drug delivery method based on light-activated disruption of intracellular vesicles after internalization of bio-functionalized mesoporous silica nanoparticles loaded with cargo. We developed an improved synthesis method to optimize size control of mesoporous silica nanoparticles in the 20-200nm range while preserving porosity. We optimized surface bio-functionalization to obtain a highly stable and targetable nanocarrier, and used an embedded photosensitizer to combine the power of targeted delivery with the spatiotemporal control of light activation. NP-mediated endosomal disruption can be controlled at the single vesicle level. As an example, we delivered a cell-impermeable fluorescent compound exclusively to the cytosol of multidrug resistant cancer cells in a mixed population. The second part of this thesis investigated the use of both photosensitizer-coupled and magnetic nanoparticles as actuators of ion channels. While direct magnetic actuation was not found to promote activation of the receptors tested, several members of the transient receptor potential superfamily (TRP), notably TRPV3 and TRPA1, were found to be activatable by light in conjunction with photosensitizers, including the endogenously present all-trans retinal. The findings were established using a combination of calcium imaging with both chemical and genetically encoded indicators, in addition to whole cell patch clamping. Spontaneous activation of channels in heterologous expression systems was observed upon illumination at selective wavelengths, establishing an effective actuation method, potentially adding native channels to the current opto-genetic toolbox. / by Sébastien Febvay. / Ph. D.

Thesis (Ph. D.)--Harvard-MIT Division of Health Sciences and Technology, 2008. / Includes bibliographical references (leaves 125-135). / Outer hair cells (OHCs) are key elements of the mammalian hearing system, which amplify sound-evoked signals transmitted into the inner ear. OHCs are innervated by peripheral projections of olivocochlear (OC) and type-II spiral ganglion neurons. Type-II neurons innervate up to 100 OHCs, but their function is unknown. It has been suggested that type-II neurons interconnect neighboring OHCs through reciprocal synapses, which are bi-directional (afferent and efferent) synaptic interactions (Nadol, 1981). Since reciprocal synapses on the OHCs have been shown to be prevalent only in aged primates, they were thought to be a pathological finding. In addition to their interactions with OHCs, type-II neurons are also innervated by OC neurons. Synapses between OC and type-II neurons (OC/type-II synapses) have been described (Smith and Rasmussen, 1963), but these interactions have not been characterized in detail. Serial and semi-serial section transmission electron microscopy were used to study the synaptic interactions of type-II neurons with OHCs and OC neurons in a young human and in adult cats. A high prevalence of nerve terminals with reciprocal synapses was observed in the young human and in adult cats. These reciprocal terminals were processes of type-IIs, and not of OC neurons. Reciprocal type-II terminals were found in all frequency regions studied in cats, but were most prevalent below 4,000Hz. All the type-II fibers traced to more than one OHC in an adult cat had reciprocal interactions with OHCs. Type-II fibers/terminals were heavily innervated by OC neurons, which preferentially targeted terminals with reciprocal synapses that were predominantly afferent in an adult cat. / (cont.) The innervation patterns of type-IIs and OC neurons in the cat were similar to that found in comparable frequency regions of primates. Type-II neurons have reciprocal synaptic interactions with OHCs and form an "outer spiral network", which may functionally integrate the OHCs. The OC system may modulate this network through OC/type-II synapses. The outer spiral network and its innervation by the OC system seem to be relevant to OHC function, and further research is needed to determine their role in hearing. / by Fabio Albuquerque Theirs. / Ph.D.

Thesis (Ph. D.)--Harvard-MIT Division of Health Sciences and Technology, 2008. / Includes bibliographical references. / Genetic mapping by association is an unbiased approach to discover genes and pathways influencing disease traits and response to drugs and environmental exposures. There are two key obstacles to mapping in humans: (1) The full sequence of study subjects cannot yet be obtained; and (2) There are substantial limits to the phenotypes that can be safely elicited or measured. Geneticists thus rely on practically measurable sets of genotypes to proxy for the sequence and human in-vitro models that proxy for in-vivo genetics and physiology while allowing for perturbation and characterization in high throughput. This thesis presents the development of one important class of proxy genotypes, those that capture most common genetic variation, as well as an evaluation and refinement of proxy phenotypes offered by one commonly used in-vitro model, the lymphoblastoid cell-line.Capturing common human genetic variation for genome-wide association studies requires genotyping a feasible subset of proxy (or "tag") SNPs. We investigated selection and analysis of tag SNPs, examined the relationship between investment in genotyping and statistical power, and evaluated whether power is compromised when tags are selected from an incomplete resource such as HapMap. We demonstrate an efficient haplotypebased tagging approach and other methods that dramatically increase tagging efficiency. Examining all observed haplotypes for association increases power to detect rare causal alleles, while reducing power for common alleles. Power is robust to completeness of the reference panel and holds across demographically related groups.Lymphoblastoid cell lines (LCLs) are being developed into an in-vitro model where genetics of human gene expression, drug response, and other traits can be studied under controlled conditions. However, the impact of the immortalization process, the relative influence of non-genetic factors, and reproducibility of measured traits are not yet understood. / (cont.) We addressed these questions while mapping loci for response to chemotherapy and found that traits in LCLs are subject to substantial confounders and are only modestly reproducible in independent experiments. Despite this, RNA expression of many genes is affected by genetic variation and predicts response to drugs; integrating SNPs, RNA, and drug response can identify novel pharmacogenetic variation mediated by RNA. / by Roman Yelensky. / Ph.D.

Thesis (S.M.)--Harvard-MIT Division of Health Sciences and Technology, 2011. / Cataloged from PDF version of thesis. / Includes bibliographical references (p. 25). / The influence of middle-ear disease on hearing in mice is not well understood. We investigated middle-ear function and structure in a small group of dopamine beta hydroxylase (DBH)- knockout mice, which are known to be susceptible to middle-ear disease. We measured auditory brainstem response (ABR) and distortion-product otoacoustic emission (DPOAE) thresholds, as well as sound-induced umbo velocity, and used standard histological techniques to study normal and pathological middle-ear anatomy. Anatomical analyses included observation of tympanic membrane and ossicular structures, reconstruction of middle ear volume, and estimation of the volume of lightly or darkly stained middle-ear fluid. Lightly stained fluid in diseased ears was located throughout the middle ear, whereas darkly stained fluid, when present, was usually seen near the round window. The ABR and DPOAE thresholds and the umbo velocity produced by lower- and middle-frequency sound stimuli were strongly correlated with middle-ear air volume. The volume of darkly stained fluid was correlated with the DPOAE and ABR thresholds for lower- and middle-frequency sounds. The volume of lightly stained fluid was correlated with the umbo velocity in the middle and upper frequencies. Regression analyses between the thresholds and velocity show the DPOAE threshold about twice the ABR threshold in the middle frequencies, while the ABR threshold was roughly inversely proportional to the umbo velocity at lower stimulus frequencies. The correlations between histological and functional results are consistent with a decrease in middle-ear air volume and the presence of lightly stained fluid affecting the motion of the TM, while the darkly stained fluid near the round window acts to reduce the middle ear vibrations that reach the cochlea. The inverse relationship between the ABR threshold and the umbo velocity is consistent with a conductive hearing loss produced by reduced middle-ear air volume. / by Suh-Kyung Lee. / S.M.

Thesis (Ph. D.)--Harvard--Massachusetts Institute of Technology Division of Health Sciences and Technology, 2001. / Leaves 141 and 142 reversed in binding and microfilming process. / Includes bibliographical references (leaves 143-150). / Aim: To examine the effects of cellular and tissue pharmacokinetics on control released growth factors. Motivation: The resurgence of interest in controlled drug delivery reflects the increasing appreciation of the importance of local pharmacokinetics in defining the efficacy and potency of bioactive compounds. Despite promising data in vitro, growth factor use in vivo has generally failed to live up to its full potential. Without a theoretical framework to consider local pharmacology, there remains no consensus approach for improving the clinical outcomes of control released growth factors. As a result, a series of experiments incorporating cellular pharmacokinetics, computational modeling, and tissue pharmacokinetics were conducted. CellularPharmacokinetics: The effect of ligand-receptor trafficking was examined on the potency of sustained release versus bolus administration of two growth factors that differed by intracellular trafficking kinetics. Sustained delivery potency was demonstrated to be dependent on both ligand and receptor trafficking and could be predicted based on trafficking considerations. / (cont.) Computational Models: Computational models were conceptualized to describe the relationship between controlled-delivery, transport, receptor ligand pharmacokinetics, and tissue response. Theoretical predictions were made about potential approaches for optimizing local delivery and about the spatial correlation of tissue concentrations with tissue response. Tissue Pharmacokinetics: The roles of physiological transport forces and proteolytic constraints on control released growth factor were examined. Partitioning and convection were shown to be dominant forces in governing drug distribution. A novel method based on intramolecular fluorescence quenching was implemented to describe the proteolytic constraints of local fibroblast growth factor delivery in an ex vivo carotid explant model. Conclusion: Both experimental and theoretical models of cellular and tissue interactions of growth factors have suggested concepts that may be relevant for local growth factor delivery. / by David Wu. / Ph.D.

Thesis (S.M.)--Harvard-MIT Division of Health Sciences and Technology, 2006. / Includes bibliographical references (p. 54-58). / Industry-sponsored clinical research of investigational drugs (also called clinical development) has traditionally been carried out in relatively developed countries in the North American, Western European, and Pacific regions. However, lately it has been widely reported that clinical trials starting now are becoming increasingly diffused globally, with significant growth of activity in so-called emerging economies in Eastern Europe, Latin America, and Southeast Asia. This change in location of clinical development activities has numerous implications for patients, health care providers, pharmaceutical companies, regulatory agencies and governments around the globe. Even though there is much debate about the topic, a public systematic quantitative assessment of the current status of the globalization of clinical drug development phenomenon is lacking. The objective of this thesis research is to provide such objective quantification while addressing some issues that are currently in active discussion. This thesis documents that the participation of emerging countries is still relatively small (13%) and they most commonly participate in very large (involving more than five countries) phase Ilb or III trials. / (cont.) Albeit perceived as small, this participation is growing at a rapid pace (23% average annual growth rate) and the number of clinical sites of global clinical trials located in all emerging countries (11,038) is comparable with the sum of Germany, France, U.K., and Italy (11,061). Eastern European and Latin American countries have the greatest participation in clinical trials among emerging countries, but Southeast Asia is the region that is experiencing fastest growth. Meanwhile, Western Europe has experienced negative average annual growth of -8%, and North America has seemingly been stable. This thesis discusses findings and key drivers behind the globalization process. I also consider the argument that the sustainability of this model will depend on stringent protection of patients in these emerging countries and continued development of these nations, with eventual creation of an attractive market for pharmaceutical products. The extension of this process of globalization of clinical trials, if coupled with substantial improvements in health care delivery and research capacity in these emerging economies, has the potential of revolutionizing medical product development within the next two decades. / by Fabio Albuquerque Thiers. / S.M.

Includes bibliographical references (p. 197-212). / Thesis (Ph. D.)--Harvard-MIT Division of Health Sciences and Technology, 2004. / (cont.) Our methods analyze the coupling between instantaneous lung volume and heart rate and, subsequently, derive representative indices of parasympathetic and sympathetic control based on physiological and experimental findings. The validity of each method is evaluated via experimental data collected following interventions with known effect on the parasympathetic or sympathetic control. With the above techniques, this thesis explores an important topic in the field of space medicine: effects of simulated microgravity on cardiac autonomic control and orthostatic intolerance (OI). Experimental data from a prolonged bed rest study (simulation of microgravity condition) are analyzed and the conclusions are: 1) prolonged bed rest may impair autonomic control of heart rate; 2) orthostatic intolerance after bed rest is associated with impaired sympathetic responsiveness; 3) there may be a pre-bed rest predisposition to the development of OI after bed rest. These findings may have significance for studying Earth-bound orthostatic hypotension as well as for designing effective countermeasures to post-flight OI. In addition, they also indicate the efficacy of our proposed methods for autonomic function quantification. / System identification is an effective approach for the quantitative study of physiologic systems. It deals with the problem of building mathematical models based on observed data and enables a dynamical characterization of the underlying physiologic mechanisms specific to the individual being studied. In this thesis, we develop and validate a new linear time-invariant system identification approach which is based on a weighted-principal component regression (WPCR) method. An important feature of this approach is its asymptotic frequency-selective property in solving time-domain parametric system identification problems. Owing to this property, data-specific candidate models can be built by considering the dominant frequency components inherent in the input (and output) signals, which is advantageous when the signals are colored, as are most physiologic signals. The efficacy of this method in modeling open-loop and closed-loop systems is demonstrated with respect to simulated and experimental data. In conjunction with the WPCR-based system identification approach, we propose new methods to noninvasively quantify cardiac autonomic control. Such quantification is important in understanding basic pathophysiological mechanisms or in patient monitoring, treatment design and follow-up. / by Xinshu Xiao. / Ph.D.