Thesis (Ph.D.)--Harvard--Massachusetts Institute of Technology Division of Health Sciences and Technology, 2000. / Includes bibliographical references (leaves 135-139). / Detecting the metabolic state of atherosclerotic lesions is a promise of nuclear medicine imaging. Several researchers are developing radiopharmaceuticals for atherosclerosis imaging. In this thesis, we provided procedural guidelines to detect carotid lesions with single photon ,Emission Computed Tomography (SPECT). We first established a method to assess the requirements for "successful" lesion detection. Although this method was used to detect focal carotid lesions, it is also applicable to the detection of focal lesions in other arteries or veins. We measured lesion detectability using the output values of a 3D moving Non Pre-Whitening Matched Filter (30 mNPWMF) with the .Localization Receiver Operating Characteristics (LROC) paradigm. We simulated SPECT images of the neck using SimSPECT, our in-house analog Monte Carlo radiation transport code. We used 400 64x64 reconstructed images formed by 99mTc photons of a focal lesion in a carotid artery next to a jugular vein, both in a cylindrical water neck. We then applied the 3D \ mNPWMF along the large neck vessels. The NPWMF has been found to correlate well with human observers in simple ROC studies. We expect the mNPWMF operation to mimic a radiologist who already has a blood pool image which identifies the location of the large neck vessels. Using this detection method, we calculated that 1 to 6 kBq/cm were needed in the lesion. At large blood activity (4.6 times the surrounding tissue activity), the minimum radiopharmaceutical uptake increased by 1.6-2.9 times when the patient was lying down as opposed to sitting up. At this blood activity, a carotid dilation of 1 cm radius distracted the moving Matched Filter from lesion detection. We recommend that the blood activity be as low as possible to avoid any focal dilation from distracting our detector. We recommend that, at high blood activity, the patient be imaged in an upright position in which the jugular veins are collapsed, preventing their blood pool activity from obscuring the carotid arteries. Finally, we showed that a lesion needed 140% of the radiopharmaceutical when acquired with a radius of rotation (ROR) of25 cm instead of 15 cm. In conclusion, we assessed successfully the effect of the jugular veins and carotid dilation on detection of carotid lesions in SPECT images of the neck using the LROC detection paradigm. / by Marie-José Bélanger. / Ph.D.

Thesis: Ph. D. in Medical Engineering and Medical Physics, Harvard-MIT Program in Health Sciences and Technology, 2017. / Cataloged from PDF version of thesis. / Includes bibliographical references (pages 124-135). / The complex, dynamic, and responsive behavior of cells arises from integrated signaling pathways and regulatory networks. With advancement in our ability to engineer mammalian cells, we harness a novel set of molecular tools to develop synthetic biology-enabled applications that help facilitate our understanding of complex biological networks and cellular behaviors. The recent discovery of the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated (Cas) system from prokaryotic adaptive immune system demonstrated unprecedented genome editing efficiency and programmability in sequence specific genome editing of mammalian cells. In this thesis, I utilized the CRISPR-Cas system to construct a combinatorial genetic perturbation platform that enables massively parallel high throughput screening of multiple gene elements. This technology platform allows systematically interrogation of higher-order interactions of genetic regulators. The later part of the work described the establishment of a genomically encoded cellular recorder with the ability to longitudinally track and record molecular events in live animals. This cellular recorder encodes cellular memory through the quantitative accumulation of targeted genomic mutations, that allows mapping of a dynamical set of gene regulatory events without the need for continuous cell imaging or destructive sampling. Together, we envision these sets of technology and tools will offer new insights into cellular process in disease and in health. / by Cheryl H. Cui. / Ph. D. in Medical Engineering and Medical Physics

Thesis (Ph. D.)--Harvard-MIT Division of Health Sciences and Technology, 2005. / Includes bibliographical references (p. 109-119). / Eukaryotic cells are permeated by a three-dimensional network of entangled filamentous proteins termed the cytoskeleton. Like scaffolding, the cytoskeleton provides rigidity and resistance to deformation from forces transmitted to and from the cell membrane. In order to model the mechanics of the cytoskeleton, the interaction of individual structural proteins must be established. To this end the relationship between two critical proteins of the actin cytoskeleton is examined. Actin reversibly assembles into filaments that provide cells with shape and confer to the cell its mechanical properties. Filamin is an abundant actin-binding protein that efficiently cross-links actin filaments in large-angle orientations, requiring the lowest concentration to convert actin filaments into a cohesive gel. Filamin dimers are composed of two 24-repeat domains that come together like a V with an actin-binding region at each free end. Analysis reveals that the repeat domains of filamin are more flexible than the self-association region of the dimer. These findings dispute the initial claim that filamin is a rigid molecule. This thesis investigates the binding modality of filamin to actin. The structure of filamin bound to actin was compared to filamin in solution and immunogold molecules bound along the filamin rod were used to map the 3-D organization of filamin-actin junctions. There is evidence that filamin binds to actin at more sites than filamin's two established N-terminal actin-binding sites. These features, flexible repeat extensions, multiple-site binding, and a rigid self-association region, make filamin a potent cross-linking agent. / (cont.) The long flexible extensions allow filamin molecules to sample a large volume of cytoplasm in search of an actin target. The binding length of filamin along actin filaments provides a less-flexible linkage from actin to the rigid filamin self-association region, enabling reproducible large angles. At the same time, filamin brings actin filaments into close proximity, creating tight network entanglement, while filamin's angle prevents linked actin filaments from slipping into dense bundles as they do with short cross-linkers like [alpha]-actinin, instead maintaining a strong, disperse network. Tightly constrained junctions support recent entropic and enthalpic models of the cytoskeleton. / by Christopher A. Hartemink. / Ph.D.

Thesis (Ph.D.)--Harvard--Massachusetts Institute of Technology Division of Health Sciences and Technology, 1999. / Includes bibliographical references. / by Jennifer Hartt Elisseeff. / Ph.D.

Thesis (Ph. D. in Bioinformatics and Integrative Genomics)--Harvard-MIT Program in Health Sciences and Technology, 2013. / Cataloged from PDF version of thesis. / Includes bibliographical references. / One in every 88 children is diagnosed with Autism Spectrum Disorders (ASDs), a set of neurodevelopmental conditions characterized by social impairments, communication deficits, and repetitive behavior. ASDs have a substantial genetic component, but the specific cause of most cases remains unknown. Understanding gene-environment interactions underlying ASD is essential for improving early diagnosis and identifying critical targets for intervention and prevention. Towards this goal, we surveyed adenosine-to-inosine (A-to-I) RNA editing in autistic brains. A-to-I editing is an epigenetic mechanism that fine-tunes synaptic function in response to environmental stimuli, shown to modulate complex behavior in animals. We used ultradeep sequencing to quantify A-to-I recoding of candidate synaptic genes in postmortem cerebella from individuals with ASD and neurotypical controls. We found unexpectedly wide distributions of human A-to-I editing levels, whose extremes were consistently populated by individuals with ASD. We correlated Ato- I editing with isoform usage, identified clusters of correlated sites, and examined differential editing patterns. Importantly, we found that individuals with ASD commonly use a dysfunctional form of the editing enzyme ADARB1. We next profiled small RNAs thought to regulate A-to-I editing, which originate from one of the most commonly altered loci in ASD, 15q11. Deep targeted sequencing of SNORD115 and SNORD116 transcripts enabled their high-resolution detection in human brains, and revealed a strong gender bias underlying their expression. The consistent 2-fold upregulation of 15q11 small RNAs in male vs. female cerebella could be important in delineating the role of this locus in ASD, a male dominant disorder. Overall, these studies provide an accurate population-level view of small RNA and A-to-I editing in human cerebella, and suggest that A-to-I editing of synaptic genes may be informative for assessing the epigenetic risk for autism. / by Alal Eran. / Ph.D.in Bioinformatics and Integrative Genomics

Thesis: Ph. D. in Medical Engineering and Medical Physics, Harvard-MIT Program in Health Sciences and Technology, 2016. / Cataloged from PDF version of thesis. / Includes bibliographical references (pages 137-148). / Patterning complex 3D features at the nanoscale offers potential applications for a wide range of fields from materials to medicine. While numerous methods have been developed to manipulate nanoscale materials, these methods are typically limited by their difficulty in creating arbitrary 3D patterns. Self-assembly of nucleic acids has emerged as a promising method for addressing this challenge due to the predictability and programmability of the material and its structure. While a diversity of DNA nanostructures have been designed by specifying complementarity rules between strands, creation of 3D nanostructures requires careful design of strand architecture, and patterns are often limited to a volume of 25 x 25 x 25 nm³ Here, we address the challenges in structural DNA nanotechnology by developing a modular DNA "brick" approach. These bricks are short, single-stranded oliogomers that can self-assemble in a single-pot reaction to a prescribed 3D shape. Using this modular approach, we demonstrate high efficiency in 3D design by generating 100 distinct, discrete 3D structures from a library of strands. We also created long-range ordering of channels, tunnels, and pores by growing micron-sized 3D periodic crystals made from DNA bricks. Finally, we applied this approach to control over 30,000 unique component strands to selfassemble into cuboids measuring over 100 nm in each dimension. These structures were further used to pattern highly complex cavities. Together, this work represents a simple, modular, and versatile method for 3D nanofabrication. This unique patterning capability of DNA bricks may enable development of new applications by providing a foundation for intricate and complex control of an unprecedented number of independent components. / by Luvena Le-Yun Ong. / Ph. D. in Medical Engineering and Medical Physics

Thesis (S.M.)--Harvard-MIT Division of Health Sciences and Technology, 2011. / Cataloged from PDF version of thesis. / Includes bibliographical references (p. 58-61). / Interviews were conducted to establish a baseline for how orphan drug forecasting is currently undertaken by financial market and industry analysts with the intention of understanding the variables typically accounted for in such a model. A literature search formed the basis of subsequent interviews conducted with experts from industry, payers, providers, legislators, patient groups, and the FDA. Discussion then focused on elements of the market which are poised to change in the short-term, how such changes might be reflected in existing models, and/or how these models may instead need to be modified to adapt to the new environment. We hypothesized that impending changes in the healthcare sector would indeed impact the legitimacy of current forecasting models, and that significant changes would need to be introduced to account for these new market forces. Our hypothesis, however, was not confirmed, in that although much of the literature and, indeed, public outcry over rising healthcare costs in general and drug prices in particular make a strong case for implementing changes in the orphan market via payers, government, or other actors, an assessment of healthcare experts regarding market changes over the next five years revealed a general consensus that meaningful change will likely not occur during this timeframe for orphan products, with the exception of a possible increase in pharmacoeconomic requirements for drugs which are only marginally effective. Thus, current orphan drug forecasting models constructed for use by financial and industry analysts correctly avoid discounting for these potential changes, as they will likely not face significant changes in the US until closer to a ten year time horizon. Potential exceptions to this conclusion depend on implementation and regulatory treatment of the fields of personalized medicine and gene therapy, as developments in these areas may closely interact with existing orphan drug legislation. Our results have significant implications for all companies and stakeholders entering or currently operating in the orphan market, and open the door for further quantitative and qualitative analysis. / by Joshua Gottlieb. / S.M.

Thesis (Ph. D.)--Harvard-MIT Division of Health Sciences and Technology, 2005. / Includes bibliographical references. / Many brain tumors are localized deeply and are currently surgically inaccessible without causing severe damage to the overlying structures of the brain. The current spectrum of non-invasive methods for treating such tumors includes radiotherapy, which requires exposure to ionizing radiation, and chemotherapy, which is systemically toxic. However, these tumors may also potentially be attacked by focusing highly intense ultrasound onto them. Focused ultrasound surgery is without the side effects of radiotherapy and chemotherapy, and the therapeutic effect of ultrasound therapy can be monitored in real- time using the proton chemical shift MRI technique. However, in order for brain tumors to be treated non-invasively, the ultrasound must be focused onto the targeted brain tissue through the intact cranium. Transcranial focusing of ultrasound is a longstanding and difficult problem as skull is a highly heterogeneous material. As the ultrasound field propagates through the bones of the skull, it undergoes substantatial distortion due to the variations in density and speed of sound therein. There is substantial individual variation in skull size, thickness and composition. Furthermore, the acoustic attenuation coefficient in bone is high, so the skull may also be heated by the ultrasound propagating through it. This thesis contains novel simulation techniques for analyzing transcranial acoustic propagation and for analyzing the temperature changes so produced in the brain, skull and scalp. These techniques have also been applied to modeling non-invasive treatment of the liver, and to producing therapeutic ultrasound fields that harness non-linear acoustic effects advantageously. / (cont.) The thesis also contains unified models for the speed of sound and the acoustic attenuation coeffiecient in human skull. These models were generated by combining genetic optimization algorithms, acoustic propagation modeling and empirical measurement of intracranial ultrasound fields; they are valid across the full range of trabecular and cortical cranial bone. / by Christopher W. Connor. / Ph.D.

Thesis (S.M.)--Harvard-MIT Division of Health Sciences and Technology, 2006. / Includes bibliographical references (leaves 63-64). / The United States population is aging, and the need for novel approaches to treat and manage disease continues to grow. Among the diseases that will impact this population, cancer remains a therapeutic area with significant unmet need. Pharmaceutical and biotechnology industries must continue to meet revenue and income growth expectations and will become increasingly dependent on novel drugs in their pipelines. In order for the pharmaceutical and biotechnology industries to meet the demands of both patients and shareholders, productivity in the research and development process will need to improve significantly. In order to understand how best to improve the drug discovery and development process, it is important to identify potential sources of innovation throughout the process. Among these, an important consideration is to understand the paths that molecules take through the discovery and development process. / (cont.) This thesis used the marketplace of oncology drugs in development to test the hypothesis that novel molecules largely originate in academia, are developed by biotechnology companies and ultimately are licensed to pharmaceutical companies for commercialization. This thesis analyzed a database of 364 unique oncology small molecules and biologics entering Phase I clinical development between 1991 and 2002. / by Jorge Cesar Conde. / S.M.

Thesis (Ph. D.)--Harvard-MIT Division of Health Sciences and Technology, 2009. / Includes bibliographical references (p. 107-121). / The clinical application of chemotherapy to brain malignancies has been severely limited because many potential therapeutic agents are typically unable to penetrate the blood-brain barrier (BBB). A novel approach to overcome this barrier uses focused ultrasound to induce localized BBB disruption in a targeted region of the brain and magnetic resonance imaging (MRI) to guide and monitor the procedure. The purpose of this thesis was to develop a technique using MRI-guided focused ultrasound for trans bbb drug delivery applications. This thesis demonstrates that MRI-guided focused ultrasound can be used to achieve consistent and reproducible BBB disruption without invasive craniotomy in rats, to enable doxorubicin to accumulate in normal brain at clinically therapeutic levels, and to increase the antitumoral efficacy of doxorubicin in a rodent model of aggressive glioma. Using a microbubble-based ultrasonographic contrast agent, focal BBB opening was consistently achieved using transcranial focal pressures of 1.2 MPa or greater; locations in the posterior brain exhibited consistent BBB disruption with applied focal pressures of 0.8 MPa or greater. When combined with systemic administration of liposomal doxorubicin, we achieved local drug concentrations of 900 ± 300 ng/g tissue in the brain with minimal tissue effects, and up to 5400 ± 700 ng/g tissue with more significant tissue damage, while accumulation in non-targeted contralateral brain tissue remained significantly lower (p < 0.001). In addition, MRI signal enhancement in the sonicated region correlated strongly with doxorubicin concentration in tissue (r = 0.87), suggesting that contrast-enhanced MRI may provide useful feedback on drug penetration. / (cont.) Finally, glioma-bearing rats treated with ultrasound-enhanced chemotherapy exhibited significantly longer median survival times (31 versus 25 days; p = 0.0007) and slower tumor growth (average tumor volume doubling time, 3.7 ± 0.5 days, versus 2.3 ± 0.3 days) than nontreated rats; rats which received standard intravenous chemotherapy showed no significant difference in survival or tumor growth rate. In sum, this thesis research provides pre-clinical data toward the development of MRI-guided focused ultrasound as a noninvasive method for the delivery of agents such as doxorubicin across the BBB to treat patients with diseases of the central nervous system. / by Lisa Hsu Treat. / Ph.D.