Thesis: Ph. D., Harvard-MIT Program in Health Sciences and Technology, 2013. / This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections. / Cataloged from student-submitted PDF version of thesis. / Includes bibliographical references (pages xviii-xxxvi). / Contamination of food, water, medicine and ingestible household products is a public health hazard that episodically causes outbreaks worldwide. Existing laboratory methods are often expensive, require a laboratory environment and/or trained staff to perform manual steps. The aim of this PhD thesis was to create and test methods and instruments for affordable diagnostic tests for contaminants and pathogens. To achieve this goal, the LabReader was introduced, which employs a LED-based detection scheme for four simultaneous fluorescence- and UV-measurements. Assays were developed to detect (di-)ethylene glycol in consumables ≥0.1wt% and alcohols ≥1ppb. Pathogens in water, foods and blood were detected at ≥104 CFU/ml using nonspecific intercalating dyes. To gain sensitivity and specificity for cell-based analysis, biochemical amplification methods had to be incorporated. To be deployable outside a laboratory, sample preparation needed to be automated. Automation was achieved by combining the LabReader with the already developed LabTube, a disposable platform for automated DNA extraction inside a standard centrifuge. Performing DNA amplification/readout in an external optical reader, made the LabSystem broadly deployable and flexible. DNA extraction of food bacteria (E.coli and Alicyclobacillus) was optimized inside the LabTube for 102-109 inserted DNA copies. The extracted DNA was amplified using the qualitative isothermal LAMP method and semi-quantitative, real-time PCR inside the LabReader. The combined extraction and amplification detection limit of the LAMP-LabSystem and the quantitation limit of the PCR-LabSystem were as low as 102 copies. Performing extraction and amplification inside the centrifuge/LabTube was also outlined, which may be preferable when contamination risks are high. After theoretically evaluating heating methods, a battery-driven heated LabTube was designed, in which 102-108 DNA copies of VTEC E.coli were extracted, LAMP-amplified and visually readout within 1.5 hrs. The major contribution of this thesis is the full system integration of versatile, automated sample preparation and detection systems. They offer great flexibility as they may be used with each other or in combination with other analytic methods, depending on the application. At the same time, they are frugal and deployable at low-to-medium throughput - even outside a traditional laboratory. Whilst the focus was put on food safety, the systems were also used for medical, environmental or consumer product quality applications, hence demonstrating their broad applicability. / by Melanie Margarete Hoehl. / Ph. D.

Thesis (Ph. D.)--Harvard-MIT Program in Health Sciences and Technology, 2012. / Pages 157 and 158 blank. Cataloged from PDF version of thesis. / Includes bibliographical references (p. 150-156). / Vertebrate hair cells respond to mechanical stimuli with an inward current that is carried by extracellular cations through mechanically-gated transmembrane ion channels called transduction channels, located in the hair cell's specialized apical surface called the stereocilia. The current is characterized as having a rapid onset and adapting exponentially with a fast and slow time constant. The fast component is usually attributed to calcium binding directly to the transduction channels to promote channel reclosure. Myosin-1C, an unconventional myosin motor protein that is also modulated by calcium, adjusts the tension applied to the transduction channel to cause slow adaptation. Neither adaptation typically acts completely to restore the transduction current back to the baseline level. Recent evidence has suggested that the transduction channel is further away from myosin-IC than previously believed, creating a spatial separation that changes the nature of the calcium feedback. I developed a computational model to explore the motion of vertebrate hair cells simultaneously with calcium diffusion within the cell. The model is also capable of simulating many other experimental techniques that are commonly applied to hair cells. The results of the model suggest a fundamentally different viewpoint for understanding adaptation in vertebrate hair cells. Calcium can create unique responses from different transduction channels within the same hair cell. The implications of these findings help to explain the incompleteness of adaptation as well as implicate myosin-1C for fast adaptation as well as slow adaptation. In addition, groundwork for better understanding stereocilia-based amplification in the mammalian cochlea was developed. Experimental predictions were created to test these theories. / by Paul D. Niksch. / Ph.D.

Thesis: Ph. D., Harvard-MIT Program in Health Sciences and Technology, 2015. / This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections. / Vita. Cataloged from PDF version of thesis. / Includes bibliographical references (pages 111-127). / Pancreatic cancer is responsible for nearly 40,000 deaths in the U.S. annually, with a dismal 5-year survival rate below 7%. The poor therapeutic outcomes reflect a paucity of new approaches targeting the genomic underpinnings of pancreatic ductal adenocarcinomas (PDAC, the vast majority of pancreatic cancers) as well as our inability to overcome the desmoplastic stromal barrier characteristic of PDAC. RNA interference through siRNA holds promise in targeting key mutations driving PDAC, such as oncogenic KRAS; however, a nucleic acid delivery vehicle that homes to PDAC and breaches the stroma does not yet exist. Noting that the novel cyclic peptide iRGD mediates tumor targeting and penetration through interactions with [alpha][upsilon][beta]3/5 integrins and neuropilin-1, we hypothesized that "tandem" peptides combining a cell-penetrating peptide and iRGD can complex with siRNA to form tumor-penetrating nanocomplexes (TPNs) effective in delivering siRNA to PDAC. Such a nanoscale carrier could provide a practical means of bridging our understanding of PDAC as a genetic disease to the clinic. Furthermore, the modular aspect of these self-assembled particles permits them to accommodate alternate cargoes or targeting domains, and we have proposed that tandem peptide complexes could be extended to applications outside of RNA interference, particularly for the delivery of components for CRISPR/Cas9-mediated gene editing. This delivery system could be applied to generate improved animal models of pancreatic cancer. In this work, we first designed, characterized, and optimized iRGD-based TPNs for RNAi in pancreatic cancer cells in vitro, showing robust knockdown of single and multiple targets. In order to stabilize these nanoparticles for systemic administration, we then devised and compared diverse, non-covalent materials for formulating TPNs with polyethylene glycol (PEG). The best material in this capacity, a peptide-PEG conjugate, reduced accumulation of TPNs in off-target organs and improved circulation kinetics, while preserving functional knockdown capacity. Incorporating this approach with the iRGD tandem peptides, we studied the translational potential of PEGylated iRGD TPNs to deliver siRNA to various models of pancreatic cancer and have begun therapeutic testing with siRNA targeting KRAS. Finally, we have adapted the tandem peptide platform to mediate delivery of CRISPR/Cas9 components. Particle configurations to deliver guide RNA alone, guide RNA with a DNA template for homology-directed repair, and guide RNA with Cas9 protein have all shown efficacy in gene editing in vitro, important steps toward creating sporadic mutations to model PDAC. Thus, we have established a versatile approach to the delivery of nucleic acids for studying and treating pancreatic cancer. / by Justin Han Je Lo. / Ph. D.

Thesis (S.M.)--Harvard-MIT Division of Health Sciences and Technology, 2006. / Includes bibliographical references (leaves 37-40). / Since the Bayh-Dole Act of 1980, the commercialization of ideas generated in academia has driven significant startup activity and expansion in the life sciences. This commercial transformation has been shown by others to be concentrated among a relatively small number of elite academic institutions. However, within these institutions, we find that a small number of prestigious scientists are disproportionately responsible for entrepreneurial and commercial activity. To date, limited research has been conducted which aims to understand the characteristics of such serial scientist-entrepreneurs or their significance in early commercial ventures. This study identifies and characterizes 18 serial scientist-entrepreneurs (defined as faculty who have founded or served on the board of directors of 3 or more startups) on the basis of academic impact, patenting, and social network centrality, as compared to their first-time entrepreneur (i.e., faculty who founded or directed 1-2 companies) and noncommercial peers. These individuals constitute a subset of 66 scientist-entrepreneurs from a population of the 493 scientists who served as faculty in life sciences-related departments at MIT, during the period of 1981 to 2005 (representing the primary commercialization period for biotechnology). / (cont.) The thesis highlights three key findings. First, the subset of 18 serial scientist-entrepreneurs founded or directed two-thirds of all startup ventures associated with the entire population thus underscoring the significant "skew" in commercial activities. Furthermore, empirical analyses revealed that these serial scientist-entrepreneurs had significantly higher academic impact (i.e., "academic prestige"), as measured by citations to their work, as compared to first-time entrepreneurs and noncommercial scientists. Perhaps not surprisingly, they also had significantly higher numbers of issued U.S. patents, compared to first-time entrepreneurs. Second, the serial scientist-entrepreneurs developed robust relationships with a small group of venture capital investors, who have repeatedly funded their companies. Several of these serial scientist-entrepreneurs retained central positions in the social network of faculty entrepreneurs, potentially brokering and accelerating entrepreneurial activity, including scientific advisory board membership, within the community. These findings suggest that serial scientist-entrepreneurs play a vital role in contributing reputation, deep technical insight, access to intellectual property, and relationship networks to startup life sciences ventures. / (cont.) It remains for additional research to determine whether the active involvement of serial scientist-entrepreneurs has resulted in enhanced startup value or performance. / by Eugene Chiu. / S.M.

Thesis (Ph. D.)--Harvard-MIT Division of Health Sciences and Technology, 2009. / Cataloged from PDF version of thesis. / Includes bibliographical references. / The current clinical standard for cervical cancer diagnosis is colposcopy, a procedure that involves visual inspection and biopsy of at-risk tissue, followed by histopathology. The major objective of colposcopy is detection of high-grade squamous intraepithelial lesions (HSIL), which are precancerous lesions with high risk of progression. Colposcopy, even when conducted by experts, is subject to significant diagnostic variability. The aim of the work presented in this thesis was to develop a non-invasive clinical tool for detection of cervical HSIL and for guiding the biopsy during colposcopy. Previously we have developed a contact-probe portable instrument for tissue reflectance and fluorescence collection, and spectral analysis models to extract and quantify biochemical and structural features of tissue to provide disease state assessment. In this thesis we further refine the instrumentation and spectral analysis models and conduct the clinical in vivo studies. The clinical in-vivo study showed cervical anatomy was a confounder to diagnostic algorithms that treat cervix as spectroscopically uniform. We used complex instrumentation to comprehensively study cervical tissue and found that scattering alone was sufficient to identify HSIL. We developed an accurate algorithm based on tissue scattering for detection of HSIL in the cervical transformation zone, an area where vast majority of cervical lesions arise. We further successfully extended our point-probe technique into the imaging mode, to provide the wide-area surveillance capability. / (cont.) The ongoing imaging clinical in-vivo feasibility study demonstrates spectroscopic contrast between cervical HSIL and non-HSIL tissue and is consistent with findings of the contact-probe study. The future steps include diagnostic accuracy assessment of the imaging technique, and if proven successful, a clinical study to evaluate the performance of spectroscopy-guided biopsy. / by Jelena Mirković. / Ph.D.

Thesis (Ph. D.)--Harvard-MIT Division of Health Sciences and Technology, 2010. / Cataloged from PDF version of thesis. / Includes bibliographical references (p. 53-58). / There is a strong correlation between the membrane fluctuations and the material properties of living cells. The former, consisting of submicron displacements, can be altered by changing the cells' pathophysiological conditions. It is our hypothesis that the material properties of cells can be retrieved when we quantify cell membrane fluctuation and combine that result with an appropriate physical model. We have developed: (1) an optical imaging technique to noninvasively quantify membrane fluctuations in red blood cells at the nanometer and millisecond scales; and (2) a model to retrieve the material properties of red blood cell membrane. The technique employs laser interferometry and provides full-field quantitative topographical information of living cells with unprecedented stability. Integration with the mathematical model provides the specific material properties from individual cell membrane fluctuations: shear modulus of the membrane; bending modulus; and viscosity of the cytoplasm. Employing these methods, we have systemically studied the material properties of human red blood cells altered by various pathophysiological conditions: morphological transition of red blood cell; parasitization by the P. falciparum parasites; and metabolic remodeling of the membrane driven by Adenosine-5'- triphosphate (ATP). We envision that this investigation could offer a means to link cell membrane fluctuations with the pathological conditions that lead to human disease states by quantitatively providing the alternation in material properties. A clear understanding of the mechanical alteration of red blood cells is important to studying the human diseases which cause their infection. / Ph.D.

Thesis (S.M. in Science Writing)--Massachusetts Institute of Technology, Dept. of Humanities, Program in Writing and Humanistic Studies, 2003. / Vita. / Includes bibliographical references (p. 69-76). / by Sorcha McDonagh. / S.M.in Science Writing

Thesis (Ph. D.)--Harvard-MIT Division of Health Sciences and Technology, 2004. / Includes bibliographical references (p. 191-203). / The importance of the cochlear active mechanism in the reception of speech in different types of noise was explored. The perceptual effects of loudness recruitment, a consequence of loss of the active mechanism, were assessed in simulated-loss listeners using a multiband expansion algorithm that models abnormal cochlear linearity. While this algorithm, which derives the expansion characteristic from absolute hearing thresholds, can accurately simulate the mean speech intelligibility results of hearing-impaired listeners, its ability to simulate the performance of individual listeners is limited. Given the relationship between loudness perception and the active mechanism, deriving the expansion characteristic from estimates of cochlear compression should provide a more accurate model of an individual listener's impairment. Towards this aim, the reliability of two psychoacoustic methods used to estimate the magnitude of compression (growth of masking and temporal masking) was assessed. Results suggest that growth of masking is a more reliable measure of compression in listeners with both normal and impaired hearing. The relationship between the compressive characteristics of the auditory system and speech perception in complex acoustic backgrounds was also evaluated. The operational status of the active mechanism was assessed behaviorally using three independently derived measures: (1) slope ratio of off- and on-frequency growth of masking functions, (2) equivalent rectangular bandwidth of auditory filters, and (3) masker-phase masking differences. These measures were correlated with speech reception thresholds (SRTs) in backgrounds with and without spectral and/or temporal-modulations. The relationship between slope ratios, filter bandwidths, and the maximum / (cont.) SRT difference (SRT in steady noise minus SRT in temporally modulated, spectral gap noise) was significant. These results indicate that the ability to take advantage of momentary fluctuations in the amplitude or frequency spectrum of background noise requires an intact active mechanism. The speech reception performance of two hearing-impaired listeners was modeled using a customized version of the expansion algorithm. The algorithm was customized to an individual's impairment based on psychoacoustic measures used to evaluate the integrity of the active mechanism. The maximum SRT difference in the simulated-loss listeners more closely matched the results of their hearing-impaired counterparts, compared to SRTs measured using the original algorithm. These results provide further evidence of the importance of the active mechanism to the perception of speech in modulated noise. / by Peninah S. Rosengard. / Ph.D.

Thesis (Ph. D.)--Harvard-MIT Division of Health Sciences and Technology, 2008. / Includes bibliographical references (p. 155-174). / The bone marrow contains a multipotent stromal cell, commonly referred to as a mesenchymal stem cell (MSC). There has been recent interest in the clinical use of MSCs for cell-based therapy because: (1) bone marrow aspiration is a routine method used in medicine thereby allowing for easy accessibility to human MSCs; (2) MSCs are easily isolated and can expand to clinical scales in a relatively short period of time; (3) MSCs can be biopreserved without loss of potency and stored for point-of-care delivery; and (4) human trials of MSCs thus far have shown no adverse reactions to allogeneic versus autologous MSC transplants suggesting that therapy can cross histocompatibility barriers. This thesis describes the development of new modalities and indications for MSC-based treatments by leveraging the endogenous functions of these cells for therapeutic purposes. First, it is known that marrow stromal cells support hematopoiesis by secreting bioactive molecules that aid in the growth, differentiation, function and migration of hematopoietic cells within the marrow cavity. We show that these same secreted molecules derived from MSCs ex vivo can be formulated as an intravenous drug. In a D-galactosamine model of acute liver failure, a bolus injection of a concentrated form of MSC conditioned medium (MSC-CM) led to a significant survival benefit with a one week study endpoint. We employed in vitro and in vivo assays to demonstrate the effect of MSC-CM on leukocytes and resident liver cells. Traditional biochemical approaches were performed to identify active fractions within MSC-CM that were responsible for its therapeutic efficacy. As a corollary to an injectable drug, we developed MSCbased extracorporeal devices to serve as a dynamic source of MSC-CM in a dialysis-like setting. / (cont.) Liver injured rats supported by extracorporeal bioreactors seeded with MSCs had significant improvements in liver serologies and survival in the short-term, whereas a composite device containing both MSCs and hepatocytes was shown to have a long-term survival benefit after 30 days. The second natural function of MSCs that was exploited for therapy concerns recent evidence that stromal cells can present antigens in lymphoid organs. We discovered that MSCs can express peripheral tissue antigens similar to other specialized antigen presenting cells in the thymus and lymph nodes - a process known to induce tolerance to self-reactive T cells in vivo. We show that MSC transplantation can be an effective treatment of intestinal autoimmunity in a chemically-induced model of colitis and a mouse model deficient in regulatory T cells. In addition, we demonstrate that MSC grafts increase the endogenous population of suppressor cells in vivo, which can potentially amplify and sustain the immunosuppression of the original transplant. The proposed work is significant, as development of such therapies for acute liver failure and inflammatory bowel disease would potentially treat an estimated 100,000+ newly diagnosed patients or ones who are refractory or contraindicated to standard-of-care medical/surgical procedures. These studies may empower the future use of MSCs in other organ failure syndromes and autoimmune conditions. Finally, exploration of the therapeutic functions of MSCs is expected to enhance our understanding of the mechanisms involved in cell therapy and give further insight to the natural functions of MSCs during health and disease. / by Biju Parekkadan. / Ph.D.

Thesis (Ph. D. in Speech and Hearing Bioscience and Technology)--Harvard-MIT Division of Health Sciences and Technology, 2010. / Cataloged from PDF version of thesis. / Includes bibliographical references (p. 165-180). / Speakers use auditory feedback to monitor their own speech, ensuring that the intended output matches the observed output. By altering the acoustic feedback signal before it reaches the speaker's ear, we can induce auditory errors: differences between what is expected and what is heard. This dissertation investigates the neural mechanisms responsible for the detection and consequent correction of these auditory errors. Linguistic influences on feedback control were assessed in two experiments employing auditory perturbation. In a behavioral experiment, subjects spoke four-word sentences while the fundamental frequency (FO) of the stressed word was perturbed either upwards or downwards, causing the word to sound more or less stressed. Subjects adapted by altering both the FO and the intensity contrast between stressed and unstressed words, even though intensity remained unperturbed. An integrated model of prosodic control is proposed in which FO and intensity are modulated together to achieve a stress target. In a second experiment, functional magnetic resonance imaging was used to measure neural responses to speech with and without auditory perturbation. Subjects were found to compensate more for formant shifts that resulted in a phonetic category change than for formant shifts that did not, despite the identical magnitudes of the shifts. Furthermore, the extent of neural activation in superior temporal and inferior frontal regions was greater for cross-category than for within-category shifts, evidence that a stronger cortical error signal accompanies a linguistically-relevant acoustic change. Taken together, these results demonstrate that auditory feedback control is sensitive to linguistic contrasts learned through auditory experience. / by Caroline A. Niziolek. / Ph.D.in Speech and Hearing Bioscience and Technology