Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2019 / "DOCTOR OF PHILOSOPHY IN BIOLOGICAL ENGINEERING With a focus in Polymers and Soft Matter (PPSM)." Cataloged from PDF version of thesis. / Includes bibliographical references (pages 106-112). / Osteoarthritis is a debilitating joint disease that affects over 30 million people and has no disease-modifying therapies. The current standard of care for the disease is merely palliative until joint replacement is necessary. Disease-modifying osteoarthritis drugs have been tested in the clinic, but all have been unsuccessful in clinical trials. A key point of failure for several of these drugs has been inefficient and inadequate delivery to target cartilage cells. Cartilage is avascular and thus cannot be targeted efficiently through the systemic circulation. Due to the localized nature of osteoarthritis, direct injection of therapeutics into affected joints is an attractive solution to this problem. However, delivery via this approach remains impeded by rapid turnover of the synovial fluid within joints and the dense, highly charged nature of cartilage tissue. / To overcome this biological barrier, we took advantage of a recently demonstrated phenomenon in which positively charged nanomaterials electrostatically interact with anionic cartilage, both avoiding joint clearance and facilitating diffusion through the tissue in the process. This work describes two strategies using such polycationic materials to deliver insulin-like growth factor 1 (IGF-1), a promising anabolic growth factor for osteoarthritis that has known delivery challenges. The first approach used an electrostatic assembly of IGF-1, poly(L-glutamic acid), and poly(L-arginine) into a nanoscale complex coacervate, or nanoplex, for delivery of unmodified, bioactive IGF-1. The second approach involved a densely charged polyamidoamine (PAMAM) dendrimer, end-grafted with poly(ethylene glycol) (PEG) of various molecular weights at various % end group functionalization. / From this panel of nearly 50 PEGylated dendrimers, an optimally charged dendrimer was selected based on criteria of cartilage uptake and nontoxicity. The selected dendrimer was covalently modified with IGF-1. Both systems were tested to ensure that they could deliver bioactive IGF-1, penetrate human thickness cartilage tissue, extend joint residence time in vivo, and mitigate the progression of early traumatic osteoarthritis in rats. Both the nanoplex and optimally PEGylated dendrimer-IGF-1 achieved these goals, suggesting that polycationic nanocarriers could potentially improve pharmacokinetics and efficacy of disease-modifying osteoarthritis drugs in the clinic. / by Brett Charles Geiger. / Ph. D. / Ph.D. Massachusetts Institute of Technology, Department of Biological Engineering

This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections. / Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2019 / Cataloged from student-submitted PDF version of thesis. / Includes bibliographical references (pages 153-172). / Mathematical modeling is essential to the understanding and design of biological systems. Modeling uncertainty, which variously represents lack of data, variability between individuals and between different measurements of a single individual, ambiguity in the proper model form, and others, is essential to explaining the limitations of our understanding and constraining the confidence of our predictions. Current methods for modeling uncertainty provide a rich mathematical means of analyzing simple forms of uncertainty in self-contained models. However, real biological systems of interest exhibit many forms of uncertainty simultaneously and may require the composition of multiple levels of models to create useful predictions. I develop and test new methods for characterizing and propagating uncertainty through multi-level models in order to better make clinically relevant predictions. These methods are applied to three systems. / First, a selenium chemoprevention clinical trial's patients were modeled at the cellular metabolic, mutation accumulation, and cancer detection levels. Metabolite, demographic, and epidemiological data were integrated to produce predictions of prostate cancer risk and putative trial outcomes. The value of information - from doing experiments to reduce uncertainty in a targeted manner - was evaluated on trial design. Second, a population pharmacokinetics/ pharmacodynamics model was created to guide preclinical studies of antibody-drug conjugates targeting breast cancer. An optimal experimental design method was created to efficiently reduce uncertainty in estimates of drug-related parameters of interest. The contributions of inter-individual variability and parameter uncertainty are specially handled by sampling and propagating ensembles of models. / Third, a two-level drug efficacy and cellular dynamics model was created to analyze the efficacy of targeted liposomal-doxorubicin in multiple nucleolin-overexpressing cell lines. A single model topology (but with selected species- and cell line-independent parameters) adequately described the measured behavior in all cell lines. These were then used predict drug uptake and cell killing as a function of surface receptor density. In each system, a modeling framework that integrates data from multiple sources and different forms of uncertainty is applied to make predictions, quantify gaps in knowledge (and help fill them), and guide decision making in controlling clinically important outcomes. / by Kevin Shi. / Ph. D. / Ph.D. Massachusetts Institute of Technology, Department of Biological Engineering

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2016. / 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. / Microbial ecology has benefited from the decreased cost and increased quality of next-generation DNA sequencing. In general, studies that use DNA sequencing are no longer limited by the sequencing itself but instead by the acquisition of the samples and by methods for analyzing and interpreting the resulting sequence data. In this thesis, I describe the results of three projects that address challenges to interpreting or acquiring sequence data. In the first project, I developed a method for analyzing the dynamics of the relative abundance of operational taxonomic units measured by next-generation amplicon sequencing in microbial ecology experiments without replication. In the second project, I and my co-author combined a taxonomic survey of a dimictic lake, an ecosystem-level biogeochemical model of microbial metabolisms in the lake, and the results of a single-cell genetic assay to infer the identity of taxonomically-diverse, putatively-syntrophic microbial consortia. In the third project, I and my co-author developed a model of differences in the efficacy that stool from different donors has when treating patients via fecal microbiota transplant. We use that model to compute statistical powers and to optimize clinical trial designs. Aside from contributing scientific conclusions about each system, these methods will also serve as a conceptual framework for future efforts to address challenges to the interpretation or acquisition of microbial ecology data. / by Scott Wilder Olesen. / Ph. D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biological Engineering, 2011. / Cataloged from PDF version of thesis. / Includes bibliographical references (p. 133-153). / Traumatic brain injury (TBI) is a significant cause of death and morbidity in both the civilian and military populations. The major causes of TBI, such as motor vehicle accidents, falls, sports concussions, and ballistic and explosive blast threats for military personnel, are well established and extensively characterized; however, there remains much to be learned about the specific mechanisms of damage leading to brain injury, especially at the cellular level. In order to understand how cells of the central nervous system (CNS) respond to mechanical insults and stimuli, a combined modeling/experimental approach was adopted. A computational framework was developed to accurately model how cells deform under various macroscopically imposed loading conditions. In addition, in vitro (cell culture) models were established to investigate damage responses to biologically relevant mechanical insults. In order to develop computational models of cell response to mechanical loading, it is essential to have accurate material properties for all cells of interest. In this work, the mechanical responses of neurons and astrocytes were quantified using atomic force microscopy (AFM) at three different loading rates and under relaxation to enable characterization of both the elastic and viscous components of the cell response. AFM data were used to calibrate an eight-parameter rheological model implemented in the framework of a commercial finite element package (Abaqus). Model parameters fit to the measured responses of neurons and astrocytes provide a quantitative measure of homogenized nonlinear viscoelastic properties for each cell type. In order to ensure that the measured responses could be considered representative of cell populations in their physiological environment, cells were also grown and tested on substrates of various stiffness, with the softest substrate mimicking the stiffness of brain tissue. Results of this study showed both the morphology and measured force response of astrocytes to be significantly affected by the stiffness of their substrate, with cells becoming increasingly rounded on soft substrates. Results of simulations suggested that changes in cell morphology were able to account for the observed changes in AFM force response, without significant changes to the cell material properties. In contrast, no significant changes in cell morphology were observed for neurons. These results highlight the importance of growing cells in a biologically relevant environment when studying mechanically mediated responses, such as TBI. To address this requirement, we developed two model systems with CNS cells grown in soft, 3D gels to investigate damage arising from dynamic compressive loading and from a shock pressure wave. These damage protocols, coupled with the single cell computational models, provide a new tool set for characterizing damage mechanisms in CNS cells and for studying TBI in highly controllable in vitro conditions. / by Kristin Briana Bernick. / Ph.D.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2017. / Page 322 blank. Cataloged from PDF version of thesis. / Includes bibliographical references (pages 295-321). / Living cells naturally use gene regulatory networks termed "genetic circuits" to exhibit complex behaviors such as signal processing, decision-making, and spatial organization. The ability to rationally engineer genetic circuits has applications in several biotechnology areas including therapeutics, agriculture, and materials. However, genetic circuit construction has traditionally been time- and labor-intensive; tuning regulator expression often requires manual trial-and-error, and the results frequently function incorrectly. To improve the reliability and pace of genetic circuit engineering, we have developed biomolecular and computational frameworks for designing genetic circuits. A scalable biomolecular platform is a prerequisite for genetic circuits design. In this thesis, we explore TetR-family repressors and the CRISPRi system as candidates. First, we applied 'part mining' to build a library of TetR-family repressors gleaned from prokaryotic genomes. A subset were used to build synthetic 'NOT gates' for use in genetic circuits. Second, we tested catalytically-inactive dCas9, which employs small guide RNAs (sgRNAs) to repress genetic loci via the programmability of RNA:DNA base pairing. To this end, we use dCas9 and synthetic sgRNAs to build transcriptional logic gates with high on-target repression and negligible cross-talk, and connected them to perform computation in living cells. We further demonstrate that a synthetic circuit can directly interface a native E. coli regulatory network. To accelerate the design of circuits that employ these biomolecular platforms, we created a software design tool called Cello, in which a user writes a high-level functional specification that is automatically compiled to a DNA sequence. Algorithms first construct a circuit diagram, then assign and connect genetic "gates", and simulate performance. Reliable circuit design requires the insulation of gates from genetic context, so that they function identically when used in different circuits. We used Cello to design the largest library of genetic circuits to date, where each DNA sequence was built as predicted by the software with no additional tuning. Across all circuits 92% of the output states functioned as predicted. Design automation simplifies the incorporation of genetic circuits into biotechnology projects that require decisionmaking, control, sensing, or spatial organization. / by Alec A.K. Nielsen. / Ph. D.

Thesis (S.M.)--Massachusetts Institute of Technology, Dept. of Biological Engineering, 2012. / Cataloged from PDF version of thesis. Page 104 blank. / Includes bibliographical references. / Tissue engineering can be applied to develop therapeutic techniques for osteoarthritis, a degenerative disease caused by the progressive deterioration of cartilage in joints. An inherent goal in developing cartilage-replacement treatments is ensuring that tissue-engineered constructs possess the same properties as native cartilage tissue. Biochemical assays and imaging techniques can be used to study some of the main components of cartilage and assess the value of potential therapies. Agarose and self-assembling peptides have been used to make hydrogels for in vitro culture of bovine bone marrow stromal cells (BMSCs) which can differentiate into chondrocytes, undergo chondrogenesis, and produce cartilage tissue. So far, differences in cell morphology that characterize chondrogenesis had been observed in peptide hydrogels like KLD and RAD but not in the 2.0% agarose hydrogels typically used for culture. A tissue engineering study was conducted to determine if a suitable environment for cell proliferation and differentiation could be obtained using different agarose compositions. BMSCs were cultured in 0.5%, 1.0%, and 2.0% agarose hydrogels for 21 days following TGF-p1 supplementation. Results indicate that the 0.5% agarose hydrogels are clearly inferior scaffolds when compared to the 1.0% and 2.0% agarose hydrogels, which are generally comparable. Since agarose gels appear to be suboptimal in promoting chondrogenesis, self-assembling peptides should be used in future studies. In addition to the biochemical assays traditionally used in cartilage tissue engineering studies, atomic force microscopy (AFM) can be used to image aggrecan, one of the main components of cartilage. Imaging studies were carried out using fetal bovine epiphyseal aggrecan to optimize previous extraction and sample preparation procedures, as well as an AFM imaging protocol, for samples containing aggrecan. Experiments were conducted with 10, 25, and 50 ptg/mL aggrecan solutions to find the minimum concentration needed to create aggrecan monolayers on APTES-mica that would yield acceptable AFM images (25 [mu]g/mL). AFM instrument and software parameters were optimized to find the working range of the integral and proportional gains (0.2 - 0.4 and 0.6 - 0.8, respectively) and to increase the resolution, showing fields at the 800 nm level. Finally, an image processing protocol relevant to these molecules was established. / by Nicole Casasnovas Ortega. / S.M.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biological Engineering, 2012. / Cataloged from PDF version of thesis. / Includes bibliographical references. / DNA damage contributes to cancer, aging, and heritable diseases. Ironically, DNA damaging agents are also commonly used in current cancer treatment. We therefore need robust, high throughput, and inexpensive tools for objective, quantitative DNA damage analysis. The single cell gel electrophoresis (comet) assay has become a standard method for DNA damage analysis, however, it is not well suited for use in clinical and epidemiological settings due to issues of low throughput, poor reproducibility, and a laborious image analysis requirement. To overcome these limitations, we applied microfabrication techniques to engineer an arrayed cell comet platform that maximizes the number of analyzable cells and provides spatial encoding for automated imaging and analysis. Additionally, we developed complementary software that eliminates the inherent bias of manual analysis by automatically selecting comets from the defined array. In its 96-well format, the so-called CometChip integrates with high throughput screening technologies, further increasing throughput and removing user error. This improved approach enables multiple cell types, chemical conditions, and repair time points to be assayed in a single gel with improved reproducibility and processing speed, while maintaining the simple protocol and versatility of the comet assay to assess a wide range of DNA damage. Using the CometChip, we evaluated a variety of DNA damaging agents, revealing repair profiles that can be used to gain insight into biological mechanisms of damage sensitivities. We confirmed the ability of the CometChip to identify deficiencies in four major DNA repair pathways, supporting the use of the assay in determining pathway sensitivities that may be useful in guiding treatment strategies that more selectively target cancerous cells and reduce side-effects. We also used the platform to evaluate potential inhibitors of DNA repair, which are emerging as promising adjuvants in cancer management. Taken together, the CometChip enables high throughput genotoxic evaluation of chemical exposures, discovery of novel chemotherapeutic strategies, and measurement of DNA repair kinetics for identification of susceptible populations and disease prevention. The CometChip is a significant advancement in DNA damage and repair technology, providing high throughput, objective, and quantitative measurements that have the potential to become a new standard in DNA damage analysis. / by David McGregor Weingeist. / Ph.D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biological Engineering, February 2013 / Cataloged from PDF version of thesis. / Includes bibliographical references (p. 116-129). / The emergent field of synthetic biology is different from many other biological engineering efforts, in that its roots, design principles, and forward engineering perspective have been adopted from electrical engineering and computer science. Synthetic biology is uniquely poised to make great contributions to numerous fields such as bio-fuel, energy production, agriculture and eco-remediation, national defense, and biomedical and tissue engineering. Considerable progress has been made in engineering novel genetic circuits in many different organisms. However, not much progress has been made toward developing a formal methodology to engineer complex genetic systems in mammalian cells. One of the most promising areas of research is the study of embryonic and adult stem cells. Synthetic biology has the potential to greatly impact the progression and development of research in this area of study. A critical impediment to the development of stem cell engineering is the innate complexity, little to no characterization of parts, and limited compositional predictive capabilities. In this thesis, I discuss the strategies used for constructing and optimizing the performance of signaling pathways, the development of a large mammalian genetic part and circuit library, and the characterization and implementation of novel genetic parts and components aimed at developing a foundation for mammalian synthetic biology. I have designed and tested several orthogonal strategies aimed at cell-cell communication in mammalian cells. I have designed a characterization framework for the complete and proper characterization of genetic parts that allows for modular predictive composition of genetic circuits. With this characterization framework I have generated a small library of characterized parts and composite circuits that have well defined input-output relationships that can be used in novel genetic architectures. I also aided in the development of novel analysis and computational tools necessary for accurate predictive composition of these novel circuits. This work collectively provides a foundation for engineering complex intracellular transcriptional networks and intercellular signaling systems in mammalian cells. / by Noah Davidsohn. / Ph.D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biological Engineering, 2011. / 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. / Gastric cancer is the 2nd leading cause of cancer death worldwide and the 4th most commonly diagnosed cancer worldwide. Helicobacter pylori infection is the major risk factor of gastric cancer, and as such, this bacterium has been classified as a type 1, or definite, carcinogen by the International Agency for Research on Cancer. H. pylori infects the gastric mucosa of more than half of the world's population and promotes gastric carcinogenesis by inducing chronic inflammation. Over decades of persistent H. pylori infection and chronic inflammation, the stomach goes through a well characterized pathological progression involving chronic gastritis, atrophy, intestinal metaplasia, dysplasia, and ultimately cancer. Interestingly, there are strong gender differences in the development of gastric cancer, as men are twice as likely to develop the disease than women. Given the importance of H. pylori and chronic inflammation in gastric carcinogenesis, this thesis investigated the role of gender in modulating host immune responses to H. pylori. The aims of this thesis explored 1) the effect of gender on H. pylori's ability to induce mutations and 2) the effect of estrogen and the anti-estrogen, Tamoxifen, on H. pyloriinduced gastric cancer. For the first aim, the gpt delta mouse model, a murine mutational analysis model, was used to study chronic infection with H. pylori. Increased frequency of point mutations was observed in infected female mice at 12 months post infection. These mutations were not observed in infected male mice. Further analysis revealed that H. pylori induced a greater immune response in female mice in this model, as measured by increased severity of gastric lesions, decreased bacterial counts and the higher levels of Th1 antibodies for H. pylori. The spectra of mutations pointed towards oxidative damage as the underlying cause of induction. This study revealed that gender differences in mutagenesis were mediated by the severity and duration of the immune response. In the second aim, 17[beta]-estradiol prevented the formation of gastric cancer in the INSGAS mouse model, which develops gastric cancer in a male-predominant manner. Unexpectedly, this study led to the discovery that Tamoxifen may act as an agonist in this model of gastric cancer, as it was able to prevent gastric cancer using mechanisms similar to 17[beta]- estradiol. Both compounds downregulated pathways associated with cellular movement and cancer. CXCL1, a murine homolog of IL-8, was downregulated by treatment at both local and systemic levels, which led to a decreased neutrophilic infiltrate. 17[beta]-estradiol and Tamoxifen mediated the disruption of a positive feedback loop coupling CXCL1 secretion with neutrophil recruitment, which dampened the activation of proinflammatory and oncogenic pathways, leading to protection against gastric cancer. In conclusion, these studies provide further insight into the role of gender modulation of host immune response in H. pylori-induced mutagenesis and carcinogenesis. / by Alexander Sheh. / Ph.D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biological Engineering, 2011. / Cataloged from PDF version of thesis. / Includes bibliographical references (p. 191-207). / Articular cartilage is avascular, and defects have limited capacity for spontaneous healing. Angiogenesis may interfere with maturation of naturally avascular tissues. Our rationale is that the use of endostatin, a potent angiogenesis inhibitor, will facilitate the formation of hyaline cartilage during regeneration. The objective of this thesis was to develop a system with a novel approach for treating cartilage defects, namely endostatin-producing cartilaginous constructs. The constructs were engineered using nonviral gene therapy, through evaluation of select variables, including regulators (culture media, endostatin plasmid load, method of pEndo lipoplex incorporation, and oxygen tension), scaffold formulation, and cell type. We also investigated select aspects of the in vivo cartilage defect model in which the construct can be implanted, including the post-surgical rehabilitation protocol and the use of osteogenic protein (OP)- 1. The principal achievement was the engineering of endostatin-expressing cartilaginous constructs in vitro using chondrocytes and mesenchymal stem cells, collagen sponge-like scaffolds and hydrogels, and chondrogenic medium. Peaks in endostatin protein were observed during the first few days of culture, followed by decreases. The endostatin levels were comparable to therapeutic levels in vitro and physiological levels in vivo. Most of the endostatin protein was released into the expended medium; little retention was observed, including in scaffolds supplemented with heparan sulfate, chondroitin sulfate, and heparin. In vivo work examining chondral defects in the goat knee demonstrated that long-term post-operative immobilization, even with periodic passive motion exercise, resulted in significant joint degeneration. Cell-seeded scaffolds were observed in the defect 2 months following implantation and short-term immobilization, and yielded results at least as good as historical data obtained using other treatment techniques, including autologous chondrocyte implantation and microfracture, suggesting that a cell-seeded scaffold is a viable option for cartilage repair. There was no significant benefit of multiple treatments of OP-I on chondral defects. Neovascularization was observed in the largely fibrous reparative tissue filling the chondral defects, providing further rationale for the use of endostatin. A notable finding was the observation of laminin and type IV collagen, 2 common basement membrane molecules, in both in vitro engineered cartilaginous constructs and in vivo cartilage repair samples. / by Lily Jeng. / Ph.D.