Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2017. / Cataloged from PDF version of thesis. / Includes bibliographical references. / The DNA in every cell accrues nearly 100,000 lesions daily from both endogenous and exogenous sources. The accumulated damage, e.g. strand breaks and base lesions, can lead to mutations, cell death, and cancer if not repaired efficiently. To protect genome integrity, organisms have evolved multiple DNA repair processes. A deeper comprehension of DNA damage and repair during disease pathogenesis can aid the development of novel therapeutics to reduce the damage and ameliorate the disease. Here, we studied DNA damage and repair in two inflammatory contexts. First, we investigated the role of DNA damage and repair during influenza infection, a common viral respiratory disease with an active inflammatory response. Second, we examined the effects of S-nitrosation, a post-translational modification that is common in inflammatory regions, on repair of alkylation damage. Influenza induces an excessive inflammatory response in the host and a reduction in inflammation reduces morbidity. While inflammation can cause DNA damage and induce DNA repair in other inflammatory contexts, there has been minimal analysis on the existence and function of DNA damage and repair during influenza infection. Utilizing immuno-fluorescent analysis of double strand break markers, we observed an increase in strand breaks both in vitro and in vivo. Influenza infected mice also displayed a significant increase in homologous recombination (HR) gene and protein expression during the recovery phase of infection in multiple virus and mouse backgrounds. Moreover, influenza infected mice deficient in DNA repair proteins AAG, ALKBH2, and ALKBH3, displayed increased morbidity and HR protein expression when compared to wild type. Together, these results raise the possibility of a role for DNA repair and more specifically HR during influenza infection. To study the effects of inflammation on DNA repair protein function, we analyzed the capacity of cells treated with S-nitrosoglutathione (GSNO), a nitrosating agent, to repair alkylation damage. GSNO-exposed cells displayed dysregulation in the activities base excision repair (BER) proteins. Following challenge with an alkylating agent, GSNO-exposed cells had an increase in repair intermediates and reduced viability, suggesting that GSNO exposure inhibits BER completion. The knowledge gained from these studies lays the groundwork for new prevention strategies and novel therapeutics. / by Marcus Curtis Parrish. / Ph. D.

Thesis: M. Eng., Massachusetts Institute of Technology, Department of Biological Engineering, 2017. / Cataloged from PDF version of thesis. / Includes bibliographical references (pages. 49-52). / Post-traumatic osteoarthritis (PTOA) is a subtype of OA associated with cartilage defects caused by traumatic joint injury. Because articular cartilage has a limited innate healing response, due to its avascular, aneural, and alymphatic nature, these defects lead to chronic degenerative joint disease if left untreated. Current treatments to repair articular cartilage generally result in fibrocartilage that is mechanically and biochemically inferior to native hyaline tissue. This has motivated the development of tissue engineering strategies for cartilage defect repair. Hydrogel approaches have shown promising results in their ability to induce chondrogenesis, proliferation, and cartilage-like matrix production, but are often very soft at early time points and at risk of damage from joint articulation. Solid scaffolds solve this mechanical problem, but often sacrifice bioactivity and integration with native tissue. In order to avoid the drawbacks of each of these approaches, we proposed a composite scaffold approach using a synthetic solid scaffold, made of bioabsorbable polyglycolic acid:trimethylene carbonate (PGA:TMC) or expanded polytetrafluoroethylene (ePTFE), loaded with a chondrocyte-seeded self-assembling peptide hydrogel, [KLDL]₃. We hypothesized that these composite scaffolds would benefit from the mechanical protection of the solid scaffolds as well as the pro-chondrogenic and proliferative effects of the KLD hydrogel, allowing chondrocytes to produce cartilage-like extracellular matrix in a protected mechanical environment. To test the potential of these composite scaffolds for use in cartilage repair, we measured cell distribution, viability, matrix production and accumulation, and static and dynamic mechanical properties. We found that cells could be evenly distributed through at least one of the solid scaffolds tested, with all showing proliferation and maintenance of viability over four-week culture. Per-cell matrix production was an order of magnitude higher than in KLD hydrogels alone. Mechanical properties of composite scaffolds appeared to be dominated by the solid scaffolds, showing that they offered mechanical protection to the soft hydrogel within. Use in a cartilage defect model showed potential for integration with native tissue given optimization of gel-casting methods. Overall, our results show that these composite scaffolds are a viable tissue engineering strategy for articular cartilage repair. / "Funded by the National Science Foundation and W.L.Gore & Associates, Inc."--Page 3. / by Jennifer R. Ibañez. / M. Eng.

Thesis (M. Eng.)--Massachusetts Institute of Technology, Dept. of Biological Engineering, 2009. / Cataloged from PDF version of thesis. / Includes bibliographical references (p. 60-64). / Oceanic cyanobacteria are amongst the most populous species on the planet and have been found in every ocean around the world. These photosynthetic organisms play a major role in the global carbon cycle. They have adapted to a number of different temperature, light, and nutrient niches. However, as important primary producers in the oceans, these organisms play a vital role which may be threatened by global climate change and pollution. As research on cyanobacterial species progresses, these organisms have been found to show promise as potential sources of biofuel, renewable energy, and agents for bioremediation. In order to utilize these organisms for future engineering applications and basic scientific research, it is important to be able to grow the organism in a stable and reproducible manner. This research characterizes the growth of Synechococcus WH8109 in the laboratory. In the laboratory, cell culture densities of greater than 109 cells/mL with a doubling time of approximately 24 hours were achieved when grown at 28'C with a 24 hour light cycle in sea water and artificial salt water media. Not only did cyanobacteria evolve long before their distant enteric cousins, but they harness nearly all of their energy through photosynthesis. The photosystem is constantly subjected to photo-oxidative damage and degradation. Interesting insight may be gained by studying this complex repair process in the bacterial counterpart to plants, prior to applying these concepts to higher order plant species. Chaperones have been implicated in this repair process. In order to better characterize the stress response of WH8109, I have also isolated the Synechococcus homologue of GroEL using anion exchange and gel filtration chromatography and sucrose gradient centrifugation. The expression levels of this chaperone were analyzed under normal and stress conditions and they have been shown to respond to heat shock and infection. / by Erika M. Erickson. / M.Eng.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2014. / Cataloged from PDF version of thesis. Vita. / Includes bibliographical references (pages 132-153). / The Plcsmodium liver stage is an attractive target for the development of antimalarial drugs and vaccines, as it provides an opportunity to interrupt the life cycle of the parasite at a critical early stage. However, targeting the liver stage has been difficult due to a lack of human liver models that robustly recapitulate host-pathogen interactions in a physiologically relevant cell type. Through the application of hepatic tissue engineering concepts and techniques, this thesis sought to develop advanced models of liver-stage malaria that will allow the facile interrogation of potential antimalarial drugs in primary human hepatocytes. In the first part of this work, we established liver-stage Plasmodium infection in an engineered microscale human liver platform based on micropatterned cocultures of primary human hepatocytes and supportive stromal cells, enabling medium-throughput phenotypic screens for potential antimalarial drugs in a more authentic host cell, and demonstrated the utility of this model for malaria vaccine testing. We further hypothesized and showed that recapitulation of a more physiologically relevant oxygen tension that is experienced by hepatocytes in vivo improved infection rates and parasite growth in vitro. Next, we demonstrated the feasibility of establishing liver-stage malaria infections in human induced pluripotent stem cell-derived hepatocyte-like cells (iHLCs), thus enabling the study of host genetic variation on liver-stage malaria infection and antimalarial drug responses. We also applied recently discovered small molecules to induce further hepatic maturation, thus increasing the utility of using iHLCs for antimalarial drug development. Finally, we designed and provided a proof-of-concept for a humanized mouse model of liver-stage malaria that involves the fabrication and ectopic implantation of PEG-cryogel-based engineered human artificial livers, and can be generated in a facile, rapid and scalable fashion for future preclinical antimalarial drug testing in vivo. The results of this research represent a three-pronged approach towards engineering scalable human liver models that recapitulate liver-stage malaria infection which may ultimately facilitate antimalarial drug discovery at various stages of the drug development pipeline. / by Shengyong Ng. / Ph. D.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2016. / Cataloged from PDF version of thesis. / Includes bibliographical references (pages 198-215). / Focal cartilage defects caused by joint injury have a limited capacity to self-repair, and if left untreated, can lead to the early onset of osteoarthritis. The current gold standard of care, microfracture surgery, induces an endogenous repair response, but typically results in poorly integrated fibrocartilage, rather than native hyaline cartilage. The objective of this thesis was to test the hypothesis that a self-assembling peptide hydrogel functionalized with chemotactic and pro-anabolic growth factors and placed into the defect during surgery could induce migration of endogenous progenitor cells into the repair tissue. Since these progenitors are naturally accessed during microfracture surgery, clinical translation of this approach could ultimately steer repair to a more hyaline-like response. Poor cartilage repair is believed to be the result of an insufficient number of progenitor cells at the defect site. We hypothesized that the addition of a single dose combination of chemotactic growth factors (such as platelet derived growth factor-BB (PDGF-BB), transforming growth factor-P 1 (TGF-[beta]1), and heparin-binding IGF-l (HB-IGF-1)) premixed into a hydrogel scaffold could stimulate bone-marrow progenitor cell migration into the hydrogel. A novel 3D gel-to-gel migration assay, using (KLDL)₃ self-assembling peptide gels, demonstrated that the combination of PDGF-BB and TGF-[beta]1 induced significant migration into the gel compared to growth-factor free controls. Importantly, these growth factors were retained in the hydrogel and exhibited a slow release over 1-2 weeks. We also hypothesized that a brief enzymatic pre-treatment of the defect site could release proteoglycans from the walls of the surrounding native cartilage in a controlled manner, and thereby create space for newly synthesized repair tissue to anchor and integrate with this adjacent host cartilage. We used an in vitro model in which a cylindrical annulus of native cartilage was pre-treated with trypsin over a 2-minute period and then filled with a chondrocyte-seeded (KLDL) ³ hydrogel ftnctionalized with pro-anabolic HB-IGF-I that had been premixed into the gel. (This procedure was deemed to be clinically tractable by collaborating equine surgeons now using this approach in parallel animal studies.) Trypsin pre-treatment depleted proteoglycan content of adjacent cartilage in a controlled manner, and HB-IGF-l was found to be delivered to the surrounding cartilage from the peptide gel. HB-IGF-I was found to stimulate matrix biosynthesis both in the surrounding cartilage and the chondrocyte-seeded KLD scaffold, and to enhance mechanical integration. We further explored the uptake and diffusive transport properties of HB-IGF-l into cartilage motivated by the need to understand the pharmacokinetics of delivery from the repair construct to surrounding cartilage. The positively charged heparin-binding domain of HB-IGF-l results in high uptake into cartilage, making it an effective method of delivering the pro-anabolic attributes of IGF-1, which in its native form would be rapidly cleared from the joint. The observed high and rapid uptake of HB-IGF-l into cartilage will enable characterization of dosing for HB-IGF-l delivery to cartilage by either intra-articular injection or from implanted hydrogel scaffolds. In summary our results show that of (KLDL)₃ peptide hydrogel scaffolds can foster growth-factor induced progenitor cell migration to increase progenitor cell recruitment into a cartilage defect. Thus, the use of a peptide gel premixed with PDGF-BB and HB-IGF-l to enhance progenitor migration into the scaffold, combined with a trypsin pre-treatment to help promote subsequent integration, is a promising strategy towards improving integrative repair. The combination of these approaches is currently being tested in an in vivo rabbit model. / by Paul Hancock Liebesny. / Ph. D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biological Engineering, 2011. / Cataloged from PDF version of thesis. / Includes bibliographical references. / Recent evidence demonstrates a correlative relationship between metabolic disorders and cancer prevalence. In addition, cholesterol lowering statins and the antidiabetes medication metformin both act as chemopreventive agents in prostate and other cancers. The natural compound resveratrol has similar properties: increasing insulin sensitivity, suppressing adipogenesis, and killing cancer cell lines in vitro. However, in vivo tumor xenografts acquire resistance to resveratrol by an unknown mechanism, while mouse models of metabolic disorders still respond to the compound. Given the metabolic implications of these data and the role of metabolism in prostate cancer incidence, we evaluated resveratrol in an in vitro disease progression model of prostate cancer and found that castration-resistant human prostate cancer C4-2 cells are more sensitive to resveratrol-induced apoptosis than isogenic androgen-dependent LNCaP cells. Inhibiting downstream pro-survival signaling with the MEK inhibitor U0126 rescued the C4-2 cells from resveratrol-induced death, however other MEK inhibitors did not recapitulate this response. In fact, U0126 acted independently of MEK, inhibiting mitochondrial function and shifting cells to aerobic glycolysis. Mitochondrial activity of U0126 arose through decomposition, producing both mitochondrial fluorescence and cyanide, a known inhibitor of complex IV. Applying U0126 mitochondrial inhibition to C4-2 cell apoptosis, we investigated the role of mitochondrial metabolism and focused on how glutamine supplementation of citric acid cycle intermediate a-ketoglutarate may be involved. Suppression of the conversion of glutamate to a-ketoglutarate with the transaminase inhibitors cycloserine and amino oxyacetate rescued C4-2 cells from resveratrol-induced death. In addition, reducing extracellular glutamine concentration in the culture medium also inhibited apoptosis. These results imply resveratrolinduced death is dependent on glutamine metabolism, a pathway dysregulated in a variety of cancers linked to oncogenic signaling. Further work on resveratrol and metabolism in cancer is warranted to ascertain if the glutamine dependence has clinical implications. / by Amy Marie Nichols. / Ph.D.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2018. / Cataloged from PDF version of thesis. / Includes bibliographical references. / Survival rates for ovarian cancer haven't meaningfully improved in thirty years. Ovarian cancer is particularly difficult to treat because it is usually discovered after it has metastasized and it quickly develops resistance to the few drugs that are initially effective at controlling it. Nanomedicine has the potential to change the paradigm for ovarian cancer treatment by delivering complex combinations of conventional drugs plus next-generation therapies like small interfering RNA (siRNA) and immunotherapy. However, nanoparticles must be tailored to the particular drug-delivery challenges and opportunities posed by ovarian cancer. In this thesis, we designed layer-by-layer (LbL) nanoparticles (NPs) to target ovarian cancer using library-based approaches. Using this approach, we identified promising formulations for developing an advanced nanotheranostic that both treats and detects ovarian cancer. In order to develop LbL NPs for treating ovarian cancer, we identified and overcame process engineering and fundamental materials challenges, thereby improving synthesis robustness, throughput and scale. Chapter 2 describes how modern tangential flow filtration significantly improves throughput and scalability in colloidal LbL assembly. Chapter 3 implements this improved synthetic approach to generate a small library of LbL NPs that screen for tumor-targeting properties on ovarian cancer cells, both in vitro and in vivo. Our results demonstrate that ovarian cancer cells have a high affinity to carboxylated LbL NPs, and we report several tumor-targeting formulations with distinct subcellular trafficking patterns. Chapter 4 explores the role of salt in LbL colloidal assembly, and we develop strategies for robustly synthesizing LbL-modified liposomes with high loading of siRNA. Chapter 5 advances a promising formulation identified by our surface chemistry screen, which we developed into an advanced nanotheranostic device that delivers siRNA and mediates urinary-based tumor detection. Future work that continues to improve the synthesis of LbL NPs will be essential to generate larger and more ambitious LbL NP libraries. In turn, these libraries will facilitate systematic studies that further tailor the LbL platform to specific diseases and biomedical applications. / "This material is partly based upon work supported by the National Science Foundation Graduate Research Fellowship under Grant No. 1122374. This material is partly based upon work supported by the National Science Foundation Graduate Research Fellowship under Grant No. 1122374"--Page 187. / by Santiago Correa. / Ph. D.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2014. / 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. / Mass spectrometry has become the tool of choice for proteomics. Its unrivaled coverage and reproducibility has positioned it head and shoulders above competing techniques for analyzing protein expression post-translational modification. With the increased popularity comes a flood of new research applications, each with its own biological motivations and goals. To ensure that mass spectrometry-based proteomics can be useful to as many biological questions as possible, it is of utmost importance to ensure high data quality. This research focuses on two general stages of the typical proteomics workflow and introduces tools to facilitate effective target screening, follow-up analysis, as well as more precise measurements. This new pipeline is then demonstrated in a case study of Epidermal Growth Factor Receptor (EGFR) signaling and phenotype prediction. The quantity of proteomic mass spectrometry data available from a single analysis has increased exponentially as new generations of instruments become quicker and more sensitive. This deluge of data leaves many tempted to forego time-intensive manual validation of database identified targets in favor of global data set quality statistics. Particularly in the realm of post-translational modifications, long lists of putative matches are often reported with little or no scan-specific validation. Such practices no longer provide assurance that any single identified target is indeed correct, leaving researchers vulnerable to expending vast resources chasing false positives. The argument is that manual validation is too time-intensive to be carried out for each and every identification. To remedy this problem we have introduced the Computer Assisted Manual Validation (CAMV) software package to expedite the procedure by preprocessing the database results so as to remove the tedious steps associated with the validation task and only recruit human judgment for the final quality decision. This approach has drastically decreased the time required for manual validation; a task that used to take weeks now is completed in hours. Another focus of this research is the development of a multiplex, multisite absolute quantification method, which has improved the quality of quantitative proteomic mass spectrometry data. Absolute site-specific data allows many more biological hypotheses to be directly tested with a single mass spectrometry experiment, including phosphorylation stoichiometry. This technique has been applied to the EGFR system to better understand signaling downstream of three distinct ligands. These ligands all bind the same receptor yet elicit different phenotypes, suggesting differential information processing. The analysis showed unique patterns of receptor phosphorylation present following sub-saturating ligand treatment. However, at saturating doses the same pattern of phosphorylation is produced regardless of ligand, but the magnitude of that pattern is still ligand-dependent. In this regime, the adaptor proteins were still able to retain ligand-specific phosphorylation patterns presumably responsible for differential phenotypes. The data set also permitted the identification of signals important for the regulation of only one of the two phenotypes examined. / by Timothy Gordon Curran. / Ph. D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biological Engineering, 2012. / Cataloged from PDF version of thesis. Page 175 blank. / Includes bibliographical references (p. 161-174). / Immunostimulatory therapies that activate immune response pathways are of great interest for overcoming the immunosuppression present in advanced tumors. Agonistic antibodies against the co-stimulatory receptors CD40 and CD137, Toll-Like Receptor (TLR) ligands such as CpG oligonucleotides, and immunostimulatory cytokines such as IL-2 have all previously demonstrated potent, synergistic anti-tumor effects. However, the clinical use of such therapies is significantly hampered by the severe, dose-limiting inflammatory toxicities provoked upon systemic exposure. We hypothesized that by anchoring immunomodulatory agents to lipid nanoparticles we could retain the bio-activity of therapeutics in the local tumor tissue and tumordraining lymph node, but limit systemic exposure to these potent molecules. We first prepared liposomes bearing surface-conjugated anti-CD40 and CpG and assessed their therapeutic efficacy and systemic toxicity compared to soluble versions of the same immuno-agonists, injected intratumorally in established solid tumors in mice. Anti-CD40/CpG-coupled liposomes significantly inhibited primary tumor growth and induced a survival benefit similar to locally injected soluble anti-CD40+CpG. Biodistribution analyses following local delivery showed that the liposomal carriers successfully sequestered anti-CD40 and CpG in vivo, reducing leakage into systemic circulation while allowing draining to the tumor-proximal lymph node. Contrary to locally administered soluble immunotherapy, anti-CD40/CpG liposomes did not elicit significant increases in serum levels of ALT enzyme, systemic inflammatory cytokines, or overall weight loss, confirming that off-target inflammatory effects had been minimized. Thus, these results confirmed the development of a delivery strategy capable of inducing robust antitumor responses concurrent with minimal systemic side effects. We next assessed the dissemination of the tumor-specific immune response that had been primed by locally administered, liposome-conjugated therapy. Since anti-CD40/CpG-coupled liposomes were unable to consistently induce the rejection of a secondary distal tumor challenge, we adapted the strategy of liposome-coupled delivery for the administration of anti-CD 137 and IL-2, two potent T cell-stimulatory agents. Local intra-tumoral therapy using anti-CD137-liposomes + IL-2-liposomes induced the highly potent inhibition of primary treated tumors and achieved a majority of complete cures, while successfully minimizing systemic exposure and eliminating symptoms of inflammatory toxicity, including lethality. In addition, 100% of anti-CD 137 + IL-2 liposome-treated mice were protected against a secondary distal tumor challenge, and demonstrated a significant delay in the progression of simultaneously inoculated, distal untreated tumors. Subsequent analyses confirmed that anti-CD137-liposomes and IL-2-liposomes bound specifically to cytotoxic T cells (CTLs) within the treated tumor, and that the depletion of CTLs abrogated the therapeutic anti-tumor response. Overall, these results indicated the effective local priming of an adaptive tumor-specific response, capable of mediating local, systemic, and memory anti-tumor immunity. The versatility of this liposome conjugation strategy suggests that we have developed a generalizable tool enabling the local delivery of highly potent immunomodulatory agonists in the absence of systemic toxicity, which could substantially improve the clinical applicability of such agents in cancer therapy. / by Brandon Kwong. / Ph.D.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2016. / Cataloged from PDF version of thesis. / Includes bibliographical references. / The integrity of our DNA is challenged by roughly 100,000 lesions per cell on a daily basis. Failure to cope with DNA damage can lead to cancer, immunodeficiency and degenerative disease. Quantitating and understanding an individual's DNA repair capacity may enable us to predict and prevent disease in a personalized manner. Base Excision Repair (BER) is known for the recognition and repair of endogenous and exogenous mutagenic non-helix-distorting lesions produced by DNA base alkylation, deamination and oxidation. BER is initiated by the action of one of eleven DNA glycosylases known-to-date. Many studies have shown that levels of these glycosylases can vary between individuals, suggesting a basis for inter-individual differences in DNA repair capacity. Moreover, the methods for measuring DNA repair capacity used so far are cumbersome, time consuming, low throughput and only allow for the analysis of one glycosylase at a time. We have taken a fluorescence-based multiplex flow-cytometric host cell reactivation assay wherein the activity of several DNA glycosylases and their immediate downstream endonuclease (APE1) can be tested simultaneously, at single-cell resolution, under physiological conditions. Taking advantage of the transcriptional properties of several DNA lesions we have designed and engineered specific fluorescent reporter plasmids for OGG1, AAG, MUTYH, UNG and APE1. Inter-individual differences in DNA repair capacity of a panel of cell lines derived from healthy individuals have been measured. Regression models that incorporate these measurements have been developed in order to predict cellular sensitivity to the chemotherapeutic and DNA damaging agents 5-FU, H₂O₂ and MMS, with the interest of understanding the contributions that these differences can have on personalized disease prevention and treatment. Finally, we have conducted a pilot population study with 56 healthy subjects where we implemented all the methods developed in order to determine the feasibility of measuring DNA repair capacity variations in a healthy human population. Additionally, we report the discovery of a novel in vivo role of the TC-NER pathway in the repair of the lipid-peroxidation product, 3,N⁴-etheno-cytosine. / by Isaac Alexander Chaim. / Ph. D.