Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, February 2017. / Cataloged from PDF version of thesis. / Includes bibliographical references. / Post traumatic osteoarthritis (PTOA) refers to the progressive degradation of cartilage often triggered by a traumatic joint injury, such as a tear of the meniscus or anterior cruciate ligament (ACL). Such impact injuries lead to elevated levels of inflammatory cytokines in the synovial fluid of the joint, including IL-1, IL-6, and TNFa. In turn, these cytokines cause decreased matrix synthesis by chondrocytes and contribute to reprogramming of chondrocytes and synovial cells to increase release of matrix proteases. PTOA accounts for 12% of the OA population and typically affects younger individuals. The first part of this thesis focuses on developing a combination therapeutic which can address multiple aspects of cartilage degradation associated with the pathogenic responses to joint injury. We studied the combined use of insulin-like growth factor 1 (IGF-1) and dexamethasone (Dex) to block multiple degradative effects of cytokine challenge to articular cartilage. We found that in young bovine cartilage, the combination of IGF- 1 and Dex significantly inhibited the loss of sulfated glycosaminoglycans (sGAG) and collagen induced by IL-I. rescued the suppressed matrix biosynthesis, and inhibited the loss of chondrocyte viability caused by iL- 1 treatment. In adult human cartilage, only IGF- 1 rescued matrix biosynthesis and only Dex inhibited sGAG loss and improved cell viability. Thus, the combination of IGF-1+Dex together showed combined beneficial effects in human cartilage. Our findings suggest that the combination of IGF-I and Dex has greater beneficial effects than either molecule alone in preventing cytokine-mediated cartilage degradation in adult human and young bovine cartilage. In the second part of this thesis, a global phosphoproteomics approach was employed to determine the pathways that are activated upon cytokine challenge of adult human chondrocytes. We identified key regulatory kinases, p38, JNKI/2, ERKI/2, ERK5, JAK2, and STAT3 that were upregulated in phosphorylation as a result of inflammatory cytokine treatment. In addition, we identified 417 phosphopeptides with MAPK substrate motif that were more than 4 times upregulated in response to cytokine treatment. Using inhibitors against the key kinases, it was shown that P38, JNK1/2, ERK5 played important roles in cytokine induced cell death in bovine and human cartilage, while inhibition of JNK1/2 and ERK5 had the anti-catabolic effect of reducing GAG loss from cartilage matrix. In addition, JNK inhibition sensitized chondrocytes to IGF-1 stimulation in young bovine cartilage. These result indicate that kinase activity plays an essential role in cytokine induced cartilage catabolism and that kinase inhibitors have therapeutic potential in preventing cartilage degeneration. The third and final part of this work examined the release of matrix molecules upon mechanical injurious compression and/or cytokine treatment in long term culture to identify potential biomarkers of cartilage degeneration. A quantitative mass spectrometry approach was used to characterize the kinetics of aggrecan and collagen degradation. Although mechanical injury alone does not lead to a substantial increase in matrix degradation, mechanical injury can accelerate cytokine-induced matrix degradation and release. Additionally, we found that a collagen type III neo-epitope could be a potential biomarker for cartilage degradation. A neoepitope of cartilage oligomeric matrix protein (COMP), which was identified in the synovial fluid of acute injury patients, was also found in our ex vivo explant injury model. This makes our model physiologically relevant and it can be a valuable system for determining the effects of potential drug treatment on matrix degradation. / by Yang Wang. / Ph. D.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2017. / Cataloged from PDF version of thesis. / Includes bibliographical references. / The human tRNA methyltransferase 9-like (TRM9L) gene is a homolog of yeast Trm9 and human ALKBH8 and it has an important function in suppressing tumor growth in colorectal cancer. Loss of heterozygosity events on the Chromosome 8p22 loci, where TRM9L is located, are overrepresented in a wide variety of cancers, including prostate cancer, breast carcinoma, and hepatocellular carcinoma; downregulation of TRM9L expression is also observed in many different types of cancer. These findings implicate a general role potentially played by TRM9L in tumor suppression. A mechanistic understanding of TRM9L would have a broad impact in oncology. The broad objective of my thesis research is to connect mechanistically the biochemical function of TRM9L to its tumor suppressing activity. Of my special interest is TRM9L is regulated at the protein level. In this thesis, I demonstrated that TRM9L is a phosphoprotein, with phosphorylation dynamics at serines S214, S255, S279, S291, S306, and S380 correlated with protein-protein interactions and tumorigenicity. Phosphorylation levels were found to be modulated by stressors to which cells become resistant when TRM9L is silenced. For example, oxidative stress caused by hydrogen peroxide (H202) exposure increased phosphorylation on S255, S291, and S380, but phosphorylation was unchanged in response to ionizing radiation. Using chemical genetics approaches, I showed that phosphorylation of S380 is downstream of ribosomal protein S6 kinase (RSK), which is downstream of H202-activated extracellular signalregulated kinase (ERK). TRM9L mutations S214A, S255A and S380A significantly enhanced tumor growth, while S214A and S255A mutations also abolished a direct interaction between TRM9L and certain 14-3-3 isoforms. The results revealed a novel oxidative stress phosphosignaling regulatory mechanism underlying TRM9L's tumor suppressor behavior. I also demonstrated that TRM9L altered the ability of colorectal cancer cells to respond to stresses caused by reactive oxygen and nitrogen species. Results supported the idea that TRM9L reduces the cell's capacity to detoxify harmful reactive oxygen and nitrogen species and effectively makes them more toxic. Finally, my finding supported the notion that TRM9L expression downregulates the hypoxia-induced cell migration, presumably by controlling an aspect of epithelialmesenchymal transition (EMT). / by Chen Gu. / Ph. D.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, February 2016. / Cataloged from PDF version of thesis. "November 2015." / Includes bibliographical references (pages 95-103). / In adoptive cell therapy (ACT), autologous tumor-specific T-cells isolated from cancer patients or genetically engineered lymphocytes are activated and expanded ex vivo, then infused back into the individual to eliminate metastatic tumors. A major limitation of this promising approach is the loss of ACT T-cell effector functions in vivo due to the highly immunosuppressive environment in solid tumors. Protection of T-cells from immunosuppressive signals can be achieved by systemic administration of supporting adjuvant drugs such as interleukins, chemotherapy, and other immunomodulators, but these adjuvant treatments are often accompanied by serious toxicities and may still fail to optimally stimulate lymphocytes in all tumor and lymphoid compartments. Here we propose a two-pronged approach to address this problem, namely 1) repeatedly reloading supporting drugs to T-cells and 2) extending the initial functional lifetime of drug carriers conjugated to cell surfaces before transfer. To achieve this, we developed a novel strategy to repeatedly stimulate or track ACT T-cells, using cytokines or ACT-cell-specific antibodies as ligands to target PEGylated liposomes to transferred T-cells in vivo. Using F(ab')2 fragments against a unique cell surface antigen on ACT cells (Thyl.1) or an engineered interleukin-2 (IL-2) molecule on an Fc framework as targeting ligands, we demonstrate that >95% of ACT cells can be conjugated with liposomes following a single injection in vivo. Further, we show that IL-2-conjugated liposomes both target ACT cells and are capable of inducing repeated waves of ACT T-cell proliferation in tumor-bearing mice. These results demonstrate the feasibility of repeated functional targeting of T-cells in vivo, which will enable delivery of imaging contrast agents, immunomodulators, or chemotherapy agents in adoptive cell therapy regimens. On the other hand, we identified CD45 as a non-internalizing receptor on T-cells that could be used as an anchor to block internalization of cell surface-conjugated nanoparticles. Anti-CD45 decorated nanogels consisting of IL-15 superagonists remained on T-cells surfaces for over 12 days and induced 15-fold T-cell expansion in tumors in vivo and significant tumor regression without toxicity, while equivalent doses of free IL-15Sa were lethal. These results show that anti-CD45 can be generally employed to decorate a broad array of nanoparticles to endow them enhanced stability on cell surfaces for extracelluar drug delivery, tracking, or diagnostic purposes. We also compared the efficacy of anti-Thyl.1 liposomes and anti-CD45 liposomes in delivering SB525334, an immunosuppression-reverting drug for inhibiting TGF-[beta] signaling pathway, to ACT T-cells. In the setting of pre-loading Tcells with liposomes in vitro, binding to T-cells through the non-internalizing receptor CD45 elicited greater granzyme expression in ACT T-cells systemically and particularly led to greater donor T-cell infiltration of tumors, which correlated with greater therapeutic efficacy. Nevertheless, as a proof of concept, anti-Thyl.1 liposomes allowed specific re-arming of ACT T-cells with SB525334 by in vivo targeting and slowed down tumor growth significantly compared to equivalent dose of free drug and anti-CD45 liposomes. These might provide us with insights into designing and selecting the right targeting nanoparticles or combination of them depending on the nature of drugs. All together, these results demonstrate the efficacy and specificity of surface-ligand decorated nanoparticles in enhancing in vivo persistence of transferred T-cells. These nanoparticles may be applied to significantly improve the therapeutic index of drugs in cancer immunotherapy. / by Yiran Zheng. / Ph. D.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2016. / Cataloged from PDF version of thesis. / Includes bibliographical references (pages 128-151). / Gene interference screens are a widely adopted and popular tool for uncovering gene function but imperfections in the technology limit the power of these investigations. There are many completed and on-going RNAi investigations across a multitude of biological systems because these experiments are scalable, cost-effective, and relatively easily adapted to multiple experimental environments. The most influential disadvantage is that many of the individual reagents are non-specific and interfere with genes other than the intended target. Efforts to improve limitations in RNAi have focused on statistical models and improving reagents, yet have not explored using biological context to select gene targets. This thesis uses network modeling and data integration to provide context for gene interference studies, and demonstrates the utility of this approach in two systems: Acute Lymphoblastic Leukemia (ALL) is a disease of undifferentiated B-cells that results from accumulation of genetic lesions, yet we have an incomplete understanding of all genes contributing to the disease and how they interact. To discover genetic mediators of this disease, we employ a genome-scale shRNA screen, and complement this data with differential mRNA expression and ChIP-seq data using network integration. The integrated model identifies processes not represented in any input set and predicts novel genes contributing to disease. We specifically validate the role of Wwpl as a tumor suppressor in ALL. Aberrant growth factor pathway activity drives cancer pathology and is the target of molecular cancer therapies. Specifically, the epidermal growth factor receptor (EFGR) pathway and its ligand, transforming growth factor alpha (TGF[alpha]) are clinically relevant to gastric cancer. We use an shRNA screen and Prize Collecting Steiner Forest (PCSF) algorithm to discover the pathway regulating TGF shedding. This pathway identifies common regulators of TGF[alpha] shedding and NF[chi]B regulation, yet targeting NF[chi]B and the EGFR pathway has thus far been unsuccessful in cancer therapies. Our network identifies IRAK1 as a viable path forward for modulating both TGF[alpha] and NF[chi]B in gastric cancer. / by Jennifer L. Wilson. / Ph. D.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2014. / Cataloged from PDF version of thesis. / Includes bibliographical references (pages 96-104). / Identification of effective drug targets to intervene, either as single agent therapy or in combination, is a critical question in drug development. As complexity of disease like cancer is revealed, it has become clear that a holistic network approach is needed to identify drug targets that are specially positioned to provide desired leverage on disease phenotypes. In this thesis we develop a computational framework to exhaustively evaluate target behaviors in biochemical network, either as single agent or combination therapies. We present our single target therapy work as a problem of identifying good places to intervene in a network. We quantify a relationship between how interventions at different places in network affect an output of interest. We use this quantitative relationship between target inhibited and output of interest as a metric to compare targets. In network analyzed here, most targets show a sub-linear behavior where a large percentage of targeted molecule needs to be inhibited to see a small change on output. The other key observation is that targets at the top of the network exerted relatively small control compared to the targets at the bottom of the network. In the combination therapy work we study how combination of drug concentrations affect network output of interest compared to when one of the drugs was given alone at equivalent concentrations. By adapting the definitions of additive, synergistic, and antagonistic combination behaviors developed by Ting Chao-Chou (Chou TC, Talalay P (1984), Advances in enzyme regulation 22: 27-55) for our system and systematically perturbing biochemical pathway, we explore the range of combination behaviors for all plausible combination targets. This holistic approach reveals that most target combinations show additive behaviors. Synergistic, and antagonistic behaviors are rare. Even when combinations are classified as synergistic or antagonistic, they show this behavior only in a small range of the inhibitor concentrations. This work is developed in a particular variant of the epidermal growth factor (EGF) receptor pathway for which a detailed mathematical model was first proposed by Schoeberl et al. Computational framework developed in this work is applicable to any biochemical network. / by Nirmala Paudel. / Ph. D.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2014. / Cataloged from PDF version of thesis. / Includes bibliographical references (pages 101-106). / Despite being responsible for 90% of cancer mortality, metastasis is not well understood. This thesis is focused on the circulation step of the metastatic cascade, examining three types of mobilized tumor cells: circulating tumor cells (CTCs), intraoperatively shed tumor cells, and malignant pleural effusions (MPE). We investigated the functional behavior of mobilized tumor cells in order to explain the discrepancy between the number of tumor cells in circulation and the number overt metastases. The first part of this thesis examines the functional behavior of CTCs isolated from the peripheral blood of metastatic castration-resistant prostate cancer patients. Individual CTCs were compartmentalized using arrays of nanowells to enable clonal comparison and mapping of heterogeneity. The viability, invasiveness and secretory profiles of CTCs were measured. Only a subset of CTCs was found to possess malignant traits indicative of metastatic potential. These CTCs were resistant to anoikis, were invasive or secreted proteolytic enzymes. The second part of this thesis determines the presence of intraoperatively shed tumor cells using blood samples withdrawn from the pulmonary vein after pulmonary lobectomy procedures. Previous studies did not distinguish tumor cells from normal epithelial cells specifically or sensitively. Single-cell genetic approaches were used to compare the genotype of isolated single cells to matched tumor cells and normal adjacent tissue, thereby confirming the malignancy of shed epithelial cells. The third and last part of the thesis delineates the tumorigenic population with surface markers using MPEs. A total of 35 surface antigens were screened from four categories: 1) cancer stem cell 2) epithelial-mesenchymal transition 3) metastatic signature and 4) tyrosine kinase receptors. Surface antigen CD24 was found to be specifically and abundantly expressed in MPE, and was required for the colonization of the lung. In conclusion, metastatic inefficiency is due to the presence of inactive cells and cellular heterogeneity. Inactive cells are either normal epithelial cells or apoptotic tumor cells. Cellular heterogeneity may arise from differences in surface marker expression or functional states. Therefore, only a subset of mobilized tumor cells can give rise to metastases, and therapeutic strategies should be focused on this subset. / by Xiaosai Yao. / Ph. D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biological Engineering, February 2010. / Cataloged from PDF version of thesis. / Includes bibliographical references (p. 86-99). / Functional brain imaging technologies seek to expand our understanding of intact neural systems. Present day functional MRI (fMRI) measures the delayed hemodynamic response that is indirectly associated with neural activity. To study underlying molecular systems noninvasively but precisely, tools must be developed to modulate MRI contrast as a function of discrete molecular events within pathways of interest. In optical imaging modalities, genetically encoded sensors based on fluorescent proteins have provided an engineerable platform on which to optimize desirable device characteristics by exploiting the tools of molecular biology and protein biochemistry. Analogously, we seek to build genetically encodable sensors based on engineered metalloproteins whose effects on MRI contrast are regulated by specific biochemical interactions. In this work, we present two technological advancements toward realizing fMRI contrast sensors for molecular neuroimaging. First, a genetically encodable sensor for free calcium is described, consisting of a novel ferritin-based device that reversibly enhances NMR transverse relaxation times (T2) by Ca - dependent crosslinking. Second, we show that the T1 contrast effect of a recently proposed family of cytochrome P450-based MRI sensors can be significantly enhanced by substitution of the protein's native heme with a high spin manganese porphyrin. / by Victor S. Lelyveld. / Ph.D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biological Engineering, 2012. / Cataloged from PDF version of thesis. / Includes bibliographical references (p. 114-131). / The importance and pervasiveness of naturally occurring regulation of RNA function in biology is increasingly being recognized. A common regulatory mechanism uses inducible protein-RNA interactions to shape diverse aspects of cellular RNA fate. Recapitulating this using a novel set of protein-RNA interactions is appealing given the potential to subsequently modulate RNA biology in a manner decoupled from normal cellular physiology. We describe a ligand-responsive protein-RNA interaction module that can be used to target a specific RNA for subsequent regulation. Using the Systematic Evolution of Ligands by Exponential Enrichment (SELEX) method, RNA aptamers binding to the bacterial Tet Repressor protein (TetR) with low- to sub- nanomolar affinities were identified. This interaction is reversibly controlled by tetracycline in a manner analogous to the interaction of TetR with its cognate DNA operator. Aptamer minimization and mutational analyses support a functional role for conserved sequence and structural motifs in TetR binding. We illustrate the utility of this chemically-inducible RNA-protein interaction to directly regulate translation in both a prokaryotic and eukaryotic organism. By genetically encoding TetR-binding RNA elements into the 5'-untranslated region (5'-UTR) of a given mRNA, translation of a downstream coding sequence is directly controlled by TetR and tetracycline analogs. In endogenous and synthetic 5'-UTR contexts, this modular system efficiently regulates the expression of multiple target genes, and is sufficiently stringent to distinguish functional from nonfunctional RNA-TetR interactions. We also demonstrate engineering this TetR-aptamer module to regulate subcellular mRNA localization. This is efficiently achieved by fusing TetR to proteins natively involved in localizing endogenous transcripts, and genetically encoding TetR-binding RNA aptamers into the target transcript. Using this platform, we achieve tetracycline-regulated enhancement of target transcript subcellular localization. We also systematically examine some rules for successfully forward engineering this RNA localization system. Altogether, these results define and validate an inducible protein-RNA interaction module that incorporates desirable aspects of a ubiquitous mechanism for regulating RNA function in Nature and that can be used as a foundation for functionally and reversibly controlling multiple fates of RNA in cells. / by Brian J. Belmont. / Ph.D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biological Engineering, February 2009. / "February 2008." Cataloged from PDF version of thesis. / Includes bibliographical references (p. 110-114). / Monoclonal antibodies have emerged as a promising class of therapeutics for the treatment of human disease, and in particular human cancer. While multiple mechanisms contribute to antibody efficacy, the engagement and activation of immune effector cells - mediated by the interaction of the conserved Fc regions of the antibody with the Fc gamma receptors (Fc[gamma]Rs) on immune cells - is critical to the efficacy of several. This thesis describes the engineering of antibody Fc domain interactions with Fc[gamma]Rs, using the' yeast S. cerevisiae. In an initial step, a microbial system for the production of full-length antibodies in S. cerevisiae in milligram per liter titers has been developed, which serves as a platform for the engineering of antibody Fc domains with defined properties. The presence of a single N-linked glycan on each chain of the antibody Fc, as well as the specific composition of the glycoforms comprising it, are critical to the binding of the Fc to Fc[gamma]Rs, and have largely limited the production of therapeutic antibodies to mammalian expression systems. Using a display system that tethers full-length antibodies on the surface of yeast, we identify and characterize aglycosylated antibody variants that bind a subset of the human low-affinity Fc[gamma]Rs, Fc[gamma]RIIA and Fc'yRIIB, with approximately wildtype binding affinity and activate immune effector functions in vivo. In a separate approach, we identify aglycosylated variants that weakly bind a third low-affinity receptor, Fc[gamma]RIIIA, and through subsequent engineering generate variants that bind all of the low-affinity Fc[gamma]Rs with approximately wild-type binding affinity. By decoupling the function of the antibody from its post-translational processing, these variants have the potential to open up therapeutic antibody production to a far wider array of expression systems than currently available. Finally, in parallel work, we use a similar system to screen for glycosylated Fc variants with improved affinity and specificity for the activating receptor Fc[gamma]RIIIA compared to the inhibitory receptor Fc[gamma]RIIB, properties which have been hypothesized to lead to more potent antibody therapeutics. / by Stephen L. Sazinsky. / Ph.D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biological Engineering, 2013. / Title as it appears in MIT Degree awarded booklet, February 2013: Quantitative analysis of the Interleukin-2/15 receptor signaling in T lymphocytes. Cataloged from PDF version of thesis. / Includes bibliographical references (p. 168-183). / IL-2 and IL-15 are common y-chain family cytokines critically involved in regulation of T cell differentiation and homeostasis. Both cytokines signal through a heterotrimeric surface receptor complex (IL-2/15R) composed of an [alpha], a [beta], and the common gamma ([gamma]c) chain. Signaling occurs through the shared [beta] and [gamma]c chains, and the a-chains function as high affinity ligand capture agents. Despite sharing signaling chains and pathways, IL-2 and IL-15 have nonredundant roles in T cell biology. In vitro, although IL-2 and IL-15 function as equivalent mitogens, they greatly differ in their activities as growth factors. T cells cultured with IL-2 are larger in size, show increased protein synthesis, and increased amino-acid uptake compared to T cells cultures in IL-15. Additionally, transient exposure of T cell to IL-15, but not IL-2, results in prolonged survival and proliferation after cytokine withdrawal. In vivo, IL-2 is critical for the initial clonal expansion and contraction of activated T cells, whereas IL-15 is critical for the differentiation, and maintenance of memory T cells. The mechanisms through which IL-2 and IL- 15 trigger distinct phenotypes while initiating identical signaling pathways remain elusive. Using mass spectrometry based proteomics, we found that IL-2 and IL-15 trigger a quantitatively and qualitatively identical phosphotyrosine signaling response in T cells. The distinct effects of IL-2 and IL-15 on T cells could not be explained through activation of qualitatively distinct signaling pathways. Instead, our results show that IL-2 and IL-15 mediate distinct T cell phenotypes through differential regulation of IL-2/15R signal strength and duration. The increased survival and proliferation of T cells in response to transient IL-15 exposure is mediated through its ability to trigger prolonged signaling through the IL-2/15R after cytokine withdrawal. Furthermore, we find that T cell proliferation in response to IL-2 and IL-15 stimulation requires a constant signal input from the IL-2/15R. Additionally, T cell proliferation and metabolism are controlled in a quantitatively distinct manner through IL-2/15R signal strength independent of the cytokine identity. Proliferation is controlled by signal strength in a digital manner, whereas cell size and metabolism are controlled in an analog fashion. The differences in cytokinereceptor binding affinity, receptor expression levels, physiological cytokine levels, and cytokinereceptor intracellular trafficking kinetics can result in the differential regulation of IL-2/15R signal strength and duration by IL-2 and IL-15. These results suggest that IL-2 and IL-15 can trigger distinct phenotypes by differentially regulating the strength and duration of IL-2/15R signal transduction, without having to activate distinct biochemical signaling pathways. These results provide important insights into the function of other shared cytokine and growth factor receptors, quantitative regulation of cell proliferation and metabolism through signal transduction, and improved design of cytokine based clinical immunomodulatory therapies for cancer and infectious diseases. / by Abhinav Arneja. / Ph.D.