Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2017. / Cataloged from PDF version of thesis. / Includes bibliographical references (pages 236-275). / Glycans are an important class of biological molecules that regulate a variety of physiological processes such as signal transduction, tissue development and microbial pathogenesis. However, due to the structural complexity of glycans and the unique intricacies of glycan-protein interactions, elucidating glycan structure-function relationships is challenging. Thus, uncovering the biological function of glycans requires an integrated approach, incorporating structural analysis of glycans, and glycan-proteins interactions with functional analysis. In this thesis, I develop new tools and implement integrated approaches to study glycans and glycan-binding proteins (GBPs). I apply these approaches to study glycans and GBPs in two areas: i) the role of hemagglutinin-glycan receptor specificity in human adaptation and pathogenesis of influenza and ii) the function of glycan regulation of cell-microenvironment interaction in cancer progression. Section 1: Influenza poses a significant public health threat and there is a constant looming threat of a pandemic. Pandemic viruses emerge when avian viruses acquire mutations that enable human adaptation, leading to infection of an antigenically naive host. Influenza Hemagglutinin (HA), and HA-glycan receptor interactions, play a central role in host tropism, transmissibility, and immune recognition. In section one, I develop and apply an integrated approach comprised of structural modeling, inter-amino acid network analysis, biochemical assays, and bioinformatics tools to study the hemagglutinin-glycan interaction and, in some cases, HA's antigenic properties. Using this approach, we i) identify the structural determinants required, and potential mutational paths, for H5N1 to quantitatively switch it's binding specificity to human glycans receptors, ii) identify the mutations that enable the 2013 outbreak H7N9 HA to improve binding to human glycan receptors in the upper respiratory tract, iii) uncover H3N2 strains that are currently circulating in birds and swine that possess features of a virus that could potentially re-emerge and cause a pandemic, and iv) characterize the glycan binding specificity of a novel 2011 Seal H3N8 HA. The approaches implemented here and the findings of these studies provide a framework for improved surveillance of influenza viruses circulating in non-human hosts that pose a pandemic threat. Section 2: Glycans are abundant on the cell surface, and at the cell-ECM interface where they mediate interactions between cells and their microenvironment. Despite this, the function of glycans in cancer progression remains largely understudied. Here, I develop an integrated approach to characterize the cell surface glycome, including N-linked, 0-linked glycans, and HSGAGs. This approach integrates glycogene expression data, analytical tools, and glycan binding protein reagents. I demonstrate that this platform enables rapid and efficient characterization of the N- and 0-linked glycome in a model cell system, representing metastatic versus non-metastatic cancer cells. Next, I apply this integrated approach to uncover new roles of glycans. I study the role that HSGAGs play in regulating cancer stem cell (CSC) activity in breast cancer. Here, we report that SULF1, an HSGAG modifying enzyme, is required for efficient tumor initiation, growth and metastasis of CSCs. Furthermore, we identify a putative mechanism by which SULF1 regulates interactions between CSCs and their microenvironment. The approaches implemented here and the finding of these studies Overall, this thesis provides important tools, approaches and insights to enable and improve the study of glycans and glycan binding proteins. Together the work here provides a framework for decoding structure-function relationship of glycans. / by Nathan Wilson Stebbins. / Ph. D.

Thesis: S.M., Massachusetts Institute of Technology, Department of Biological Engineering, 2017. / Cataloged from PDF version of thesis. / Includes bibliographical references (pages 89-95). / Understanding the elastic and viscoelastic responses of biological soft tissues and engineered polymer simulants is of great interest to predicting and preventing penetrative injuries. Detailed understanding of the mechanical processes at work could aid in the development and evaluation of protective strategies such as armor and helmets, and repair strategies including robotic surgery or needle-based drug delivery. However, due to the mechanical complexity of so-called "soft tissues," including nonlinear viscoelastic behavior, surface adhesion, material failures and shock effects, the experimental characterization of various soft tissues is challenging and individual mechanical processes are often impossible to decouple without computational models and simulations. This thesis presents two finite element models designed to provide both replicate the results of indentation and impact experiments on synthetic polymers, aimed to decouple competing mechanical characteristics of contact based deformation. The first of these models describes the indentation on polydimethylsiloxane bilayer composites, with the aim of describing the relative effects of a adhesion and viscoelastic properties on the measured deformation response. That model expands on this objective via the analysis of the effects of surface adhesion commonly associated with highly compliant polymers and tissues. The second model attempts to replicate impact of a high velocity projectile on a relatively stiff material, polyurethane urea, and on a comparatively compliant polymer, gelatin hydrogel. These models provide means to simulate, predict and characterize material response, validated by comparison with available experiments. Such validated models can be used to simulate and design new materials as tissue simulants or as protective media that predictably dissipate concentrated mechanical impact. / by Kyle Geiser. / S.M.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biological Engineering, 2011. / Cataloged from PDF version of thesis. / Includes bibliographical references (p. 164-183). / Cancer causes 13% of all deaths worldwide. Inflammation-mediated cancer accounts for ~15% of all malignancies, strongly necessitating investigation of the molecular interactions at play. Inflammatory reactive oxygen and nitrogen species (RONs), including peroxynitrite and nitric oxide (NO'), may potentiate malignancy. We hypothesize that the base excision repair (BER) pathway modulates susceptibility to malignancy, by modulating the BER-intermediate levels, large scale genomic rearrangements and toxicity following exposure to RONs. We further hypothesize that DNA methyltransferases are responsible for the memory of genotoxic insult, and the epigenetic propagation of genomic instability, following exposure to genotoxins. Here, we exploited cell lines engineered to carry deficiencies in BER to study repair of DNA damage induced by RONs. Toxicity and BER-intermediate levels were evaluated in XRCC1 proficient and deficient cells, following exposure to the peroxynitrite donor, SIN-1 and to NO*. Using the alkaline comet assay, we find that while XRCC1 proficient and deficient CHO cells incur equivalent levels of SIN-1 induced BER-intermediates, the XRCC1 null cells are more sensitive to killing by SIN-1, as assessed by clonogenic survival. Furthermore, using bioreactors to expose CHO cells to NO', we found that the BER-intermediate levels measured in XRCC1 null cells were lower than in WI cells. We found that while XRCC1 can facilitate AAG-mediated excision of the inflammation-associated base lesions ethenoadenine and hypoxanthine, in vitro; XRCC1 deficient human cells were no more susceptible to NO' than WT cells. However, in live glioblastoma cells, XRCC1 is acting predominantly downstream of AAG glycosylase. This work is some of the first to assess the functional role of XRCC1, in response to RONs and suggests complexities in the role of XRCC1. We also demonstrate that the underlying basis for the memory of a genotoxic insult and the subsequent propagation of genomic instability is dependent on the DNA methyltransferases, Dnmtl and Dnmt3a. We found that a single exposure led to long-term genome destabilizing effects that spread from cell to cell, and therefore provided a molecular mechanism for these persistent bystander effects. Collectively, our findings impact current understanding of cancer risk and suggest mechanisms for suppressing genomic instability, following exposure to inflammatory genotoxins. / by James T. Mutamba. / Ph.D.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2015. / Cataloged from PDF version of thesis. / Includes bibliographical references (pages 121-132). / During the process of metastasis, tumor cells must undergo changes that enable them to detach from a tumor, exit surrounding tissue during intravasation, enter into circulation and eventually stop at a distant site for extravasation. Here, we measure the physical changes in the deformability of tumor cells, indicated by the length of time required to pass through a microfluidic constriction in a suspended microchannel resonator (SMR), as related to different stages of the metastatic process-particularly, in an epithelial-mesenchymal transition (EMT) and existence in the circulation. We find that a mesenchymal population of murine tumor cells (MMTV-PyMT) that had undergone a spontaneous EMT at the primary tumor site were more deformable than the parental population of epithelial cells. In contrast, MMTV-PyMT and Ep5 murine breast carcinoma cells that had received signaling from platelets to undergo an epithelial-mesenchymal-like transition maintained the same deformability or became less deformable, respectively. In all cases, however, epithelial and mesenchymal tumor cells both take much longer to pass through a constriction than typical blood cells, as confirmed by examining various human cancer cell lines (H1975, SKBR-3, MDA-MB231, PC3-9). Using a syngeneic mouse tumor model, we find that cells that are able to exit a tumor and enter the circulation are not required to be particularly more deformable than the cells initially injected into the mouse. However, in a limited study of prostate cancer patients, various circulating tumor cells (CTCs) can pass through a constriction quickly because some are relatively small in size, while others are more deformable than typical tumor cell lines and more mechanically similar to blood cells. Nonetheless, due to the ambiguity in cell identity when a heterogeneous sample like blood is assessed by the SMR, there was a need to correlate each cell's precision biophysical measurement to its molecular expression. I thus developed a technique whereby cells can be sorted off-chip based on their passage time and/or buoyant mass characteristics, and collected into a 96- well plate. The proof-of-principle is demonstrated by sorting and collecting cells from cell linespiked blood samples as well as a metastatic prostate cancer patient blood sample, classifying them by their surface protein expression and relating them to distinct SMR signal trajectories. Taken together, our results provide impetus for further studies on the mechanical properties of CTCs as well as the future utilization of this platform for other types of biophysical-molecular characterizations. / by Josephine W. (Shaw) Bagnall. / Ph. D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biological Engineering, 2008. / Cataloged from PDF version of thesis. / Includes bibliographical references (p. 130-145). / Nanoparticle-protein conjugates have a variety of applications in imaging, sensing, assembly and control. The nanoparticle-protein interface is made of numerous complex interactions between protein side-chains and the nanoparticle surface, which are likely to affect protein structure and compromise activity. Ribonuclease S and cytochrome c are covalently linked to nanoparticles via attachment to a specific surface cysteine, with the goal of optimizing protein structure and activity, and understanding conditions that minimize non-specific adsorption. Protein behavior is explored as a function of the nanoparticle surface chemistry and material, the density of proteins on the nanoparticle surface, and the position of the labeled site. Ribonuclease S is attached to Au nanoparticles by utilizing its two-piece structure. Enzymatic activity is determined using RNA substrate with a FRET pair. Conjugation lowers the ribonucleatic activity, which is rationalized by the presence of negative charges and steric hindrance which impede RNA in reaching the active site. Cytochrome c is linked to Au and CoFe204 nanoparticles. The protein is denatured when the nanoparticle ligands are charged, but remains folded when neutral. The presence of salt in the buffer improves folding. This indicates that electrostatic interactions of charged amino acids with the charged ligands are prone to lead to protein denaturation. The attachment site can be controlled by mutations of surface residues to cysteines. Protein unfolding is more severe for nanoparticle attached in the vicinity of charged amino acids. Molecular dynamics simulations of the conjugate reveal that electrostatic interactions with· the nanoparticle ligand lead to local unfolding of [alpha]-helices of cyt c. Furthermore, the nanoparticle induces more structural disturbance when it is attached on the N- and C-terminal [alpha]-helices foldon, which is the most stable motif of cyt c and the most essential for folding. / by Marie-Eve Aubin-Tam. / Ph.D.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2015. / Cataloged from PDF version of thesis. In title on title page, "[beta]" appear as lower case Greek letter. / Includes bibliographical references (pages 107-125). / Autologous bone marrow grafting has been shown to aid in the healing of bone defects since the 1950s. Transplantation of freshly-aspirated autologous bone marrow, together with a scaffold, is a promising clinical alternative to harvest and transplantation of autologous bone for treatment of large defects. However, survival and proliferation of the marrow-resident osteoprogenitors (CTPs) can be limited in large defects by the inflammatory microenvironment. Ligands that can improve CTP survival and other relevant upstream processes like colony formation and proliferation should advance bone healing. One such ligand is the Epidermal Growth Factor (EGF). EGF, when presented tethered (tEGF) on non-graftable synthetic polymer substrates, induced growth and colony formation of CTPs with additional cytoprotective effects not observed under soluble EGF stimulation. The objective of this thesis work was to test whether tEGF can be a viable alternative to enhance bone regeneration by tethering EGF onto a graftable, osteoconductive matrix, beta-tricalcium phosphate ([beta]TCP). Due to the lack functional groups for bioconjugation on the [beta]TCP surface, the tethering strategy involved the use of a high-affinity [beta]TCP binding peptide fused to the EGF domain. This broadly-applicable tethering strategy led to retention of tethered EGF for more than a week, while maintaining bioactivity in the bound state. Novel methods were designed in order to study the effects of EGF-tethered [beta]TCP scaffolds on marrow stromal cell proliferation and on osteoprogenitor colony formation from plated marrow. Results showed that tEGF can enhance both of these processes. This motivated a experiment designed to test the performance of EGF-tethered B-TCP scaffolds on a mid-sized, pre-clinical bone defect model (Canine Femoral Multi-Defect Model; CFMD). The CFMD model revealed that both control and EGF-tethered scaffolds promote bone formation to levels comparable to mineralized cancellous allograft, with the tEGF condition showing signs of advanced remodeling. However, due to a potential ceiling effect, a more compromised bone defect model will be needed to accurately assess differential graft performance. Altogether, this thesis demonstrates the capability of tEGF to influence important biological processes related to bone healing, which shows promise for its future use in bioactive graft formulations. / by Jaime J. Rivera Abreu. / Ph. D.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2015. / 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 95-103). / Malaria kills over 500,000 people annually, the majority of whom are children under five years old in sub-Saharan Africa. This disease is caused by several parasite species, of which Plasmodium falciparum is associated with the highest mortality. The clinical manifestations of malaria are associated with the phase of infection where parasites develop within red blood cells (RBCs). Infected RBCs can adhere to the host microvasculature, triggering inflammatory responses in affected organs that contribute to the pathophysiology of life threatening cerebral malaria and pregnancy-associated malaria. The expression of specific Plasmodium falciparum Erythrocyte Membrane Protein 1 (PfEMP1) variants on the RBC surface is associated with severe disease, such as VAR2CSA-mediated placental sequestration during pregnancy-associated malaria. While parasite proteins expressed on the surface of infected RBCs are linked to disease pathogenesis, this surface proteome is poorly characterized. Identifying parasite-derived antigens on the infected RBC surface could facilitate diagnosis, monitoring, and prevention of sequestration. To interrogate the infected RBC surface proteome, we require a panel of affinity reagents that robustly distinguish the parasite-derived proteins from the elaborate RBC surface milieu. Nucleic acid aptamers are widely used in biological applications for their high specificity and affinity to targets and are highly suitable for malaria applications. Efficiently generating aptamers against complex targets-such as whole cells-remains a challenge. Here we develop a novel strategy (I-SELEX) that utilizes inertial focusing in spiral microfluidic channels to stringently partition cells from unbound oligonucleotides. We use I-SELEX to efficiently discover high affinity aptamers that selectively recognize distinct epitopes present on target cells. Using first an engineered RBC model displaying a non-native antigen and, second, live malaria parasite-infected RBCs as targets, we establish suitability of this strategy for de novo aptamer selections. We demonstrate recovery of a diverse set of aptamers that recognize distinct epitopes on parasite-infected RBCs with nanomolar affinity, including an aptamer against the protein responsible for placental sequestration, VAR2CSA. These findings validate I-SELEX as a broadly applicable aptamer discovery platform that enables identification of new reagents for mapping the parasite-infected RBC surface proteome at higher molecular resolution to potentially contribute to malaria diagnostics, therapeutics and vaccine efforts. / by Christina M. Birch. / 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. / The work presented in this thesis explores two novel and complementary facets of endogenous DNA damage: the development of biomarkers of inflammation based on metabolites of DNA damage products and the formation of DNA adducts by electrophilic products of intermediary metabolism. From the first perspective, endogenous DNA damage generated by reactive oxygen and nitrogen species from inflammation and oxidative stress has shown strong mechanistic links to the pathophysiology of cancer and other human diseases, with the damage products reflecting all types of damage chemistries including oxidation, deamination, halogenation, nitration and alkylation. However, the use of DNA damage products as biomarkers has been limited by poor understanding of the damage actually arising in tissues and a lack of appreciation of the fate of DNA damage products from the moment of formation at the site of damage to release from cells to final excretion from the body. The goal of the work presented in the first part of this thesis was to investigate the metabolic fates of the base propenal products arising from 4'-oxidation of 2'-deoxyribose in DNA, one of the most common products of DNA oxidation, and to define base propenal metabolites as potential biomarkers of oxidative stress. This project was approached with systematic metabolite profiling, starting with prediction of potential base propenal metabolites based on a priori knowledge of its chemical reactivity as an [alpha],[beta]-unsaturated aldehyde toward glutathione (GSH) in non-enzymatic reactions and in rat liver cell extracts. Of 15 potential candidates predicted and identified from these in vitro studies, analysis of urine samples from rats given intravenous doses (IV) of thymine propenal revealed three major metabolites: thymine propenoic acid and two mercapturic acid derivatives, which accounted for ~6% of the injected dose. An additional four metabolites, including conjugates with GSH, cysteinylglycine and cysteine, were observed in bile and accounted for ~22% of the dose. One of the major metabolites detected in urine and bile, a bis-mercapturic acid adduct of reduced thymine propenal was detected as a background excretory product in saline-treated rats and was significantly elevated after oxidative stress caused by treatment with bleomycin and CCl₄. Our observations suggest that metabolism and disposition of damaged biomolecules should be considered as crucial factors in the development of biomarkers relevant to inflammation and oxidative stress. The second part of this thesis addresses the complementary hypothesis that electrophilic metabolites generated endogenously from intermediary metabolism can react with DNA to form adducts. This concept is illustrated here with glyoxylate from the glyoxylate metabolic cycle, whicvh plays a key role as an alternative to the TCA cycle in plants, bacteria, protists and fungi under changing conditions of environmental nutrients. The goal of this project was to characterize DNA adducts caused by glyoxylate in the mycobacterium M. smegmatis, with the studies motivated by the higher-than-expected mutation rate of mycobacteria during dormancy induced by nutrient deprivation and a shift to utilization of the glyoxylate cycle. Initially, in vitro reactions of 2'-deoxyguanosine (dG) with glyoxylate yielded N²-carboxyhydroxymethyl dG (N²-CHMdG) as the only adduct. However, the adduct proved to be unstable, so a reduction-based analytical method was developed to yield the stable amine derivative, N2-carboxymethyl dG (N²-CMdG). This stable adduct was used to develop an isotope-dilution chromatography-coupled tandem mass spectrometry method to quantify N²-CHMdG as N²-CMdG in calf thymus DNA treated with glyoxylate in vitro. This analytical method was then applied to quantify and compare the level N2-CMdG in (1) wild-type M. smegmatis grown in rich medium (7H9) or in minimal M9 medium supplemented with acetate, the latter inducing a switch from the TCA cycle to the glyoxylate cycle; and (2) the isocitrate dehydrogenase (ICD)-deficient mutant of M. smegmatis. Mycobacteria grown in the acetate medium experienced a 2-fold increase in the adduct compared to those grown in 7H9. Similarly, the adduct increased 2-fold in the ICD mutant compared to wild-type M. smegmatis grown in 7H9. The results support the idea that shifts in intermediary metabolism can lead to DNA damage that may cause mutations associated with nutrient deprivation in mycobacteria, with implications for the genetic toxicology of other metabolism-derived electrophiles. / by Watthanachai Jumpathong. / Ph. D.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2017. / Cataloged from PDF version of thesis. / Includes bibliographical references (pages 115-124). / Balancing protein expression is critical when optimizing genetic systems. Typically, this requires construction of a library where variants of parts (e.g. promoters) are tried for each gene, which can be expensive and time-consuming. Here, we present an approach that leverages transacting RNA regulators to explore large gene expression spaces without de novo library construction. First, we developed six sRNAs whose strengths have been optimized against a set of 15nt "target" sequences that can be inserted upstream of a ribosome-binding site to generate up to 175-fold repression when maximally expressed. By controlling sRNA expression, the targeted gene can be tunably repressed from 1.6- to 121-fold. We then built a pool where each of the six sRNAs was placed under the control of 16 promoters, yielding ~107 combinations. This pool can optimize up to six genes in any system. Only a single variant of the system is constructed, where a target sequence is placed upstream of each gene. This is then transformed with the pre-built sRNA pool and the resulting library is screened. The system is then rebuilt by rationally selecting parts that reproduce the optimal knockdown of each gene identified by the screen. We demonstrated the versatility of this tool by using the same pool to optimize a beta-carotene pathway and an XNOR circuit. In a second study, we developed tools to facilitate a similar approach in yeast using CRISPRi. We leveraged T7 RNA polymerase to produce guide RNAs (gRNA), and show that modulating gRNA levels with T7 promoters can regulate gene expression. As a proof of principle, we used this system to modulate flux in a carotenoid pathway. Together, the tools presented in this thesis drastically reduce the time and cost to optimize multi-gene systems in a variety of organisms. / by Amar Ghodasara. / Ph. D.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2014. / Cataloged from PDF version of thesis. / Includes bibliographical references (pages 70-82). / Materials that can interact with and transit membranes without toxic bilayer disruption or poration are of great interest in the drug delivery field. These materials can presumably bypass endocytosis to directly enter the cell cytosol through the plasma membrane, which is often the desired site of action for therapeutics, thereby avoiding potential and likely cargo degradation if trapped in endosomes. Alternatively, if these materials are endocytosed, they are often able to escape the endosome by interacting with and transiting the endosomal membrane, and moving into the cytosol. Here, we present a unique membrane-interacting, amphiphilic gold nanoparticle (amph-AuNP) system, which we show can interact with, embed within, and even penetrate through multiple adjacent lipid bilayers without evidence of membrane disruption or poration. By virtue of these key properties, we also present these amph-AuNPs as effective carriers for therapeutic molecules. Using a one-step reaction, we synthesized small -2-4 nm core size amph- AuNPs with an amphiphilic ligand shell comprised of one or two alkanethiols. Each amph-AuNP is coated by a mixture of long-chain mercaptoundecanesulfonate (MUS) terminated by a water-soluble sulfonate group, and in some cases, short-chain hydrophobic octanethiol (OT). First, we describe our efforts to adapt and develop a method to synthesize giant multilamellar model membranes to study amph-NP-membrane interaction in a well-defined setting. We show that giant membranes can be synthesized and fine-tuned by varying lipid composition and buffer salt concentration, can be fluorescently labeled with lipid tracers, and can be analyzed robustly with confocal microscopy and flow cytometry. Second, we describe our systematic analysis of amph- AuNP and membrane characteristics that influence mechanisms of NP association with bilayers. We study effects of general membrane properties such as electrostatics and phase that govern NP-membrane interactions, and found that NP penetration of bilayers was blocked under conditions where strong electrostatic repulsion or gel-phase lipids were employed. We further studied effects of AuNP core diameter, surface charge, and surface hydrophobicity on NP-membrane interactions at the nanoscale. We found that MUS particles with an optimal gold core size -2- 3nm in diameter and MUS:OT particles of a broader size range were capable of inducing hemifusion between liposomal membranes, while MUS:OT 2:1 particles of intermediate hydrophobicity were capable of spontaneously aggregating within the bilayer of vesicles to form Janus egg-like morphologies. Third, we built on these NP membrane-embedding properties to explore and characterize NP-embedding in erythrocyte membranes, with particular attention to the glycocalyx and membrane fluidity, for the future application of constructing therapeutic erythrocyte 'pharmacytes' in situ. Finally, we describe work in engineering amph-AuNPs to carry short antigenic peptide cargoes for in vivo vaccine applications, where immunization experiments have shown much promise for antigen-ferrying amph-AuNPs in eliciting robust and long-lasting CD8+ T cell responses. / by Prabhani U. Atukorale. / Ph. D.