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.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2017. / Cataloged from PDF version of thesis. / Includes bibliographical references (pages 226-238). / To date, there has been limited usage of biomass and agricultural residues in rural areas as a form of renewable energy, mainly due to the expensive costs involved in collecting and transporting raw biomass. A decentralized biomass torrefaction system has the potential to upgrade the quality and transportability of distributed biomass residues in situ, thereby creating additional localized economic values and mitigating the environmental consequences associated with open burning of the excess biomass residues. Nonetheless, most existing biomass torrefaction systems so far have been designed for large-scale, centralized deployment, and are unsuitable to be scaled down in decentralized applications due to their high level of sophistication and capital cost. We propose a biomass torrefaction system based on the concept of torrefaction in a low-oxygen environment. By eliminating the stringent requirements of an inert torrefaction environment, we demonstrated that we can greatly simplify the reactor design and derive a laboratory-scale system that is also scalable. We proceeded to build and validate this torrefaction system with respect to different operating conditions and types of biomass. Using a quantitative definition for torrefaction severity, we were also able to relate the various fuel user requirements in real life back to the fundamental reactor operations. By quantifying in detail the overall energy performance, pressure requirements, and transient timescales, we also demonstrated how such a reactor system can be operated at scale, as well as the various design improvements that can further boost the performance of a scaled-up system. Therefore, this work builds the foundation towards the development of a low-cost, small-scale, and portable torrefaction system that can potentially be widely deployed in rural areas. / by Kevin S. Kung. / Ph. D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biological Engineering, 2010. / Cataloged from PDF version of thesis. / Includes bibliographical references. / Osteoarthritis (OA) is the most common form of joint disorder. Individuals who have sustained an acute traumatic joint injury are at greater risk for the development of OA. The mechanisms by which injury causes cartilage degradation are not fully understood, but the elevated levels of injury-induced pro-inflammatory cytokines, such as TNFa and IL-6, have been implicated to play important roles in the pathogenesis of OA. We have used in vitro models of cartilage injury to examine the interplay between mechanical and cytokine-mediated pathways and to identify processes associated with cartilage degradation following joint injury. The overall aims of this thesis were to characterize the combined effect of TNFa and IL-6/sIL6R on matrix degradation and chondrocyte gene expression in mechanically injured cartilage, and to investigate whether cartilage degradation could be inhibited by potential therapeutic approaches. TNFa and IL-6/sIL-6R interacted to cause aggrecanase-mediated proteoglycan degradation. Importantly, the combined catabolic effects of cytokines were highly potentiated by mechanical injury. Furthermore, cartilage degradation caused by the in vitro injury model appeared to be initiated at the transcriptional level, since the gene expression of matrix proteases, cytokines and iNOS were all highly elevated in the treatment conditions. The degradative effects of TNFa in injured cartilage was due, in part, to the action of endogenous IL-6, as proteoglycan degradation was partly reduced by an IL-6 blocking Fab fragment. Interestingly, cartilage degradation induced by the combinations of proinflammatory cytokines and mechanical injury was fully abrogated by short-term treatments with dexamethasone. The results of this work are significant in that they provide evidence suggesting joint injury affects cell-mediated responses as well as the transport of cytokines and proteases in extracellular matrix, making cartilage tissue more susceptible to further degradation by biochemical mediators. / by Yihong C.S. Lu. / Ph.D.

Thesis: Sc. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2015. / Cataloged from PDF version of thesis. / Includes bibliographical references (pages 157-192). / Unlike the genomic sequence, the epigenome is dynamic, responding to and influencing the cellular state. Understanding the ways in which chromatin structure exercises control over transcriptional programs during development as well as aberrant gene expression signatures in human disease is a central challenge to biology and pathology. Despite the advance of tools to chart the chromatin landscape, much is still unknown about how epigenetic signals are integrated in the transcriptional outcome of a gene. Here, a number of systematic, genome-wide approaches result in the discovery of epigenetic patterns associated with the modulation of gene expression. We begin by examining Huntington's disease (HD), a fatal neurodegenerative disorder. The discovery of the genetic mutation that causes HD has led to the development of in-vitro and in-vivo models representative of the disease. Studies of these systems and patient samples have identified transcriptional dysregulation as a major component of the early stages of HD. Thus, HD serves as an ideal case in which to examine the modulation of gene expression in disease. First, we connect the changes in DNA methylation to the dynamics of gene expression and transcription factor binding in HD. We next identify a unique signature of the histone mark H3K4me3 at genes transcriptionally repressed in HD. Targeting this signature reverses the downregulation of genes and protects against neurodegeneration. Finally, we apply a novel, machine learning approach to 16 chromatin features in 44 human cell types. By globally examining the epigenetic states of genes in a variety of tissue contexts, we discover a small set of coordinated patterns that we term "epigenetic ensembles." Genes with particular ensembles are associated with changes in gene expression during differentiation and the control of cell-type-specific gene regulators such as super-enhancers, distal-enhancer-looping, nuclear lamina, and master transcription factors. Ensembles are also disproportionately affected in both HD and Alzheimer's disease. Together, this thesis presents a toolkit for recognizing and understanding epigenetic patterns that can further insight into the regulation of genes in development and disease. / by Christopher W. Ng. / Sc. D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biological Engineering, 2012. / Cataloged from PDF version of thesis. / Includes bibliographical references. / Human adaptation of avian influenza viruses pose an enormous public health challenge as the human population is predominantly naive to avian influenza antigens. As such, constant surveillance is needed to monitor the circulating avian strains. Of particular importance are strains belonging to H5N1, H7N7, H7N2 and H9N2 subtypes that continue to circulate in birds worldwide and have on occasions caused infections in humans. A key step in influenza human adaptation is the accumulation of substitutions/mutations in the viral coat glycoprotein, hemagglutinin (HA), that changes HA's binding specificity and affinity towards glycan receptors in the upper respiratory epithelia (referred to as human receptors). Unlike for the H1, H2, H3 and more recently H5 HA a correlation between the quantitative binding of HA to human receptors and respiratory droplet transmissibility has not been established for H9 and H7 subtypes. This thesis is a systematic investigation of determinants that mediate changes in HA-glycan receptor binding specificity, with focus on the molecular environments within and surrounding the glycan receptor binding site (RBS) of avian HAs, particularly the H9 and H7 subtypes. The glycan receptor binding properties of HA were studied using a combination of biochemical and molecular biology approaches including dose dependent glycan binding, human tissue staining and structural modeling. Using these complementary analyses, it is shown that molecular interactions between amino acids in and proximal to the RBS, including interactions between the RBS and the glycan receptor converge to provide high affinity binding of avian HA to human receptors. For the H9 HA [alpha]2-->6 glycan receptor-binding affinity of a mutant carrying Thr-189-->Ala amino acid change correlated with the respiratory droplet transmission in ferrets conferred by this change. Further, it was demonstrated for the first time that two specific mutations; Gln226-->Leu and Gly228-->Ser in glycan receptor-binding site of H7 HA substantially increase its binding affinity to human receptors. These approaches and findings contribute to a framework for monitoring the evolution of HA and the development of general rules that govern human adaption applicable to strains beyond ones currently under study. / by Karunya Srinivasan. / Ph.D.