Thesis (Ph. D.)--Massachusetts Institute of Technology, Biological Engineering Division, 2004. / Includes bibliographical references. / Complex carbohydrates such as the heparin/heparan sulfate glycosaminoglycans (HSGAGs) and the asparagine linked glycans (N-glycans) have emerged as crucial components of many human biological systems. Both classes of molecules have diverse roles in normal and pathological processes including development, cell growth, wound healing, angiogenesis, viral entry, and cancer. Taking advantage of the ability of carbohydrates to affect biological outcomes, several companies have already begun developing sugar-based structures as therapeutics for malaria and breast cancer, among others. However, the structural complexity that allows HSGAGs and N-glycans to exhibit such a wide array of activities also makes them more difficult to study than their protein and nucleotide counterparts. There are currently no effective methods for rapid synthesis of specific HSGAG or N-glycan structures, and until recently, most common analytical techniques were not useful in this field due to sensitivity constraints. Now, with advances in separation techniques and mass spectrometry, the fundamental roles of biological carbohydrates can be elucidated more efficiently. However, there is still a need for improved methodologies for studying these classes of molecules. This thesis describes the development of a novel technique for the direct isolation and sequencing of protein-binding HSGAG oligosaccharides. As a proof-of-concept, we used heparin and HSGAG-binding proteins of known specificity (fibroblast growth factor and antithrombin III), then utilized the technique to identify an oligosaccharide binder for a protein with unknown HSGAG binding specificity (endostatin). Furthermore, this technique can be readily applied to carbohydrate material from the cell surface. We have / (cont.) optimized methods for harvesting and purifying cell surface HSGAGs that are suitable for use in many experimental procedures. Taken together, these methods will allow for the rapid identification of HSGAG sequences from biologically relevant sources that bind to proteins of interest, potentially leading to new pharmaceutical targets. Not only do carbohydrates represent a huge potential drug market, they have an enormous role in the development of protein therapeutics. Since N-glycans can significantly alter the activity and half-life of proteins, it is essential to have the appropriate glycosylation for maximum effect. We therefore optimized a method for analyzing the carbohydrate component of N-glycans from mass-produced proteins, and compared the glycosylation patterns of the same protein produced in different types of bioreactors. Further, we built upon the methods for analyzing N-glycans from single proteins to obtain a complete N-glycan profile from total serum glycoproteins. Searching for patterns in these profiles may yield new information on N-glycan changes in disease states, and lead to more sensitive diagnostic testing. / by Nishla Keiser. / Ph.D.

Thesis (S.M.)--Massachusetts Institute of Technology, Biological Engineering Division, 2004. / Includes bibliographical references (leaves 36-38). / Anti-angiogenic therapy offers many benefits over traditional cytotoxic chemotherapy including fewer toxic side effects and the reduced development of drug resistance. Anti-angiogenics alone have not proven effective in inducing tumor regression in the clinic due to both the cytostatic nature of anti-angiogenic therapy and the potential formation of new regions of hypoxia within the tumor after therapy. The new therapeutic paradigm is for combining both anti-angiogenics and traditional cytotoxic agents for a synergistic effect. The efficacy of cytotoxic agents may be reduced after anti-angiogenic therapy, however, due to limited access to tumor vasculature and hypoxia-induced drug resistance. We propose that loading cytotoxic agents within the tumor prior to blood vessel collapse will enable both greater drug accumulation within the tumor as well as a reduction in the formation of therapy-induced regions of hypoxia. We present here a novel nanodelivery vehicle termed a 'nanocell' for the spatio-temporal recruitment of both anti-angiogenics and cytotoxic agents within the solid tumor to achieve this goal. Nanocells consist of a polymeric nanocore encapsulating the cytostatic agent doxorubicin surrounded by a lipid vesicle containing the anti-angiogenic agent combretastatin A4. / (cont.) Nanocell treatment resulted in an 88% reduction in tumor size in vivo, compared to a 66% reduction in tumor size after delivering combretastatin A4 lipid vesicles and doxorubicin nanocores simultaneously but separately. Nanocell treatment also resulted in a significant reduction in systemic toxicity, fewer metastases to the lung and liver, and a greater degree of tumor apoptosis. / by David A. Eavarone. / S.M.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Biological Engineering Division, 2006. / Includes bibliographical references. / Recent studies have developed preliminary wiring diagrams for a number of important biological networks. However, the design principles governing the construction and operation of these networks remain mostly unknown. To discover design principles in these networks, we investigated and developed a set of computational tools described below. First, we looked into the application of optimization techniques to explore network topology, parameterization, or both, and to evaluate relative fitness of networks operational strategies. In particular, we studied the ability of an enzymatic cycle to produce dynamic properties such as responsiveness and transient noise filtering. We discovered that non-linearity of the enzymatic cycle allows more effective filtering of transient noise. Furthermore, we found that networks with multiple activation steps, despite being less responsive, are better in filtering transient noise. Second, we explored a method to construct compact models of signal transduction networks based on a protein-domain network representation. This method generates models whose number of species, in the worst case, scales quadratically to the number of protein-domain sites and modification states, a tremendous saving over the combinatorial scaling in the more standard mass-action model was estimated to consist of more that 10⁷ species and was too large to simulate; however, a simplified model consists of only 132 state variables and produced intuitive behavior. The resulting models were utilized to study the roles of a scaffold protein and of a shared binding domain to pathway functions. / by Bambang Senoaji Adiwijaya. / Ph.D.

Thesis (M. Eng.)--Massachusetts Institute of Technology, Biological Engineering Division, 2006. / Includes bibliographical references (leaves 80-84). / While many have explored how vascular processes alter gap junction communication and composition few have analyzed the role of specific gap junction connexin proteins in regulating cellular communication and wound healing. Using RNA interference or peptide inhibitors to downregulate specific connexins we examined the role of gap junctions in intercellular diffusion, calcium excitation, and in mediating the expression of vascular regulators transforming growth factor-[Beta][ (TGF-[beta]), prostacyclin, and endothelial nitric oxide synthase (eNOS). siRNA inhibition of connexin 43 in porcine aortic endothelial cells (PAEC) significantly decreased the diffusion distance of Lucifer yellow dye and cytoplasmic calcium levels after mechanical wounding. Wound healing experiments suggested that stimulatory signals travel through gap junctions containing connexin 43, while inhibitory signal travel through gap junctions containing connexin 37. Connexin 43 and connexin 37 inhibition, alone or in combination, reduced the levels of secreted latent TGF-[beta] in confluent PAEC monolayers after 24 hours of incubation. Human umbilical vein endothelial cells (HUVEC) behaved in a similar manner. Inhibition of any one of the three connexins resulted in a marked increase in eNOS concentration. / (cont.) Yet, TGF-P was sensitive to simultaneous inhibition of connexins 37, 40, and 43 and prostacyclin was controlled by connexin 37 and/or connexin 40 but not connexin 43. We have demonstrated how selective inhibition of gap junction connexin expression can reveal the potent gap junction mediation of cellular communication, wound healing, and vascular function. We demonstrate for the first time that connexin proteins play distinct roles in vasoregulation with differential effects on TGF- [beta], eNOS and prostacyclin. This technique in general and findings in specific may help explain density-dependent control of vascular signaling and repair. / by David Morad Elihu. / M.Eng.

Thesis (S.M.)--Massachusetts Institute of Technology, Biological Engineering Division, February 2007. / Includes bibliographical references. / Background: Investigators have focused on mechano-regulation of upstream signaling and responses at the level of gene transcription, protein translation and post-translational modifications. Intracellular pathways including those involving integrin signaling, mitogen activated protein kinases (MAPKs), and release of intracellular calcium have been confirmed in several laboratories. Studies with IGF-1: Insulin-like growth factor-I (IGF-1) is a potent anabolic factor capable of endocrine and paracrine/autocrine signaling. Previous studies have demonstrated that mechanical compression can regulate the action of IGF-1 on chondrocyte biosynthesis in intact tissue; when applied simultaneously, these stimuli act by distinct cell activation pathways. Our objectives were to elucidate the extent and kinetics of the chondrocyte transcriptional response to combined IGF-1 and static compression in cartilage explants. Discussion: Clustering analysis revealed five distinct groups. TIMP-3 and ADAMTS-5, MMP-l and IGF-2, and IGF-1 and Collagen II, were all robustly co-expressed under all conditions tested. In comparing gene expression levels to previously measured aggrecan biosynthesis levels, aggrecan synthesis is shown to be transcriptionally regulated by IGF- 1, whereas inhibition of aggrecan synthesis by compression is not transcriptionally regulated. / (cont.) Conclusion: Many genes measured are responsive the effects of IGF-1 under 0% compression and 50% compression. Clustering analysis revealed strong co-expressed gene pairings. IGF-1 stimulates aggrecan biosynthesis in a transcriptionally regulated manner, whereas compression inhibits aggrecan synthesis in a manner not regulated by transcriptional activity. / by Cameron A. Wheeler. / S.M.

Thesis (M. Eng.)--Massachusetts Institute of Technology, Biological Engineering Division, 2007. / Includes bibliographical references. / Cancer science is a heavily researched and rapidly changing field. Cutting edge research consistently reveals unique features of tumors that can be exploited for treatment. For example, it is well known that cells of varying tumor types have unique molecular markers and cell-surface receptors - fingerprints of sorts - that set cancerous cells apart from healthy cells. Furthermore, the tumor microenvironment has been explored to the point that its unique fluid mechanical and biochemical properties are well understood in the context of tumor growth and survival. However, very little of this research has penetrated clinical medicine. The purpose of this thesis is to present a recent concept in cancer therapy: an anti-cancer nanocell that is capable of the spatial and temporal targeting of drugs to tumor cells. The combination of targeting mechanisms designed into the nanocell is a product of our current understanding of tumors. The design serves to improve the effectiveness of inexpensive, out-of-patent cytotoxic and anti-angiogenic drugs to standards representative of modern research. Efforts towards improving the efficiency of the nanocell, with regards to both drug loading and tumor cell targeting, are presented and discussed. / (cont.) The synthesis of various polymer-ligand conjugates for use in the improved nanocell is reported, as is the development of a prodrug consisting of a generation three polyamidoamine dendrimer conjugated to methotrexate via an ester bond; cell studies demonstrating the effectiveness of the prodrug are included. The expandability of the nanocell is also explored, because ultimately, the nanocell must be robust enough to accommodate both tumor-type and population variability. / by Samir Awasthi. / M.Eng.

Thesis (M. Eng.)--Massachusetts Institute of Technology, Biological Engineering Division, 2006. / Includes bibliographical references (p. 73-79). / Neutrophils in the pulmonary microcirculation are subjected to mechanical deformation while traveling through capillaries of sizes much smaller than the mean neutrophil diameter. This deformation has been shown to result in significant reductions in both the shear storage and shear loss moduli of the cell, with subsequent recovery towards their initial values. Also, deformation above a threshold stimulus results in neutrophil activation, evidenced by pseudopod projection from the cell. These two events are thought to occur via independent pathways, yet little is known about the mechanosensing signaling involved. Other work has demonstrated that physiological deformation of neutrophils induces a marked increase in the levels of cytosolic calcium, suggesting that this occurrence may trigger the biomechanical response observed in the cell. The aim of this thesis was to elucidate the role of calcium in the neutrophil response to the mechanical deformation experienced during transit through the pulmonary capillaries. / (cont.) Chelating intracellular calcium in neutrophils resulted in (i) decreased deformability of the cells into a microchannel, (ii) attenuation of the drop in shear storage modulus (G') observed in untreated cells upon deformation, and (iii) shorter activation times. These findings suggest that cytosolic calcium holds an important function in the neutrophil transit through the capillaries, and inhibition of normal calcium release within the cell can lead to leukostasis-like conditions. / by Jeffrey J Hsu. / M.Eng.

Thesis (S.M.)--Massachusetts Institute of Technology, Biological Engineering Division, 2004. / Includes bibliographical references (p. 33-37). / Label-free detection is the detection of biomolecules and their interactions, without the use of a molecule external to the interaction, used as a reporter to indicate presence and/or location. The suspended microchannel resonator offers the opportunity to perform such label-free measurements. The goal of this work is to open new avenues of possible applications for the suspended channel. I introduce the concept of detecting mass subtraction as a new approach, rather than the conventional detection of mass addition. In a model implementation scenario of this approach, a mass-intensifying tag bound to a small ligand molecule will be equilibrated with surface-immobilized receptors, and later displaced by an identical, but label-free, ligand molecule. This approach offers opportunities to extend the sensitivity range of the device, as well as introduces new functionality for it. It enables researchers to follow, label-free, real-time enzymatic reactions, relative affinities of different ligands to a receptor, and presence of small molecules in a solution. / by Shelly Levy-Tzedek. / S.M.

Thesis (S.M.)--Massachusetts Institute of Technology, Biological Engineering Division, 2004. / Includes bibliographical references (leaves 59-61). / One of the major challenges in liver research today lay in the understanding of the complex relationship between liver structure and function. The highly orchestrated events that take place in the liver to maintain homeostasis require the presence of all liver cell types. In vivo experiments offer only a snapshot of the liver, and usually involve perturbation of normal function through injury or experimental disease. The role of cell-cell interactions in maintaining normal liver function is far less understood than in pathological conditions. This may be because of the lack of methods in monitoring normal function in vivo. Culturing systems may capture pieces of the puzzle, but often capture only two cell types, and involve mediators presented to the cells in concentrations much higher than physiological values. In addition, the liver lobule contains a 3-dimensional metabolic zonation, and liver cell types comprise a heterogeneous population from the portal triad where the blood flows into the sinusoids to the central vein area where the blood flows out of the sinusoids. Liver cell types are dynamic responders to environmental cues from soluble factors to heterotypic cell interactions, to extracellular matrix proteins. Therefore, a system that serves to promote the health of all liver cell types through a 3-dimensional, perfused scaffold, and allows for self-organization of the liver cells in response to the engineered environment, would serve as a useful tool in understanding some of these complex, orchestrated events. In the research presented here, methods were developed to detect and characterize the heterogeneous population that makes up the hepatic stellate cell population inside the liver bioreactor (Griffith et. al). / (cont.) This cell type, comprising a small percentage of total liver cells (approximately 5-10%), rapidly change their phenotype in response to liver injury, and, similarly, upon being taken out of the liver and cultured in 2-D on tissue culture plastic. This cell type plays a major role in relaying signals to and from both parenchymal and other nonparenchymal cells; stellate cells are also in charge of maintaining the components of the Space of Disse and are the key players in the pathology of liver fibrosis. They are found to be tightly complexed with sinusoidal endothelial cells and at the same time found to be tightly interacting with hepatocytes, sometimes even penetrating the hepatic plate. Stellate cell function, is therefore, highly dependent upon its interaction with other liver cells in maintaining the tightly knit structure-function relationship. For this reason, the liver bioreactor serves as a highly useful tool, in order to better understand the hepatic stellate cell's role in these complex situations. In this dissertation, detection and characterization methods are developed with the goal of capturing the heterogeneous stellate cell population as a whole with a toolbox of characterization markers, as well as to learn more about their functionality and location within tissue structures. These tools can be used to detect and characterize the population at various timepoints during tissue formation inside the bioreactor, as well as after exposure to physiologically-relevant concentrations of toxins, viruses, pharmaceuticals, etc. ... / author: Kathryn E. Wack. / S.M.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Biological Engineering Division, 2007. / MIT Science Library copy: printed in leaves. / Also issued printed in leaves. / Includes bibliographical references (p. 127-141). / Utilizing molecular recognition and self-assembly, material-specific biomolecules have shown great promise for engineering and ordering materials at the nanoscale. These molecules, inspired from natural biomineralization systems, are now commonly selected against non-natural inorganic materials through biopanning random combinatorial peptide libraries. Unfortunately, the challenge of studying the biological inorganic interface has slowed the understanding of interactions principles, and hence limited the number of downstream applications. This work focuses on the facile study of the peptide-inorganic interface using Yeast Surface Display. The general approach is to use combinatorial selection to suggest interaction principles followed by rational design to refine understanding. In this pursuit, two material groups-II-VI semiconductors and synthetic sapphire (metal oxides)-are chosen as inorganic targets due to their technological relevance and ease of study. First, yeast surface display (YSD) was established as a broadly applicable method for studying peptide-material interactions by screening a human scFv YSD library against cadmium sulfide (CdS), a II-VI semiconductor. The presence of multiple histidine residues was found to be necessary for mediating cell binding to CdS. As a follow-up, a systematic screen with yeast displayed rationally designed peptides was performed on a panel of II-VI semiconductors and gold. Cell binding results indicated that peptide interaction was mediated by a limited number of amino acids that were influenced by locally situated residues. Interpretation of the results facilitated design of new peptides with desired material specificities. Next, the nature of peptide/metal oxide binding interface was interrogated using sapphire crystalline faces as model surfaces. / (cont.) Biopanning a random peptide YSD library and subsequent characterization of the identified binding partners revealed the importance of multiple basic amino acids in the binding event. Study of rationally designed basic peptides revealed a preference for those amino acids to be spaced in such a manner that maximized simultaneous interaction with the surface. Fusing peptides to maltose binding protein (MBP) allowed for quantitative affinity measurement with the best peptides having low nanomolar equilibrium dissociation constants. Finally, peptides were demonstrated as facile affinity tags for protein immobilization in micro-patterning and biosensor assays. / by Eric Mark Krauland. / Ph.D.