Thesis (Ph. D.)--Massachusetts Institute of Technology, Biological Engineering Division, 2002. / Includes bibliographical references (leaves 193-218). / Specific interactions between heparan sulfate glycosaminoglycans (HSGAGs) and proteins are central to a wide range of biological processes such as anticoagulation, angiogenesis and growth factor activation. The specificity involved in the HSGAG-protein interactions stems from the structural heterogeneity of HSGAGs, which are highly acidic biopolymers associated on the cell surface and in the extracellular matrix. It is believed that structural specificity in the HSGAG-protein interactions determines the biological functions mediated by HSGAG-binding proteins such as basic fibroblast growth factor (FGF2). A number of models have been proposed to account for the mode of FGF-FGFR interactions and the role of HSGAGs in modulating FGF2 signaling. It was hypothesized that one role played by HSGAGs was to stabilize FGF2 oligomers in a "side-by-side" or cis fashion for presentation to fibroblast growth factor receptor (FGFR). In this thesis research, we systematically examined different proposed modes of FGF2 dimerization and showed that extensive oligomerization of a FGF2 mutant protein could be achieved by oxidatively crosslinking. Heparin, a highly sulfated form of HSGAGs, was demonstrated to increase the extent of oligomerization. Therefore, the results reported here were consistent with the hypothesis that HSGAGs promoted FGF2 oligomerization in a "side-by-side" mode. The functional significance of a FGF2 dimer was tested using a genetically engineered dimeric FGF2 (dFGF2). Biochemical and biophysical properties of dFGF2, such as protein folding, heparin affinity and receptor / (cont.) binding, were assayed. dFGF2 was found to exhibit higher activities in stimulating cell proliferation and cell survival in vitro compared with the monomeric wildtype. An in vivo rat cornea pocket model further corroborated the in vitro findings. The functional role of HSGAGs derived from the cell surface was studied here. It was found that distinct HSGAG fragments released by heparinase treatment were capable of modulating FGF2-stimulated cell proliferation depending on the expression of FGFR isoforms. This founding is consistent with the proposal that structural specificity of distinct HSGAG fragments dictated the interaction of HSGAGs with FGF and FGFR. The role of heparinase-generated HSGAG fragments in inhibiting cell proliferation was investigated. B16 melanoma cells treated with heparinase III were found to exhibit biochemical and morphological hallmarks of apoptosis. Conditioned medium derived from heparinase-treated cells was shown to be effective in suppressing cell growth. Gene array experiments and caspase activity assays further suggested that apoptotic cell death was mediated through a caspase 8-, death receptor-dependent pathway. Thus, the present study lends further credence to the proposal that cell surface HSGAGs plays a critical role in orchestrating cell phenotype. This thesis work provides the framework for understanding the molecular mechanism of growth factor activation and the structure-function relationship of HSGAG-mediated cell signaling. Results from this study may potentially be useful for therapeutic protein engineering and carbohydrate-based drug discovery. / by Chi-Pong Kwan. / Ph.D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Biological Engineering Division, September 2006. / "July 2006." / Includes bibliographical references (leaves 123-137). / Connective tissue progenitors (CTP) can act as a pluripotent source of reparative cells during injury and therefore have great potential in regenerative medicine and tissue engineering. However, the response of CTP to most growth factors and cytokines is unknown. Many envisioned applications of CTP, such as treating large defects in bone, involve in vivo implantation of CTP attached to a scaffold, a process that creates an acute inflammatory environment that may be hostile to CTP survival. This project entails the design of a two-component polymeric implant system to aid in the healing process of bony defects by influencing cell behaviors at the implant site through the covalent modification of the implant surface with selected ligands. We investigate cellular responses of CTP on a biomaterial surface covalently modified with epidermal growth factor (EGF) and find that surface-tethered EGF (tEGF) promotes both cell spreading and survival more strongly than saturating concentrations of soluble EGF. By sustaining MEK-ERK signaling, tEGF increases the contact of CTP with an otherwise moderately adhesive synthetic polymer and confers resistance to apoptosis induced by the proinflammatory cytokine, FasL. / (cont.) We confirm that these signaling, spreading, and apoptotic responses are conserved across three sources of CTP: an hTERT-immortalized human mesenchymal stem cell (MSC) line, primary porcine bone-marrow CTP, and primary human bone-marrow-derived CTP. We conclude that tEGF may offer a protective advantage to CTP in vivo during acute inflammatory reactions to tissue engineering scaffolds. The tEGF-modified polymers described here could be used together with structural materials to construct CTP scaffolds for the treatment of hard-tissue lesions, such as large bony defects. / by Vivian H. Fan. / Ph.D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Biological Engineering Division, February 2006. / Includes bibliographical references (leaves 181-191). / Surface-based sensors take advantage of the natural high surface-to-volume ratios in microfluidic devices, low reagent consumption and high potential for integration in more complex micro total analysis systems (microTAS or pTAS). This thesis studies the fundamental limits of on-chip integrated microfluidic sensors. More specifically, it focuses on detection methods involving surface interaction in channels with thicknesses on the order of a few microns or less. Through mass transfer analysis, we demonstrate that, for thin enough channels, sample detection is limited by the convective transport of analytes, and neither by diffusion nor reaction. The results provided extend the validity of transport models to include transport in the absence of mass transfer boundary layer. All existing analytic solutions to the Graetz problem are described and compiled. The analysis, complemented by finite element simulations, successfully predicts experimental observations made for on-chip immunoassays in micron-thick fluidic channels. Subsequently, our study of on chip detection systems is carried on with emphasis on resonating cantilever sensors. In order to interpret the output signal from these devices, we develop a dynamic cantilever model to link spatially and temporally dependent mass adsorption with resonance frequency change. / (cont.) The mass adsorption is then directly related to the sensors' operating conditions via the mass transfer models previously developed. We then develop a 2D finite-element model capable of predicting the devices response and of extracting bimolecular rate constants. Finally, since hydraulic resistance severely increases as channels get shallower, we provide a structural analysis of polymer-based microsystems. Through scaling and numerical simulations we demonstrate the effect of channel deformation on the flow conditions inside the device and vice versa. Finally, channel deformation is experimentally quantified using optical methods and compared with the models developed. Throughout this thesis, the approach to physical modeling has been to use mathematical and numerical analysis as predictive tools in the design of integrated lab-on-a-chip systems. Whenever possible, scaling and analytic solutions are developed, since they provide a direct relationship between experimental observations, geometry and the multiple dependent variables in the system, and can be readily used as design criteria by the experimentalist. / by Thomas Gervais. / Ph.D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Biological Engineering Division, 2008. / Includes bibliographical references (p. 224-236). / Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease with diagnostics and treatments that are ineffective at stopping the progression. This thesis examines new ways of both diagnosing and treating ALS, including 1) a gadolinium tetanus toxin C fragment (Gd-TTC) biomarker for axonal retrograde transport, 2) TTC-conjugated biodegradable nanoparticles, and 3) poly(glycerol-co-sebacate) acrylate (PGSA) and 3D scaffolds for human embryonic stem cell (hESC) and neuronal encapsulation.A Gd-TTC conjugate was developed and characterized that was shown to be highly visible under MRI and preserved the functionality of the native TTC protein in vitro. Live animal MRI imaging and immuno fluorescent staining of the spinal cord showed that the conjugate was transported to the central nervous system (CNS) and localized in motor neurons. H&E staining and biodistribution studies showed that GdTTC was well tolerated and bio available. Quantification of MRI and staining images showed that Gd-TTC was retrograde transported and that that this rate decreased during the disease progression of ALS in a transgenic mouse model, suggesting that Gd-TTC could be used as a biomarker for neurodegenerative diseases.TTC-conjugated nanoparticles were developed by synthesizing PLGA-PEG-biotin and using biotin binding proteins (avidin, streptavidin, and neutravidin) to specifically conjugate TTC to the nanoparticle surface. TTC nanoparticles were shown to selectively target neurons and not other cell types in vitro. / (cont.) Subsequent in vivo experiments showed that nanoparticles were well tolerated and that TTC was co-localized with neurons unilaterally, suggesting that TTC-conjugated nanoparticles may be a useful drug delivery system. Porous PGSA scaffolds were prepared and characterized by porosity, swelling, mass loss, toxicity and mechanical properties, and subsequently used to encapsulated hESC and neuroblastoma cells in vitro. Neuroblastoma cells proliferated and formed matrix fibrils, and fluorescent staining of undifferentiated hESCs showed the presence of all three germ layers. In vivo experiments showed that porous PGSA scaffolds were well-tolerated and promoted vascular ingrowths. / by Seth A. Townsend. / Ph.D.

Thesis (M. Eng.)--Massachusetts Institute of Technology, Biological Engineering Division, 2007. / Includes bibliographical references (p. 53-57). / Alternative splicing is involved in numerous cellular functions and is often disrupted and involved in disease. Previous research has identified methods to distinguish alternative conserved exons (ACEs) in human and mouse. However, the cellular machinery, the spliceosome, does not use comparative genomics to decide when to include and when to exclude an exon. Human RefSeq exons obtained from the University of California Santa Cruz (UCSC) genome browser were analyzed for tissue-specific skipping. Expressed sequence tags (ESTs) were aligned to exons and their tissue of origin and histology were identified. ACEs were also identified as a subset of the skipped exons. About 18% of the exons were identified as tissue-specifically skipped in one of sixteen different tissues at four stringency levels. The different datasets were analyzed for both general features such as exon and intron length, splice site strength, base composition, conservation, modularity, and susceptibility to nonsense-mediated mRNA decay caused by skipping. Cis-element motifs that might bind protein factors that affect splicing were identified using overrepresentation analysis and conserved occurrence rate between human and mouse. / (cont.) Tissue-specific skipped exons were then classified with both a decision-tree based classifier (Random ForestsTM) and a support vector machine. Classification results were better for tissue-specific skipped exons vs. constitutive exons than for tissue-specific skipped exons vs. exons skipped in other tissues. / by Craig Jeremy Rothman. / M.Eng.

Thesis (M. Eng.)--Massachusetts Institute of Technology, Biological Engineering Division, 2005. / Includes bibliographical references (p. 77-80). / This study aims to investigate the relationships between growth parameters (agitation, glycerol concentration, salt concentration) and responses (biomass, growth rate, protein expression), by a 3-factor-3-level central composite factorial design. This experimental design involved running shake flask culture at 15 different experimental conditions with duplicates. Optical density (OD600), dry cell weight (DCW), and BCA Protein Assays were done on each experiment. Mathematical models in terms of these parameters' effects and their interactions were proposed for each of the responses. The significance of each effect and interaction, as well as the goodness-of-fit of mathematical models to data were examined by analysis of variance. It was found that biomass (with R²Adj=0.951) is a strong function of glycerol concentration (higher glycerol concentration leads to higher biomass), but it varies much less with agitation, and it is completely independent of salt concentration. Growth rate (R²Adj=0.901), however, varies strongly with agitation and salt concentration, but much more weakly with glycerol concentration. Protein production has a low R²Adj value of 0.746, implying that higher-order terms, e.g. x₁² and x₂², should be tested for significance in the model. / (cont.) Collected data were fitted to the proposed models by response surface regression, after which surface and contour plots of responses were generated to identify trends in them. High agitation (300 rpm in shaker) gave rise to both highest biomass and growth rate. In addition, biomass at high glycerol concentration (3% v/v) was almost twice as much as biomass at low glycerol concentration (1% v/v) at high agitation rate (19 g/L compared to 11 g/L). At the same agitation rate, growth rate shows the largest increase of 20.5% with increasing salt concentration from 0.7% to 2.1%. Protein production reached maximum of 7.3 mg/mL at medium agitation rate (250 rpm), high salt and glycerol concentrations. / by Joyce Chan. / M.Eng.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Biological Engineering Division, 2008. / "September 2008." / Includes bibliographical references and index. / Glioblastoma multiforme (GBM) is the most aggressive adult brain tumor and remains incurable despite multimodal intensive treatment regimens. EGFRvIII is a truncated extracellular mutant of the EGF receptor (EGFR) that is commonly found in GBMs and confers tumorigenic behavior. Although much work has been done over the past decade to elucidate pathways involved in EGFRvIII receptor signaling, the global map of the signaling networks that it activates remains incomplete, making it difficult to assess downstream components involved in EGFRvill-mediated transformation. To gain a molecular understanding of the mechanisms by which EGFRvIII acts, we have employed a mass spectrometry-based phosphoproteomic approach to quantitatively map cellular signaling events activated by this receptor. Using this approach, we have determined the major downstream pathways activated as a function of titrated EGFRvIII receptor levels. This analysis highlighted several aspects of EGFRvIII tumor biology, including crosstalk between EGFRvIII and other receptor tyrosine kinases. Specifically, we have identified the c-Met receptor as a co-target in the treatment of EGFRvIII positive GBM cells, and have shown that an EGFR and c-Met combination inhibitor strategy may be applicable in overcoming the poor efficacy of EGFR kinase inhibitor monotherapy in GBM patients. We then went on to investigate the mechanisms by which signaling networks are regulated in response to site-specific tyrosine mutations on EGFRvIII. This analysis has revealed a receptor compensation mechanism that is capable of restoring network architecture, upon the loss of a major tyrosine phosphorylation site on EGFRvIII. / (cont.) This is, to our knowledge, the first demonstration of signal compensation at the level of receptor phosphorylation and highlights an unexpected level of complexity within the signaling network. Our data also indicates that EGFRvIII fine-tunes the activity of the Erk pathway; some Erk activity is required for growth but excessive pathway activation results in cell death. We believe that the sensitivity to modulation of the Erk pathway may be exploited as a potential means of therapy for EGFRvIII positive tumors. Taken together, our study highlights the utility of quantitative phosphoproteomic analysis as a tool to gain molecular insights in cancer biology and a means for drug target discovery. / by Hua Ming Paul Huang. / Ph.D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Biological Engineering Division, 2006. / Vita. / Includes bibliographical references. / The continued success of protein therapeutics has put a strain on industry's ability to meet the large demand. Creating a more productive expression host for the manufacture of these proteins is a potential solution. Although heterologous proteins are frequently made in organisms as disparate as E. coli and bovines, the single-celled organism S. cerevisiae has emerged as a well-qualified candidate due to its approachable genetic and fermentation attributes as well as its ability to stably fold disulfide bonded and multi domain proteins. Because S. cerevisiae screens for enhanced protein secretion have traditionally utilized low-throughput and often plate-based methods, a high-throughput, liquid phase assay could offer a real advantage in secretory selection. In this thesis, yeast surface display is investigated as a potential proxy for heterologous protein secretion. Although ultimately unsuitable as a screening proxy, the surface display experiments did show a novel method of improving protein secretion by co-expressing a more stably folded protein with the protein of interest. In these studies the secretion of an scFv-Aga2p fusion was stimulated 1 0-fold by the concomitant surface expression of BPTI. / (cont.) BPTI surface expression also stimulated the secretion of secreted scFv three-fold suggesting a niche for protein coexpression as well as secretion by way of Aga2p fusions. A new screening method was developed that involves the capture of secreted protein on the surface of the cell where it can be labeled and sorted by FACS. This new method was verified to achieve thirty-five fold enrichment per pass for a three-fold enhanced protein secretor making it easily suitable for screening. The new screening methodology, the Cell Surface Secretion Assay (CeSSA), was also modeled and verified with time course data that enabled optimization of sort parameters and predicted sort outcomes based on user-derived selection parameters. The CeSSA was used to screen a library of mutant yeast alpha mating factor leader sequences for improved secretion of the scFv 4m5.3. The improved leaders imparted up to a twenty-fold improvement in scFv secretion per cell and up to thirty-fold improvement after expression tuning. These engineered leader sequences also conferred improved secretion on other scFv's and proteins including whole IgG. Moreover, the leader sequence mutants give indications of where the important residues in secretory leaders lie and the aberrations in protein traffic that result in reduced secretion. / by James A. Rakestraw. / Ph.D.

Thesis (Ph. D. in Applied Biosciences)--Massachusetts Institute of Technology, Biological Engineering Division, 2004. / Includes bibliographical references (leaves 119-130). / Tissue inhibitors of metalloproteinases (TIMPs) regulate tumor growth, progression and angiogenesis in a variety of experimental cancer models and in human malignancies. However, numerous studies have revealed important differences between TIMP family members in their ability to inhibit angiogenic processes in vitro and angiogenesis in vivo despite their universal ability to inhibit matrix metalloproteinase (MMP) activity. To address these differences, structure-function studies were conducted to identify and characterize the anti-angiogenic domains of TIMP-2, the endogenous MMP inhibitor that uniquely inhibits capillary endothelial cell (EC) proliferation and angiogenesis in vivo. Only the carboxy-terminal domain of TIMP-2 (T2C) and not the MMP-inhibitory N-terminal domain (T2N), inhibited capillary EC proliferation. Although both T2N and T2C inhibited embryonic angiogenesis, only T2C potently inhibited mitogen-stimulated angiogenesis. These findings demonstrate that TIMP-2 possesses two distinct types of anti-angiogenic activities which can be uncoupled from each other. The anti-proliferative activity of T2C was further mapped to the 24-amino acid peptide, Loop 6, which proved to be a potent inhibitor of both embryonic and nitogen-stimulated angiogenesis in vivo. Initial studies into the mechanism(s) by which Loop 6 inhibits angiogenesis revealed that the anti-proliferative effects of Loop 6 are due, at least in part, to the inhibition of cell cycle progression and not to the induction of apoptosis. This inhibition was associated with increased levels of cell cycle inhibitor p27. Although Loop 6 did not compete with bFGF for binding to its receptor, / (cont.) five potential cell surface complexes were observed in crosslinking studies of capillary EC treated with ¹²⁵I-labeled T2C or Loop 6. Finally, given the high degree of homology between TIMP-2 and TIMP-4, we hypothesized that TIMP-4 might share anti-proliferative and MMP inhibition- independent anti-angiogenic activities with TIMP-2. Our results demonstrate that although TIMP-4 inhibits capillary EC migration, it does not inhibit capillary EC proliferation. Furthermore, TIMP-4 did not result in significant inhibition of embryonic angiogenesis in the CAM. These results suggest that TIMP-2 is unique among TIMP family members in its ability to inhibit angiogenesis via two distinct pathways. One of these activities, housed within Loop 6, results in the potent inhibition of angiogenesis in vivo. / by Cecilia A. Fernández. / Ph.D.in Applied Biosciences

Thesis (Ph. D.)--Massachusetts Institute of Technology, Biological Engineering Division, February 2008. / Vita. / Includes bibliographical references. / Oxidatively damaged DNA has been strongly associated with cancer, chronic degenerative diseases and aging. Guanine is the most frequently oxidized base in the DNA, and generation of a guanine radical cation (G'") as an intermediate in the oxidation reaction leads to migration of a resulting cationic hole through the DNA n-stack until it is trapped at the lowest-energy sites. These sites reside at runs of guanines, such as 5'-GG-3' sequences, and are characterized by the lowest sequence-specific ionization potentials (IPs). The charge transfer mechanism suggests that hotspots of oxidative DNA damage induced by electron transfer reagents can be predicted based on the primary DNA sequence. However, preliminary data indicated that nitrosoperoxycarbonate (ONOOCO2"), a mediator of chronic inflammation and a one-electron oxidant, displayed unusual guanine oxidation properties that were the focus of present work. As a first step in our study, we determined relative levels of guanine oxidation, induced by ONOOCO2 in all possible three-base sequence contexts (XGY) within double-stranded oligonucleotides. These levels were compared to the relative oxidation induced within the same guanines by photoactivated riboflavin, a one-electron reagent. We found that, in agreement with previous studies, photoactivated riboflavin was selective for guanines of lowest IPs located within 5'-GG-3' sequences. In contrast, ONOOCO2" preferentially reacted with guanines located within 5'-GC-3' sequences characterized by the highest IPs. This demonstrated that that sequence-specific IP was not a determinant of guanine reactivity with ONOOCO2". Sequence selectivities for both reagents were double-strand specific. Selectivity of ONOOCO2 for 5'-GC-3' sites was also observed in human genomic DNA after ligation-mediated PCR analysis. / (cont.) Relative yields of different guanine lesions produced by both ONOOCO2" and riboflavin varied 4- to 5-fold across all sequence contexts. To assess the role of solvent exposure in mediating guanine oxidation by ONOOCO2", relative reactivities of mismatched guanines with ONOOCO2" were measured. The majority of the mismatches displayed an increased reactivity with ONOOCO2 as compared to the fully matched G-C base-pairs. The extent of reactivity enhancement was sequence context-dependent, and the greatest levels of enhancement were observed for the conformationally flexible guanine- guanine (G-G) mismatches and for guanines located across from a synthetic abasic site. To test the hypothesis that the negative charge of an oxidant influences its reactivity with guanines in DNA, sequence-selective guanine oxidation by a negatively charged reagent, Fe+2-EDTA, was assessed and compared to guanine oxidation produced by a neutral oxidant, y-radiation. Because both of these agents cause high levels of deoxyribose oxidation, a general method to quantify sequence-specific nucleobase oxidation in the presence of direct strand breaks was developed. This method exploited activity of exonuclease III (Exo III), a 3' to 5' exonuclease, and utilized phosphorothioate-modified synthetic oligonucleotides that were resistant to Exo III activity. This method was employed to determine sequence-selective guanine oxidation by Fe+2-EDTA complex and y-radiation and to show that both agents produced identical guanine oxidation pattems and were equally reactive with all guanines, irrespective of their sequence-specific IPs or sequence context. / (cont.) This showed that negative charge was not a determinant of Fe+2-EDTA-mediated guanine oxidation. Finally, the role of oxidant binding on nucleobase damage was assessed by studying sequence-selective oxidation produced by DNA-bound Fe+2 ions in the presence of H202. We found that the major oxidation targets were thymines located within 5'-TGG-3' motifs, demonstrating that while guanines were a required element for coordination of Fe+2 to DNA, they were not oxidized. Our results suggest that factors other than sequence-specific IPs can act as major determinants of sequence-selective guanine oxidation, and that current models of guanine oxidation and charge transfer in DNA cannot be used to adequately predict the location and identity of mutagenic lesions in the genome. / by Yelena Margolin. / Ph.D.