Thesis (Ph. D.)--Massachusetts Institute of Technology, Biological Engineering Division, 2007. / Includes bibliographical references. / Our understanding of glycosaminoglycan (GAG) biology has been limited by a lack of sensitive and efficient analytical tools designed to deal with these complex molecules. GAGs are heterogeneous and often sulfated linear polys accharides found throughout the extracellular environment, and available to researchers only in limited mixtures. A series of sensitive label-free analytical tools were developed to provide sequence information and to quantify whole epitopes from GAG mixtures. Three complementary sets of tools were developed to provide GAG sequence information. Two novel exolytic sulfatases from Flavobacterium heparinum that degrade heparan/heparan sulfate glycosaminoglycans (HSGAGs) were cloned and characterized. These exolytic enzymes enabled the exo-sequencing of a HSGAG oligosaccharide. Phenylboronic acids (PBAs) were specifically reacted with unsulfated chondroitin sulfate (CS) disaccharides from within a larger mixture. The resulting cyclic esters were easily detected in mass spectrometry (MS) using the distinct isotopic abundance of boron. Electrospray ionization tandem mass spectrometry (ESI-MSn) was employed to determine the fragmentation patterns of HSGAG disaccharides. These patterns were used to quantify relative amounts of isomeric disaccharides in a mixture. Fragmentation information is valuable for building methods for oligosaccharide sequencing, and the general method can be applied to quantify any isomers using MSn. Three other tools were developed to quantify GAG epitopes. Two microfluidic devices were characterized as HSGAG sensors. Sensors were functionalized either with protamine to quantify total HSGAGs or with antithrombin-III (AT-III) to quantify a specific anticoagulant epitope. / (cont.) A charge sensitive silicon field effect sensor accurately quantified clinically relevant anticoagulants including low molecular weight heparins (LMWH), even out of serum. A mass sensitive suspended microchannel resonator (SMR) measured the same clinically relevant HSGAGs. When these two sensors were compared, the SMR proved more robust and versatile. The SMR signal is more stable, it can be reused ad infinitum, and surface modifications can be automated and monitored. The field effect sensor provided an advantage in selectivity by preferentially detecting highly charged HSGAGs instead of any massive, non-specifically bound proteins. Lastly, anti-HSGAG single chain variable fragments (scFv) were evolved using yeast surface display towards generating antibodies for HSGAG epitope sensing and clinical GAG neutralization. / by Jonathan Robert Behr. / Ph.D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Biological Engineering Division, 2007. / Includes bibliographical references. / The potential of gene therapy to treat disease and improve human health is tremendous. The failure of viral gene therapy clinical trials due to toxicity, immunogenicity, and carcinogenicity has been tragic and strongly motivates a non-viral approach. However, non-viral gene delivery is currently ineffective. Here, we show that polymeric nanoparticles composed of poly([beta]-amino esters) (PBAEs) and DNA can be formulated to be stable in the presence of serum proteins and have high gene delivery without toxicity to human primary cells. The biophysical properties of PBAE/DNA nanoparticles have good correlation to transfection efficacy when tested in the appropriate media conditions. We also show that electrostatic interactions can drive peptide coating of nanoparticles and enable ligand-specific gene delivery. A biphasic efficacy relationship exists for peptide weight ratio, overall charge ratio, and ligand length, with intermediate values of coating being optimal. A balance is required when seeking to design nanoparticles that have reduced nonspecific uptake, but increased ligand-specific uptake. We develop a high-throughput assay to quantify polymer/DNA binding as a gene delivery bottleneck and find that a biphasic relationship exists between polymer/DNA binding constant, Ka, and delivery efficacy. We also show that end-modified PBAEs can be as effective as adenovirus for gene delivery. In comparison to the previous "gold standard" for polymeric transfection, 25 kDa polyethylenimine, the PBAE nanoparticles presented here have 100x higher efficacy while simultaneously having 100x lower toxicity. Small structural changes were found to have dramatic effects on multiple steps of gene delivery including the DNA binding affinity, nanoparticle size, intracellular DNA uptake, and final protein expression. / (cont.) We show that small modifications to the termini of a polymer can significantly increase its in vivo activity and demonstrate potential utility of these polymers in the fields of cancer therapy, genetic vaccines, and stem cell engineering. As the enhanced polymeric gene delivery nanoparticles described here have many attractive properties over a virus including high safety, low immunogenicity, high nucleic acid cargo capacity, ease in manufacture, a coating method for targeted delivery, and flexibility for future design improvements, we believe that these polymeric nanoparticles may be promising alternatives for therapeutic gene delivery applications. / by Jordan Jamieson Green. / Ph.D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Biological Engineering Division, 2006. / Includes bibliographical references (leaves 108-122). / Many liver physiological and pathophysiological behaviors are not adequately captured by current in vitro hepatocyte culture methods. A 3D perfused microreactor previously demonstrated superior hepatic functional maintenance than conventional 2D cultures, and was hypothesized to provide an environment favorable to endothelial cell maintenance and morphogenesis. This dissertation focuses on characterizing the 3D perfused co-culture of primary hepatocyte fraction with primary rat liver endothelial isolate. Scanning electron microscopy revealed significantly higher numbers of pore-like structures on the co-culture tissue surface resembling liver sinusoids compared to cultures containing only the hepatocytes fraction (mono-culture). EGFP-labeled endothelial cells proliferated moderately and organized into microvessel-like structures as observed by in situ multi-photon microscopy. By mixing female endothelial cells with male hepatocytes, the female cell population increased from initially -7% on day 1 to -12% on day 13, as determined by quantitative PCR on genomic DNA. The maintenance and morphogenesis of endothelial cells were not observed in parallel 2D collagen gel sandwich cultures. Immunohistochemistry further confirmed the presence of sinusoidal endothelia within the 3D co-culture tissue, as well as other non-parenchymal cells in both 3D mono-culture and co-culture. / (cont.) Global transcriptional profiling confirmed the loss of endothelia in 2D culture as the comparison between mono-culture and co-culture showed substantial differential expression levels only in the 3D format. The majority of the genes expressed substantially higher in 3D co-culture than mono-culture was found to be endothelia-specific. A group of key liver metabolism genes, however, do not show significant expression differences between the 3D cultures. This study concludes that the 3D perfused microreactor maintains non-parenchymal cells better than the 2D format, and the retention of non-parenchymal cells in the primary hepatocyte fraction likely contributes to the maintenance of key hepatic function gene expression. Additional endothelial cells organize into microvessel-like structures in this environment, but exert little influence on the gene expression of most key liver transcription factors and metabolism enzymes. Therefore 3D cultures may eliminate the need of co-cultures for applications focusing on metabolic behaviors of hepatocytes, and 3D endothelial-hepatocyte co-cultures may prove useful in studies where proper endothelium structure is required, such as cancer metastasis. / by Albert J. Hwa. / Ph.D.

Thesis (Ph. D. in Genetic Toxicology)--Massachusetts Institute of Technology, Biological Engineering Division, 2003. / Includes bibliographical references. / Although homologous recombination provides an efficient means for repairing and tolerating DNA damage, mitotic recombination between misaligned sequences can lead to loss of genetic information (e.g. deletions, translocations and loss of heterozygosity). Given that such genetic changes may promote tumorigenesis, it is critical to identify those genetic and environmental factors that render cells susceptible to homologous recombination. Our goal is to elucidate the mechanisms of DNA damage-induced recombination and to determine the role of DNA repair enzymes in modulating homologous recombination in eukaryotic cells. Alkylating agents are abundant in our environment and are generated endogenously as normal metabolites. In addition to their mutagenic and cytotoxic effects, alkylating agents stimulate homologous recombination in eukaryotic cells. Removal of alkylated bases by DNA glycosylases, such as the Magl 3-methyladenine (3MeA) DNA glycosylase, initiates the base excision repair (BER) pathway. To investigate the molecular basis for methylation-induced homologous recombination in S. cerevisiae, intrachromosomal recombination was measured under conditions where MAGI expression levels were varied. Cells lacking Magl show increased susceptibility to methylation-induced recombination, suggesting that unrepaired 3MeA lesions induce recombination. Overexpression of M4GI also elevates recombination levels, presumably due to the accumulation of recombinogenic BER intermediates. / (cont.) To study the relative importance of specific DNA repair enzymes in modulating recombination in mammals, we have engineered transgenic mice that make it possible to quantify homologous recombination events in primary somatic cells, both in vitro and in vivo. The FYDR (fluorescent yellow direct repeat) mice carry two different mutant copies of an expression cassette for enhanced yellow fluorescent protein (EYFP) arranged in a direct repeat. Homologous recombination between these truncated sequences restores expression of EYFP. Using flow cytometry, spontaneous and DNA damage-induced recombination events were quantified in primary fibroblasts cultured from embryonic and adult tissues. In addition, recombination events that occurred in vivo were detected directly in disaggregated skin cells. Currently, FYDR mice are being crossed with mice carrying engineered defects to determine how specific gene traits modulate susceptibility to mitotic recombination. Ultimately, this tool will help us better understand how environmental agents and specific genes influence cellular susceptibility to cancer-promoting recombination events in mammals. / by Carrie A. Hendricks. / Ph.D.in Genetic Toxicology

Thesis (Ph. D.)--Massachusetts Institute of Technology, Biological Engineering Division, 2006. / "June 2006." / Includes bibliographical references. / Glycans are chemically heterogeneous macromolecules that have profound importance in a variety of biological processes. Located at the surfaces of cells, deposited in the extracellular matrix, or attached to soluble signaling agents, these molecules are characterized by a structural complexity that has thus far prevented their widespread exploitation in biomedicine. Insight into the fine structure and sequence of these complex biomolecules provides a novel niche for the development of therapeutic interventions. Such an understanding is rapidly accumulating via the molecular cloning, recombinant genetic expression, and protein purification of glycosaminoglycan-degrading enzymes. To this end, enzymes from the chondroitinase family of Pedobacter heparinus and Proteus vulgaris were developed. This library of enzymatic tools will reveal glycoconjugate structure-function motifs, allowed for by structural elucidation of glycan species using coupled bioinformatics/analytical chemistry techniques centered on mass spectrometry, nuclear magnetic resonance, and capillary electrophoresis. / (cont.) Biochemical characterization of these enzymes has allowed for the rational genetic manipulation of substrate recognition and binding site amino acid residues, producing site-directed protein engineered mutants with altered action pattern and substrate specificity. Progress in these areas will allow for the elucidation of critical roles of glycans in the biological regulation of growth factors, morphogens, cytokines, and cell-surface proteins. This extension of the capabilities of glycan analytical biotechnologies will help to translate basic science glycobiology to applied glycomedicine and drug discovery. / by Vikas Prabhakar. / Ph.D.

Thesis (Ph. D. in Genetic Toxicology)--Massachusetts Institute of Technology, Biological Engineering Division, 2004. / This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections. / Includes bibliographical references (p. 139-161). / It is a widely-held hypothesis that environmental mutagens play an essential role in human somatic and germinal cell mutagenesis. In particular, the finding of small amounts of chemical mutagens in cigarette smoke has led to the general hypothesis that mutagens in cigarette smoke induce oncomutations and thus account for the carcinogenic effect of cigarette smoking in human lungs. However, this hypothesis has not been tested by an assay of nuclear point mutations in lungs of smokers and nonsmokers. Mismatch amplification mutation assay (MAMA), an effective form of allele-specific PCR, was applied for detection of point mutations in TP53 bp742, bp746 and bp747, K-ras bp35 and HPRT bp508 from a total of 291 tracheal-bronchial epithelial sectors from six smokers and nine non-smokers, yielding 949 individual mutational assays. The conditions of MAMA for each target point mutations were optimized such that the sensitivity of each was equal to or below 10⁻⁵. Lung epithelial sectors of 2.3x10⁶ cells in average contained 0-200 mutant cells in general, equivalent to mutant fractions (MFs) of 0-10⁻⁴ with an exception of rare sectors with MF larger than 4x 10⁻⁴ (4.6%). Noticeably, the distributions of the MFs among sectors did not vary appreciably with the donor's smoking status. The mean MFs per lung were very similar between smokers and non-smokers for all five target mutations assayed (p >> 0.05). The mean MFs were slightly higher in females than males (p = 0.015). The mean MFs increased with age of the subjects although the correlation did not reach statistical significance due to large variances within the same age group. The distributions of MF among sectors of smokers and non-smokers did not differ significantly by Kolmogorov-Smirnov test / (cont.) for all target mutations but HPRT. By using hypothetical turnover unit sizes and Poisson distribution, the turnover unit size of human tracheal bronchial epithelium was estimated as 64 cells (p = 0.05). These observations do not support the widely-held hypothesis that cigarette smoking causes lung cancer through its induction of point mutations in nuclear genes. The current findings demonstrate the necessity of investigation on alternative mechanisms for tobacco smoke in lung carcinogenesis. / by Hiroko Sudo. / Ph.D.in Genetic Toxicology

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.