Thesis (Ph. D. in Bioengineering)--Massachusetts Institute of Technology, Biological Engineering Division, 2003. / Leaf 117 blank. / Includes bibliographical references. / A goal for gene delivery research is to design vectors capable of (a) delivering transgenes to target cells, (b) yielding efficient gene expression, and (c) minimizing any immune, inflammatory, or cytotoxic response. Current research has focused on developing such vehicles using end gene expression as the benchmark. While transgene protein production is the overall objective of successful gene delivery, such qualitative treatment of gene delivery, especially for non-viral vectors, may result in unoptimized vectors and potential rate limiting steps unidentified. Quantitative analysis of the gene delivery pathway is essential for the characterization, comparison, and design of vectors. The complex nature of the mechanisms for gene delivery, particularly at the cellular level, contains multiple potentially rate limiting steps to successful gene expression. Through quantitative methodologies, vector efficacy can be related to molecular characteristics and specific processes within the gene delivery pathway. These potentially rate limiting steps include, but are not limited to, cell surface association, subcellular trafficking, endosomal escape, nuclear translocation, vector unpackaging, and gene expression. Design of synthetic gene delivery vectors seeks to develop molecular systems mimicking virus-like infection behavior, including cell membrane attachment and rapid internalization followed by endosomal escape, nuclear localization, and finally gene expression. To explore such opportunities for vector optimization and design, a model human hepatocellular carcinoma cell line by sets of transfection agents complexed with a plasmid. Time courses of plasmid numbers were determined both from whole cells and from isolated nuclei by real-time quantitative PCR. / (cont.) This enabled determination of values for parameters characterizing the key intracellular trafficking processes for a validated mass-action kinetic model of cellular gene delivery, in concert with model parameter values obtained from literature data. Quantitative parameter sensitivity analyses were performed for the individual gene delivery vectors, permitting elucidation of the particular rate-limiting processes specific to each vector. The resulting model predictions were then extended to test effect of increased delivery by polyethylenimine based gene delivery vectors and thus model utility. Additionally, viral vector performance was measured, providing insight into the extreme efficiency of such vectors. As no single process was found to be rate-limiting for all vectors, nor was the rate-limiting process necessarily the kinetically-slowest process. Thus, a single design factor will not likely improve all types of vectors, but rather each vector must be improved with respect to its own specific rate-limiting process(es) and improvements in vehicle design may best arise from quantitative analysis of the contributions of each to the integrated system operation. / by Csanad M. Varga. / Ph.D.in Bioengineering

Thesis (Ph. D. in Applied Biosciences and Biotechnology)--Massachusetts Institute of Technology, Biological Engineering Division, 2003. / Includes bibliographical references (p. 113-123). / Glycosaminoglycans (GAGs) are complex polysaccharides that reside in the extracellular matrix and on the surfaces of all cells. The same complexity that contributes to the diversity of GAG function has also hindered their chemical characterization. Recent progress in coupling bacterial GAG-degrading enzymes with sensitive analytical techniques has led to a revolution in understanding the structure-function relationship for an important subset of GAGs, namely heparin/heparan sulfate-like glycosaminoglycans (HSGAGs). The study of chondroitin sulfate and dermatan sulfate (CS/DS), an equally important subset of GAGs, has lagged behind partially due to a lack of enzymatic and analytical tools akin to those developed for HSGAGs. The Flavobacterial heparinases have proven indispensable in characterizing the fine structure of HSGAGs responsible for their different biological functions. As a continuation of ongoing research, a combination of chemical modification, peptide mapping, and site-directed mutagenesis was employed to explore the role of histidine in the activity of heparinase III. Of the thirteen histidines in the enzyme, His295 and His510 were found to be critical for the degradation of heparan sulfate by heparinase III. As a first step to developing the chondroitinases as enzymatic tools for the characterization of CS/DS oligosaccharides, recombinant expression and purification schemes were developed for chondroitinase AC and B from Flactobacterium heparinum. The recombinant enzymes were characterized using biochemical techniques and kinetic parameters were determined for their respective CS/DS substrates. / (cont.) By combining the modeling a tetrasaccharide substrate into the active site of chondroitinase B with site-directed mutagenesis studies, a variety of residues were identified as critical for substrate binding and catalysis. A subsequent co-crystal structure of chondroitinase B with DS-derived hexasaccharide revealed a catalytic role for a calcium ion and provided further clarity into the role of individual active site amino acids. Additionally, using a variety of defined DS-derived oligosaccharides coupled with sensitive analytical techniques, chondroitinase B was identified as an endolytic, non-random, non-processive enzyme that preferentially cleaves longer oligosaccharides compared to shorter ones. Taken together, these studies represent a critical step in developing the chondroitinases as enzymatic tools for the characterization of CS/DS oligosaccharides in a fashion akin to the use of the heparinases to characterize HSGAGs. / by Kevin R. Pojasek. / Ph.D.in Applied Biosciences and Biotechnology

Thesis (Ph. D.)--Massachusetts Institute of Technology, Biological Engineering Division, 2008. / This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections. / Includes bibliographical references (leaves 148-156). / Mechanical stimulation of 3-D chondrocyte cultures increases extracellular matrix (ECM) production and mechanical stiffness in regenerating cartilage. The goal of this study was to examine the progression of chondrocyte signaling responses to mechanical stimulation in 3-D culture during tissue regeneration. To investigate the role of integrins in chondrocyte mechanotransduction, function-blocking antibodies and small-molecule antagonists were used to disrupt integrin-matrix interactions during dynamic compression of chondrocytes in 3-D agarose culture. At early days in culture, blocking [alpha]v[beta]3 integrin abolished dynamic compression stimulation of proteoglycan synthesis, independent of effects in free-swell culture, while blocking [alpha]5[beta]1 integrins abolished the effect of compression only when blocking in free-swell increased proteoglycan synthesis. This suggests that disrupting [alpha]v[beta]3 and [alpha]5[beta]1 interactions with the ECM influences proteoglycan synthesis in distinct pathways and that [alpha]v[beta]3 more directly influences the mechanical response. To further distinguish individual mechanotransduction pathways, we investigated the temporal gene transcription response of chondrocytes to ramp-and-hold compression on Days 1, 10, and 28 in 3-D agarose culture. Clustered and individual gene expression profiles changed temporally and in magnitude over time in culture. Day 1 cultures differed from Days 10 and 28, reflecting changes in cell microenvironment with development of pericellular and extracellular matrices. Comparisons with the response of intact tissue to compression suggested similar regulatory mechanisms. We further investigated MAPkinase (ERK1/2, p38, JNK) and Akt activation on Days 1 and 28 in agarose culture through phosphorylation state-specific Western blotting. / (cont.) Compression induced transient ERK1/2 phosphorylation on both days, with Day 28 levels similar to intact tissue. Unique from tissue behavior, only slight transient p38 phosphorylation was observed on Day 28, and SEK phosphorylation was undetected. Akt was uniquely regulated in intact cartilage compared to MAPks, with decreased total Akt levels over time under static compression. In contrast, compression transiently decreased pAkt levels in agarose cultures, with no changes in total Akt. Changes in the chondrocyte responses to compression with time in agarose culture suggest that cells sense different forces and respond differently with time; further studies may help optimize mechanical loading for tissue-engineering purposes. These studies provide a basis for further examination of mechanotransduction in cartilage. / by Diana H. Chai. / Ph.D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Biological Engineering Division, 2006. / "June 2006." / Includes bibliographical references (leaves 175-187). / Molecular markers for adult stem cells (ASCs) are highly demanded for research and clinical applications. The development of specific molecular markers for ASCs has been difficult mainly due to the technical barriers in the identification and isolation of rare ASCs. Previously, reported transcriptional profiling studies for defining molecular features of ASCs were compromised by the use of impure ASC preparations. Thesis for this research was that the study of asymmetric self-renewal, a defining property of ASCs, might provide key clues to understanding ASC function and lead to discovery of novel molecular markers for ASCs. Fifty two self-renewal pattern associated (SRPA) genes were identified by cDNA microarray analysis with cell culture models whose self-renewal pattern could be reversibly regulated, instead of using heterogeneous ASC-enriched populations. From evaluation of whole genome transcript levels to expand the SRPA gene pool, 543 SRPA genes were discovered. Both microarray studies showed that asymmetric self-renewal associated (ASRA) genes were highly represented in ASC-enriched populations but not in embryonic stem cells. The SRPA gene expression signature successfully distinguished isolated ASC-enriched populations from non-stem cell populations by principal component analysis (PCA). / (cont.) The SRPA gene signature clustered and classified putative epidermal stem cell-enriched populations better than reported stemness gene signatures in PCA. Therefore, gene microarray analyses for studying self-renewal pattern per se confirmed for the first time that asymmetric self-renewal is an essential molecular feature of ASCs in vivo. Chromosome mapping of the SRPA genes identified two SRPA chromosome gene cluster regions. One chromosome cluster contained primarily ASRA genes, whereas the other contained primarily symmetric self-renewal associated (SSRA) genes. These two SRPA chromosome cluster regions are frequently rearranged or deleted in particular human cancers. Functional and expression analysis of several selected ASRA and SSRA gene-encoded proteins implicated them in control of asymmetric self-renewal and non-random chromosome co-segregation, respectively. Moreover, one plasma membrane bound ASRA protein, CXCR6, had properties of one of the most specific molecular markers for ASCs described to date. In conclusion, this research strongly supported the precept that asymmetric self-renewal is a unique molecular feature for understanding ASCs, their relation to cancer, their unique function, and for their eventual exclusive identification. / by Minsoo Noh. / Ph.D.

Thesis (M. Eng.)--Massachusetts Institute of Technology, Biological Engineering Division, 2007. / Includes bibliographical references (p. 57-60). / Cells respond to the application of force with a variety of biochemical responses modulating their shape, structure, function, and proliferation. Two force-responsive links between the inside and outside of a cell are integrin proteins, which link a cell to the extracellular matrix (ECM), and cadherin proteins, which link neighboring cells to each other. The strength of integrin-ECM bonds has been noted to increase in response to the application of force. However, the strengthening of cadherin-cadherin bonds in response to force has not been studied. Here, we use magnetic trapping to probe adhesion strengthening at cadherin adherens junctions, using cadherin-coated magnetic beads to simulate neighboring cells and apply force at adherens junctions. 43% of beads exposed to a high force (2.1 nN) detached, compared to 31% of those exposed to a low-to-high force ramp followed by high force. This indicates that adherens junctions are strengthened by force application. The actin cytoskeleton and vasodilator-stimulated phosphoprotein (VASP) both associate with adherens junctions, so their role in adhesion strengthening at adherens junctions was also studied. Cells treated with actin-inhibitor cytochalasin D showed no difference in bead detachment from constant high force and from ramped followed by high force, indicating that the actin cytoskeleton is crucial in the adhesion strengthening response. Beads attached to cells expressing GFP-VASP, which behave like VASP-overexpressing cells, detached in 24% of trials when exposed to constant high force, compared to 39% of trials in response to ramped force. Cells expressing GFP-MITO-FPPPP, which behave like VASP-downregulated cells, showed no difference in bead detachment between application of high force and ramped force followed by high force. / (cont.) These experiments indicate that VASP is necessary for the adhesion strengthening response, but high levels of VASP may slow actin restructuring and diminish the ability of the cytoskeletal linkages to respond to increasing force. The importance of VASP in cells' responses to forces from other cells suggest that modulation of VASP activity may play a role in tissue development, where cell-cell force responses are important, and the pathogenesis of certain diseases, where cell-cell adhesion is affected. / by Anita S. Kris. / M.Eng.

Thesis (M. Eng.)--Massachusetts Institute of Technology, Biological Engineering Division, 2004. / Includes bibliographical references (p. 70-72). / Self-assembling peptides are a promising new area of research with usage in numerous areas, from tissue engineering to membrane protein biology. This work is to further study the characteristics of the peptides KLD12 and RID12 and to generate new ways to control the properties of them. Peptide structures in solution were studied with circular dichroism and dynamic light scattering, and material properties of the peptide solutions were studied with rheology. Nanofiber structure was studied through atomic force microscopy. Sonication was found to have a minimal effect on KLD12, while pH could alter the length of the fibers formed. The presence of a second type of peptide in solution interacted with nanofibers of another peptide and resulted in a decrease of average fiber length. Peptide solutions transitioned from a viscous solution to a gel as the concentration of peptide increased, both alone and in mixture with another peptide. Understanding the properties of these peptides will help researchers design new biomaterials and improve applications of self-assembling peptides. / by Jessica Dai. / M.Eng.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Biological Engineering Division, 2007. / Includes bibliographical references (p. 141-153). / Creating a physiologically relevant in vitro liver model requires reproducing the cellular heterogeneity of in vivo liver in a functional state. However differentiated sinusoidal endothelial cells (SECs), marked by SE-1 expression are difficult to maintain in culture while stellate cells easily activate and over-proliferate. We hypothesized that recreating a liver tissue system that captured in vivo like paracrine influences would foster survival of these cells, and predicted that stimuli resulting from flow and oxygen gradients close to physiological conditions would preserve the delicate balance between the cell types. Spheroids containing hepatoctyes with incorporated non-parenchymal cells (NPCs) were seeded into capillary bed sized channels in polycarbonate scaffolds, housed in a three-dimensional perfused system, and maintained for two weeks. Micro-flow rates of different media through the formed tissue units in scaffolds were controlled using pneumatic pumps and microfluidics. Staining and confocal imaging of endpoint tissue showed that lower flow rates closer to physiological regimes allowed the survival of SE-1+ SECs, regardless of exogenously added growth factors in the medium. Higher flow rates, exogenous growth factors, and scaffold contact were associated with activation of stellate cells (alpha-smooth muscle actin staining). / Since oxygen measurements in the system coupled low flow rates with hypoxic tissue outlet concentrations, we parsed out these variables by repeating flow experiments in low oxygen environments. Retention of SE-i staining cells even in higher flow rates demonstrated that hypoxic conditions in the tissue could play a role in aiding their survival by overcoming negative effects brought about by high flow. The relationship of stellate cells with flow rate was unaffected by oxygen concentrations. To explore if the negative effects of high flow on SE-i expression were mediated by transforming growth factor-beta (TGF-[beta]), we added a TGF-[beta] inhibitor SB-431542 in our cultures, and found that it greatly enhanced the presence of SE-1 staining SECs at high flow rates. In conclusion we successfully created a three-dimensional flow controlled hepatic culture system that allows balanced survival of hepatocytes and non-parenchymal cells, making it useful as a potential model for studies such as cancer metastasis that require interactions between tumor cells and heterotypic host tissue. Key Words: Liver, In vitro, co-culture, sinusoidal, endothelial, stellate, oxygen, flow, shear. / by Ajit Dash. / Ph.D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Biological Engineering Division, 2005. / Includes bibliographical references (p. 131-142). / The mechanical properties of articular cartilage are associated with the extracellular matrix network of type II collagen and the proteoglycan, aggrecan, which in combination provide the tensile, shear, and compressive stiffness of the tissue. While the collagen network mainly provides resistance to tensile and shear deformation, aggrecan enmeshed within this network contributes significantly to the tissue's compressive and shear properties under equilibrium as well as dynamic loading conditions. Aggrecan has a "bottle-brush" structure that includes -100 negatively charged chondroitin sulfate glycosaminoglycan (CS-GAG) chains attached covalently to a core protein. Electrostatic interactions between these GAGs contribute to the compressive and shear stiffness of the tissue. Variations in the structure of aggrecan and its GAG constituents are known to exist as a function of tissue age, disease, and species. Using atomic force microscopy (AFM), we directly visualized the nanometer scale structure of aggrecan deposited on a 2-D substrate, including the first high resolution imaging of individual GAG chains along the core protein. We also visualized and quantified the differences in structure between aggrecan obtained from fetal epiphyseal and mature nasal bovine cartilages. / (cont.) A combination of AFM, biochemical, and polymer statistical methodologies was used to better understand the dependence of aggrecan structure and stiffness on the properties of its constituent GAG chains. The fetal epiphyseal aggrecan had a denser GAG brush region and longer GAG chains, which correlated with a higher effective persistence length of fetal core protein compared to that of mature nasal aggrecan. The effect of increasing the concentration of aggrecan on the substrate resulted in a decrease in molecular extension, suggesting a flexible protein core backbone, which allowed aggrecan to entangle and interact with neighboring molecules. AFM imaging of the conformation of aggrecan that had been deposited on substrates from solutions of varying ionic strength (IS), from DI water to the hysiological IS of 0.1 M NaCl, allowed for direct visualization of the collapse of the molecule on the substrate at the highest IS, due to charge shielding of the CS-GAGs by by Na+ counter-ions. Lastly, the nanomechanical properties of cartilage cells (chondrocytes) and their aggrecan-collagen-rich pericellular matrix (PCM) were probed via AFM nanoindentation using both a sharp nano tip and a larger micro-colloidal tip to better understand the deformation of cells in cartilage. / (cont.) The properties of cells freshly isolated from cartilage tissue, devoid of PCM, were compared to that of cells isolated and then cultured for selected times in 3-D alginate gel to obtain cells surrounded by their newly developed PCM. Using Hertzian contact mechanics as well as finite element analyses, material properties were estimated from the AFM force-indentation curves measured with these cell preparations. We also studied the effects of culture conditions on the resulting PCM properties, comparing 10% fetal bovine serum vs. medium containing a combination of insulin growth factor-i (IGF-1) + osteogenic protein-i (OP-1). While both systems showed increases in matrix stiffness with time in culture between days 7 to 28, the IGF-1 + OP-1 combination resulted in a higher effective modulus for the cell-PCM composite. These AFM cell indentation studies were enabled by the use of microfabricated chips containing wells designed to immobilize the spherical chondrocytes during testing. Due to the nonconventional but known geometry of the microfabricated wells, finite element analysis was used to include the effects of the cell-well boundary conditions and tip geometries on the calculated cell-PCM material properties. / (cont.) Taken together, these studies examining cartilage mechanics at the molecular and cellular levels give insight into the intricate roles that proteoglycans and collagen play in governing tissue-level mechanical properties. / by Laurel Jean Ng. / Ph.D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Biological Engineering Division, 2007. / Includes bibliographical references (p. 161-172). / Gene therapy has the potential to cure thousands of diseases caused by genetic abnormalities, provide novel combination therapies for cancers and viral infections, and offer a new and effective platform for next generation vaccines. However, after more than three decades of research and development efforts, clinical success has yet to be realized. Successful delivery of DNA is a crucial first step in attaining safe and effective gene therapeutics. While vectors based upon recombinant viruses have shown high delivery and transfection efficiencies, they may also pose certain health risks to patients, can be difficult to target to cell or tissue types of interest, and present difficulties for large-scale manufacturing. Non-viral vectors look to offer a safer alternative and can be engineered to more effectively treat a specific cell type, tissue, or pathology, but these vectors are still plagued with low transfection levels and cannot provide adequate and sustained levels of gene expression. Continued efforts focus on producing next generation non-viral vectors that safely deliver therapeutic transgenes with the efficiency of their viral counterparts. Many barriers exist in the successful trafficking of these non-viral complexes to the nucleus. / (cont.) Current evaluations of non-viral gene delivery treatments in more clinical settings often focus on a single barrier at a time, and as a result, may not lead to an overall improvement in gene delivery. Concurrently, more quantitative or systematic in vitro experiments may not correlate well with in vivo data. Our combined approach of quantitative vector trafficking and expression experiments coupled with computational simulation of vector specific mathematical models that describe every step of the gene delivery process has shown that a systems level approach can glean insight into the most rate-limiting steps for a given vector and generate hypotheses for future vector development and improvement. These studies have been extended to primary liver cultures, coupled with device development to attain a more clinically relevant model system and more spatial resolution to study intracellular vector trafficking and localization. A larger perfused 3-D liver bioreactor has been built that allows for long-term culture of primary hepatocytes that more closely mimic hepatic phenotype than in conventional 2-D cultures and for multiplexed quantitative measurement that is not possible in animal models. / (cont.) A newly constructed density gradient electrophoresis device can separate vesicular organelles and track vector dynamics throughout the cell. These systems have provided more comprehensive data sets which show that vectors behave differently in different culture systems and that different vectors show unique cell trafficking dynamics. These results lend insight for future vector screening methodologies and provide vector specific mathematical models for primary cell transfection that can lead to further optimization of the polymer vectors studied in this work, which can contribute to the development of more efficient next generation in vivo delivery agents. / by Nathan C. Tedford. / Ph.D.

Thesis (M. Eng.)--Massachusetts Institute of Technology, Biological Engineering Division, 2007. / Includes bibliographical references (leaves 60-61). / Idiosyncratic drug reactions are a subset of adverse reactions frequently targeting the liver, which become obvious only in large sample populations. Drug-induced hepatotoxicity, occurring in a very small fraction of patients, poses a major challenge to pharmaceutical companies due to its unknown mechanism(s) of action and deficient models for study. In vitro model systems may have the potential to predict this liver injury by generating conditions possibly representing key processes involved, both directly and indirectly, in drug effects on cellular physiology. Our ultimate goal is to develop an in vitro model effectively mimicking certain relevant aspects of the in vivo response of the human liver. In our initial effort described herein, we have designed a novel cell-based system using alternatively in both a human hepatoma cell line and primary rat hepatocytes to study toxic effects in a background reflecting in vivo inflammatory conditions. This background incorporates bacterial lipopolysaccharide (LPS) administration along with inflammatory cytokines (tumor necrosis factor, interferon y, interleukin-1 a, interleukin-113, and interleukin-6) previously shown to increase in LPS-administrated rats. Our study began with an investigation of toxicities that are induced by combinations of five cytokines and LPS in HepG2 and C3A human hepatoma cell lines and in primary rat hepatocytes. Informed by the results of these experiments, we selected representative cytokine/LPS treatments and cell systems to examine drug-cytokine synergies in vitro and were able to identify multiple idiosyncratic hepatotoxins that induced synergistic toxicity in either the HepG2 cell line or primary rat hepatocytes. / (cont.) Finally, we measured the sensitization of these cell systems to a panel of these drugs, given an inflammatory background induced by an abbreviated set of cytokine treatments including four cytokines and LPS. Analysis of this multivariate drug-cytokine toxicity data set yielded a subset of representative cytokine treatments for future drug-cytokine synergy investigations. This subset will be used to characterize the differences between cell systems, including cultured human hepatocytes, and to hopefully develop a data-driven partial least squares regression model that predicts idiosyncratic liver injury. The implications are two-fold. First, this model could provide direction to pharmaceutical companies in focusing their drug discovery and development. Second, it could help physicians design better treatment plans for their patients. / by Maya Hasan. / M.Eng.