Thesis (M. Eng.)--Massachusetts Institute of Technology, Biological Engineering Division, 2005. / "June 2005." / Includes bibliographical references (p. 40-43). / Traditional vaccines consisting of live attenuated pathogens or inactivated toxins cannot be readily applied to the more challenging diseases of the present e.g. hepatitis C and the human immunodeficiency virus. As such, there is a need to develop new methods of priming the immune system against such foreign invaders. Recombinant protein subunits and peptides are relatively safe alternatives to live attenuated pathogens. However, these antigens are poorly immunogenic when administered alone in solution form and thus require the use of an adjuvant. To this end, we have developed a hydrogel-based nanoparticulate system to encapsulate protein antigen and to co-deliver it with DNA/RNA-based adjuvants to dendritic cells, the key antigen presenting cells in primary immune responses. Using CpG oligonucleotides or siRNA as adjuvants, we observed via enzyme-linked immunosorbent assays for interleukin 12 and interferon-[alpha], respectively, that DCs were activated by CpG oligonucleotide- and siRNA-functionalized nanoparticles [approx.]10-fold more potently than by soluble CpG or siRNA ligands. / by Jonathan Woon Teck Yap. / M.Eng.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Biological Engineering Division, 2005. / Includes bibliographical references (p. 152-167). / Chondrocytes respond to in vivo mechanical loads by regulating the composition of the cartilage extracellular matrix. This study utilized three loading protocols that span the range of forces and flows induced by in vivo loading. Constant (static) compression of cartilage explants induces a transient hydrostatic pressure buildup and fluid exudation from the compacted matrix until relaxation leads to a new equilibrium compressed state. Dynamic compression induces cyclic matrix deformation, hydrostatic pressures, fluid flows, and streaming currents. Dynamic tissue shear causes cyclic matrix deformation only. After applying these loading protocols to intact cartilage explants for 1 to 24 hours, we used real-time PCR to measure the temporal expression profiles of selected genes associated with cartilage homeostasis. In concurrent experiments, we assessed the involvement of intracellular signaling pathways using molecular inhibitors. In order to interpret the results we developed two techniques that reliably clustered intermediate-sized datasets using principal component analysis and k-means clustering. Mechanical loading regulated a variety of genes including matrix proteins, proteases, protease inhibitors, transcription factors, cytokines, and growth factors. Static compression transiently upregulated matrix proteins, however, mRNA levels were suppressed by 24 hours. / (cont.) Dynamic compression and dynamic shear increased matrix protein transcription particularly after 24 hours. In contrast, matrix proteases were upregulated by all 24 hour loading regimes, particularly static compression. Taken together these results demonstrate the functionally-coordinated regulation of chondrocyte gene transcription in response to mechanical forces, and support the hypothesis that dynamic loading is anabolic for cartilage and static loading is anti-anabolic. Intracellular calcium release, cAMP activation of protein-kinase-A, and the phosphorylation of MAP kinases (ERK1/2, p38), were all identified as signaling events necessary for mechanically-induced transcription. In addition, we measured the immediate, transient increase in mRNA levels of transcription factors downstream of the MAP kinase pathway (c-Fos and c-Jun), in response to all three loading types. The prevention of protein synthesis during static compression suppressed mechanically-induced transcription suggesting that signaling molecules are synthesized in response to mechanical forces. Comparison of this well characterized model of normal cartilage mechanotransduction to what occurs within diseased cartilage will hopefully provide insight into the mechanisms driving the progression of osteoarthritis. / by Jonathan Basil Fitzgerald. / Ph.D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Biological Engineering Division, 2006. / Includes bibliographical references. / Alkyl-substituted anilines have been implicated as important etiological agents in human carcinogenesis. Specifically, 2,6-dimethylaniline (2,6-DMA), 3,5-dimethylaniline (3,5-DMA), and 3-ethylaniline (3EA) have been associated with an increased risk of human bladder cancer, independent of cigarette smoking, in a published case-control study. Understanding the metabolic activation of and DNA adduct formation by these chemicals is an important first step in elucidating their mechanisms of carcinogenesis and toxicity. Cytochrome P450-mediated metabolism was profiled based on the hypothesis that N-hydroxylated metabolites are critical intermediates in the formation of DNA adducts. This work was extended to assess in vitro DNA adduct formation with the cell-free and cell-based assays. Accelerator Mass Spectrometry (AMS) was used for detection and semi-quantification of DNA adducts formed by 14C-labeled alkylanilines. Data indicated 3,5-DMA formed high levels of DNA adducts, suggesting that it is a potent carcinogen. Additionally, the levels of adducts exhibited inter-species variation. The effects of phase II metabolism on adduct formation were evaluated by comparing the results obtained from the two types of assays and by assessing the effects of phase II enzyme cofactors on the results of cell-free assay. / (cont.) Results implied that sulfotransferase-mediated metabolism promotes cytotoxicity and mutagenicity of all three alkylanilines; however, glucuronidation may provide a protective mechanism. The effects of N-acetyltransferase-mediated metabolism on DNA adduct formation differed for the three alkylanilines; acetyl-CoA enhanced adduct formation by 3-EA and 2,6-DMA, but it reduced 3,5-DMA adduct formation. Human CYP2A6 universally catalyzed the oxidation of all structural isomers of dimethylanilines and ethylanilines, except 3-EA. In the present work, the hypothesis that 3-EA is a mechanism-based inactivator toward human P450 2A6 through covalent binding was examined by using AMS. 3-EA was characterized as a mechanism-based inactivator with a Ki of 34 !iM and a kinact of 0.055 min'. Results suggest that 3-EA might be involved in more than one biological effect in the human body through multiple pathways. Adduct formation and inhibition of CYP 2A6 by 3-EA might shift the biological effects of other compounds activated by CYP 2A6 dynamically and kinetically while appearing in the biological systems simultaneously. / by Hsiao-Lan Patty Sun. / Ph.D.

Thesis (S.M. in Bioengineering)--Massachusetts Institute of Technology, Biological Engineering Division, 2002. / Includes bibliographical references (leaves 51-52). / This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections. / One solution to the increasing need for liver transplants is to grow implantable liver tissue in the lab. A tissue-engineered liver for transplantation will require complex structures to support cell differentiation and integration with surrounding vasculature. Recent developments in 3D-printing (3DP™) technology allow the construction of such geometrically complex scaffolds out of biodegradable polymers. These artificial tissues should maintain healthy, functional hepatocytes in proper contact with supporting cell types in the context of proper flow cues. This project comprises three major efforts. First, the design and development of a 3D-printed scaffold, constructed of a porous biodegradable polymer matrix, for flow bioreactor culture. Second, the development of protocols for the production, preparation, and flow support of these scaffolds. And third, the employment of standard cell culture methodologies to test the ability of these scaffolds to support liver tissue cultures. Initial cell culture experiments showed similar rates of albumin production in the polymer disk scaffolds compared to cells in silicon-chip scaffolds under appropriately scaled flow conditions, indicating that the polymer scaffolds maintain functioning liver tissue. Further, histology sections of liver tissue grown on these polymer scaffolds show organization of cells into structures reminiscent of in vivo liver. The results of this study show that 3D-printed porous polymer scaffolds have great potential for use as biodegradable tissue culture support devices. It is believed that, combined with printing technologies now under development, the technologies developed in this thesis will help facilitate the construction of an implantable tissue engineered liver. / by James Camp. / S.M.in Bioengineering

Thesis (Ph. D.)--Massachusetts Institute of Technology, Biological Engineering Division, 2005. / Includes bibliographical references. / Chondroitin sulfate is a critical component of articular cartilage due to its contribution to the tissue's resistance to compressive deformation. Alterations in the biosynthesis of this molecule over time could impact the ability of the tissue to perform its necessary functions. Several factors have been shown to alter the biosynthesis of chondroitin sulfate in cartilage; among them are age, disease, depth of tissue, and mechanical compression. Specifically, mechanical compression has been shown to have a significant effect on the sulfation pattern and chain length and number in cartilage explant studies. The mechanisms that govern these alterations, however, have not been determined. The purpose of this study is to examine the effects of mechanical compression on chondroitin sulfate biosynthesis and analyze the roles of two possible mechanisms; enzyme transcription and organelle deformation. The effects of mechanical compression on the transcription rates of enzymes associated with the biosynthesis of chondroitin sulfate have not been previously studied. To perform this study in a bovine model, portions of the bovine genome had to be sequenced, PCR primers designed, and bulk expression levels determined. Static compression resulted in the significant up-regulation of two genes of interest: chondroitin sulfate and GalNAc 4S,6-sulfotransferase. / (cont.) Dynamic compression resulted in the significant up-regulation of the three sulfotransferases responsible for the bulk of sulfation in cartilage tissue. These results indicate a transient mechanotransduction reaction that differs based on the load regime. The effect of mechanical loading on the biosynthesis of chondroitin sulfate has been studied previously, however, this study seeks to examine more comprehensive loading regimes. Static compression and release resulted in an increase in 6-sulfation and a decrease in 4-sulfation that lasted to 48 hours after release of compression. Dynamic compression and release had the opposite effect on sulfation ratio, with an increase in 4-sulfation compared to 6-sulfation. The transcription changes seen in this study do not indicate the changes that occur in the end products of synthesis. Other factors may play a larger role, such as precursor availability or transport through the Golgi apparatus. Intracellular organelles react to static compression of the surrounding tissue in one of two manners. The majority of organelles deform much as the nucleus, proportionally in volume and shape to the cell. The Golgi apparatus appears to retain a significant portion of its volume relative to the cell and other organelles. In addition, it reforms structurally into a highly ordered stacked appearance. / (cont.) Osmotic forces within the Golgi may allow it to balance the osmotic load in the cytoplasm and resist compression and altered trafficking of the Golgi may in turn produce the altered appearance. Recent microscopy experiments on the Golgi apparatus utilizing two-photon microscopy have allowed us to examine the reaction of live tissue to static compression. These results illustrate the significant, but differing, effects of static and dynamic compression on the biosynthesis of chondroitin sulfate. The effects of these compression types on the transcription of enzymes responsible for this biosynthesis cannot fully explain the changes seen in newly synthesized chondroitin sulfate. Organelle reorganization has been shown to occur in response to static load and it is possible that altered organelle trafficking plays a role in this altered biosynthesis. Further studies are necessary to determine the final effect of the altered transcription and organelle structure on the manufacture of this important cartilage molecule. / by Jon D. Szafranski. / Ph.D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Biological Engineering Division, 2005. / Includes bibliographical references. / Acute traumatic joint injury in young adults leads to an increased risk for the development of osteoarthritis (OA) later in life irrespective of surgical intervention to stabilize the injured joint. Although the mechanism by which injury leads to joint degeneration remains to be elucidated, several injury-related factors may contribute to the development of OA. These factors include but are not limited to altered mechanical loading and initiation of a cellular response in cartilage or other joint tissues at the time of the injury. Three in vitro models of joint injury were investigated to separately evaluate the effects on cartilage of mechanical overloading and damage to joint capsule and synovial lining. Models of injury included (1) mechanical injury lto cartilage explants alone, (2) co-culture of normal cartilage explants with an excised specimen of joint capsule tissue, and (3) co-culture of mechanically injured cartilage explants with excised joint capsule tissue. These models have been shown previously to result in matrix damage and decreased biosynthesis by the chondrocytes. We measured gene expression levels of matrix molecules and matrix proteases and found them to be expressed in control cartilage at levels ranging over five orders of magnitude, and to be differentially regulated in these three models of joint injury. / (cont.) Expression of matrix molecules including collagen II and aggrecan were unaffected by injurious compression or co-culture with joint capsule tissue during the first 24 hours; however, the combination of injurious compression followed by co-culture resulted in a -50% decrease in expression by 24 hours. Matrix proteases aggrecanase-2 (ADAMTS-5) and stromelysin (MMP-3) showed increased expression of 40-250-fold by 12 hours following injurious compression and 6-12-fold during 24 hours of co-culture with joint capsule tissue. Aggrecanase-1 (ADAMTS-4) and collagenase-3 (MMP-13) showed larger magnitude increases in expression during co-culture (6-8-fold; 6-24 hours) compared to injurious compression (2-4-fold; 6-24 hours). Expression of transcription factors, c-fos and c-jun, was rapidly increased by injurious compression (40-100-fold within one hour) but was less affected by co-culture with joint capsule tissue (increased 3-5-fold; 1-24 hours). Expression level results displayed a general trend toward matrix degradation in the models of joint injury with specific differences apparent between the models. Analysis of matrix protein fragments in the same injury models showed cleavage of aggrecan at the aggrecanase site in the interglobular domain by 16 days following injurious compression and during co-culture with joint capsule tissue. / (cont.) Equilibrium and dynamic stiffness of cartilage explants were decreased by 30-35% immediately after injurious compression but were unaffected through 16 days of co-culture with joint capsule tissue. Specific changes in gene expression and activity of matrix proteases observed in these injury models may be indicative of some of the molecules responsible in the initial phase of cartilage degradation observed clinically following joint injury. / Jennifer H. Lee. / Ph.D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Biological Engineering Division, 2008. / Includes bibliographical references (p. 82-92). / The practical viability of biomolecule-nanostructure hybrids depends critically on the functional and structural stability of biomolecules in application environments. Noncovalent interactions of biochemical functional groups with nanostructure surfaces can significantly disrupt biomolecular structure and function. We report a systematic study of the effect of DNA sequence on the binding interaction between gold nanoparticles and thiolated DNA (AuNp-DNA). Base specific noncovalent nucleotide adsorption on gold surfaces can affect nucleotide bioavailability in solution. Systematic investigation of DNA oligonucleotide sequence, the location of specific sequence motifs, and the effect of nanoparticle size was performed. Sequence effects on DNA coverage and oligonucleotide adsorption affinities.were studied by Langmuir isotherm analysis. The nanoparticle coverage at saturating concentrations of thiolated DNA varied with oligonucleotide sequence. Saturation coverages correlated well with complement hybridization efficiency. From this we concluded that noncovalent interactions between nucleotides and the particle surface effect both hybridization and DNA coverage and adsorption. This hypothesis was confirmed by chemical treatment of the particle surface to eliminate noncovalent interactions. Upon treatment the effect of sequence on hybridization efficiency was removed. The effect of sequence is not consistent across nanoparticle sizes. Different bases show the highest saturation coverages and hybridization efficiencies on different AuNp sizes. These results allow for sequence selection guidelines based on AuNp size for sizes ranging from 4-11nm. For smaller particles (<5nm) adenine rich sequences show the highest saturation coverage and hybridization efficiency. / (cont.) For mid-sized particles (~7.5nm), guanine sequences show the highest saturation coverage and hybridization efficiency. Larger particles (>10nm) show little sequence dependent behavior and are likely the best choice for uses where sequence choice is limited. Sequence selection based on these guidelines will provide AuNp-DNA conjugates with the highest possible oligonucleotide bioavailability, maximizing their utility in biotechnology applications. / by Katherine A. Brown. / Ph.D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Biological Engineering Division, 2006. / Includes bibliographical references. / Ex vivo expansion of hematopoietic stem cells (HSCs) is a long-standing challenge faced by both researchers and clinicians. To date, no robust, efficient method for the pure, ex vivo expansion of human HSCs has been demonstrated. Previous methods primarily induced the expansion of committed hematopoietic progenitor cells (HPCs), yielding even less pure populations of HSCs. This research was based on the hypothesis that, like for other adult stem cells (ASCs), the major barrier to expanding HSCs ex vivo is in preferentially regulating the asymmetric self-renewal of HSCs without loss in their ability to produce differentiated committed HPCs. This laboratory has shown that a p53-dependent pathway specifically controls the self-renewal pattern of several types of ASCs and thereby provides an effective means for expansion of ASCs in culture. The method, which involves the use of purine metabolites to achieve suppression of asymmetric cell kinetics, is referred to as SACK. The utility of the p53-dependent pathway was investigated for directing expansion of human HSCs. In order to support this investigation, the proliferation of HPCs in in vitro cultures was repressed by culturing cells without hematopoietic growth factors and cytokines. / (cont.) This allowed the in vitro detection of SACK-effects on a small sub-population of cells, predicted to include HSCs. In order to determine the self-renewal capacity and multilineage potential of SACK- cultured cells, they were transplanted into non-obese diabetic/severe combined immunodeficient (NOD/SCID) mice. In vivo transplantation investigations exhibited 1.9- fold to 4.5-fold increased engraftment efficiency with SACK-agents compared to SACK-free controls, suitable for clinical applications. This result suggests that SACK-culture expands a population of SCID-repopulating cells (SRCs) that yields self-renewal and multilineage engraftment in NOD/SCID mice. Accordingly, increased engraftment efficiency for successful clinical applications may be achieved after additional optimization of HSC expansion. To obtain the full therapeutic potential of expanded HSCs, development of methods for independently marking putative ASCs for future analyses and gene therapy was explored. This early success with human HSCs supports the basic hypothesis that the SACK approach may be applicable to expansion of many types of ASCs. / by Rouzbeh R. Taghizadeh. / Ph.D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Biological Engineering Division, 2004. / Includes bibliographical references. / Biological cells such as endothelial or muscle cells respond to mechanical stimulation with activation of specific intracellular and extracellular signaling pathways and cytoskeletal remodeling, a process termed mechanotransduction. Intracellular mechanosensors are thought to be activated by conformational changes induced by local cellular deformations. Since these mechanosensors have been speculated to be located in several cellular domains including the cell membrane, the cytoskeleton, and the nucleus, it is necessary to achieve a detailed understanding of subcellular mechanics. In this work, we present novel methods to independently quantify cytoskeletal displacements, mechanical coupling between the cytoskeleton and the extracellular matrix, and nuclear mechanics based on high resolution tracking of cellular structures and receptor bound magnetic beads in response to applied strain or microscopic forces. These methods were applied to study the effects of several human disease associated mutations on subcellular mechanics and to examine the interaction between known protein function and specific changes in cellular mechanical properties and mechanotransduction pathways. Initial experiments were targeted to the role of membrane adhesion receptors. Experiments with cells expressing a mutant form of the integrin-associated molecule tetraspanin CD151 revealed that CD151 plays a key role in selectively strengthening α6βl integrin-mediated adhesion to laminin-1. We then studied cytoplasmic behavior using cells from mice with an αB-Crystallin mutation (R120G) that causes desmin-related myopathy. These studies showed impaired passive cytoskeletal mechanics in adult mouse cardiac myocytes. Finally, we studied cells deficient in the nuclear envelope / (cont.) protein lamin A/C and showed that lamin A/C deficient cells have increased nuclear deformation, defective mechanotransduction, and impaired viability under mechanical strain, suggesting that the tissue specific effects observed in laminopathies such as Emery-Dreifuss muscular dystrophy or Hutchinson-Gilford progeria may arise from varying degrees of impaired nuclear mechanics and transcriptional regulation. In conclusion, our methods provide new and valuable tools to examine the role of subcellular biomechanics on mechanotransduction in normal and mutant cells, leading to improved understanding of disease mechanisms associated with altered cell mechanics. / by Jan Lammerding. / Ph.D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Biological Engineering Division, 2006. / Includes bibliographical references. / Endogenous and exogenous oxidants react with cellular macromolecules to generate a variety of electrophiles that react with DNA produce cytotoxic and mutagenic adducts. One source of such electrophiles is deoxyribose in DNA itself. Oxidation of each position in deoxyribose generates a unique spectrum of products, many of which are highly reactive with DNA bases and lead to formation of adducts. The objective of this thesis was to clarify the chemistry of deoxyribose oxidation, with a focus on C4'-oxidation that gives rise to 3'- phosphoglycolate residues on the DNA backbone and releases base propenal or malondialdehyde, and to investigate the role of base propenals in the formation of an important endogenous DNA adduct, MI dG. First, an index of total deoxyribose oxidation was developed, one that provides a means to compare different oxidizing agents. This method exploits the reaction of aldehyde- and ketone-containing deoxyribose oxidation products with 14C-methoxyamine to form stable oxime derivatives that are quantified by accelerator mass spectrometry. Sensitive GC/MS methods were developed to quantify 3'-phosphoglycolate residues from deoxyribose C4'-oxidation and HPLC/post-column derivatization methods were developed to quantify the corresponding base propenal or malondialdehyde. / (cont.) Combined with the quantification of total deoxyribose oxidation and the alternative product of C4'-oxidation, the 4'-ketoaldehyde abasic site, under the same conditions, these results offered direct insights into the partitioning of C4'-oxidation and the chemical mechanisms of deoxyribose oxidation in DNA. With a foundation of deoxyribose oxidation chemistry and analytical methods, the in vitro DNA oxidative damage induced by y-irradiation, Fe2+/EDTA, bleomycin and peroxynitrite was explored. The results revealed that malondialdehyde was neither sufficient nor necessary for the formation of MldG, while base propenal was effective in generating MldG. These observations were extended to an E. coli cell model in which the membrane content of polyunsaturated fatty acids was controlled. The results revealed that lipid peroxidation caused by y-irradiation was insufficient to produce MldG in cells and the level of MldG adducts was inversely correlated with the quantity of membrane polyunsaturated fatty acids when cells were treated with peroxynitrite. Finally, M1dG showed a moderate (-50%) increase in tissues from a mouse model of inflammation, while etheno-adducts induced by lipid peroxidation increased -3- fold. These results are again consistent with lipid peroxidation as a minor source of MldG. / by Xinfeng Zhou. / Ph.D.