Thesis (Ph. D.)--Massachusetts Institute of Technology, Biological Engineering Division, 2007. / "June 2007." / Includes bibliographical references (leaves 118-126). / Molecular springs have recently emerged as the basis for the fastest and most powerful movements at the cellular level in biology. The spasmoneme of the protozoan, Vorticella convallaria, is a model molecular spring, relying on energy stored in protein interactions to power contraction over a few hundred micrometers in a few milliseconds. While basic characteristics of Vorticella contraction are known, the underlying biochemical mechanism is unclear. The studies outlined here define and measure key parameters of spasmoneme performance which enable discrimination between proposed movement schemes and identification of new model parameters. Recent work has classified the spasmoneme as a power-limited machine (Upadhyaya, Baraban et al. 2007), where increases in viscous load correspond to decreases in velocity; in this work the maximum load at minimum velocity (the stall force), is measured. Work done by the stalk in contraction is shown to be dependent on its fractional change in length. This energy dependence arises from the basic underlying mechanism, and a major goal of this thesis was to characterize that mechanism by imaging the underlying structure. In the case of the Vorticella spasmoneme, imaging methods like birefringence imaging and electron microscopy, which do not require preexisting knowledge of protein identity, are particularly helpful. / (cont.) High-speed measurements of live Vorticella movements show the persistence of birefringence throughout the contraction-extension cycle. Orientation-independent measurements taken with an LC Pol-Scope show strong birefringence with slow axis parallel to the stalk long axis in both the extended and contracted states. Quantification of textural differences between the two states reveals slight structural disordering upon contraction. Transmission electron micrographs show a correlation between nanometer-scale filaments and the distribution of birefringence within the spasmoneme. As a whole these measurements indicate that any model of the contractile mechanism should consider the interactions of filamentous proteins at high concentrations which lead to longitudinal microscopic alignment in both the extended and contracted states. Implications of a proposed model are considered in the context of how they may be tested in vitro with purified constituent and homologous recombinant proteins, and how they can inform the development of biomimetic, nanoscale actuators. / by Danielle Cook France. / Ph.D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Biological Engineering Division, 2006. / MIT Science Library copy: printed in pages versus leaves. / Also issued in pages. / Includes bibliographical references (leaves 183-197). / The cardiac ventricular muscle cell (myocyte) is a key experimental system for exploring the mechanical properties of the diseased and healthy heart. The myocyte experimental model provides a higher level of physiological relevance than molecular or myofibril studies while avoiding problems inherent to multicellular preparations including heterogeneity of cell types and diffusion limited extracellular spaces. Millions of primary myocytes that remain viable for four to six hours can be readily isolated from animal models. However, the mechanical properties of only a few physically loaded myocytes can be explored in this time period using current, bulky and expensive instrumentation. In this thesis, a prototype instrument is described that is modular and inexpensive and could form the basis of an array of devices for probing the mechanical properties of single mammnalian myocytes in parallel. This would greatly increase the throughput of scientific experimentation and could be applied as a high content screening instrument in the pharmaceutical industry providing information at the level of a critical cellular phenotype, myocyte mechanical properties, for drug development and toxicology studies. / (cont.) The design, development and experimental verification of the modular instrument are presented here. The mathematical, mechanical and electrical characteristics of the novel force sensor and actuator system, Ho control implementation and data processing methodology are discussed. Finally, the functionality of the instrument is demonstrated by implementing novel methodologies for loading and attaching healthy, single mammalian ventricular myocytes to the force sensor and actuator and measuring their isometric twitch force and passive dynamic stiffness at varied sarcomere lengths. / by Michael G. Garcia-Webb. / Ph.D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Biological Engineering Division, February 2008. / MIT Science Library copy: issued as 1 v. / Also issued in 1 v. with pagination as pages. / Includes bibliographical references. / Approximately one in five Americans is affected by arthritis, making it one of the most prevalent diseases and the leading cause of disability in the United States. Post-traumatic arthritis occurs after joint injury (e.g., ACL rupture or intraarticular fracture) and makes up a substantial proportion of the population with arthritis. In previous clinical studies, patients suffering from a traumatic joint injury have shown an increased risk in osteoarthritis (OA), independent of surgical intervention to stabilize the joint. Thus, the early events post-injury have an important effect on tissue within the joint in the long term. To understand the processes involved in the onset of OA and factors leading to OA post-traumatic injury, in vitro models have been developed to isolate components of the complex processes occurring in vivo. While in vitro models do not mimic true physiologic conditions in vivo, by isolating the effects of mechanical compression, cytokine treatment, and cartilage co-cultured with adjacent tissue, in vitro models can give insight into key biological and mechanical pathways occurring in vivo. This study focuses on changes in cartilage gene and protein expression and associated cartilage matrix degradation in response to static or injurious compression of the tissue in the presence or absence of cytokines including TNF-a and IL-6. In addition, normal or injuriously compressed cartilage explants were co-cultured with injured (excised) joint capsule tissue, another in vitro model of post-traumatic cellular behavior. Both young bovine cartilage and human cartilage from a wide range of ages were used. The growth factors insulin-like growth factor-1 (IGF-1) and Osteogenic protein-i (OP-1), as well as the antioxidant, superoxide dismutase mimetic (SODm), were tested to examine if they had the capability to abrogate the negative effects of these injury models. / (cont.) Taking a systems approach, the effects of these stimuli on expression of over 48 genes (in cartilage as well as joint capsule) were quantified, along with measures of chondrocyte viability, biosynthesis, protein expression, and GAG loss. Chondrocyte gene expression was differentially regulated by 50% static compression or IGF- 1 treatment or the combination of compression and IGF- 1. Results showed that IGF- 1 stimulated aggrecan biosynthesis in a transcriptionally regulated manner, whereas compression inhibited aggrecan synthesis in a manner not regulated by transcriptional activity. The injury plus co-culture model was examined in detail, and OP-1 and IGF-1 were unable to rescue changes in transcriptional expressions due to injury. However, these growth factors were able to rescue cells from apoptosis, and slightly increase biosynthesis rates. Human tissue was used to further validate the model of mechanical injury (INJ) combined with co-culture (Co). Immunohistochemical analysis of human cartilage explants after INJ+Co treatment revealed changes in versican and aggrecan protein expression, as well as changes in surface tissue morphology, that mimicked certain changes observed in human osteochondral plugs taken from patients at the time of notchplasty surgery (post ACL reconstruction) at 1, 3, or 57 months post- ACL rupture. The oxidative stress involved in a cytokine plus injury model showed that SODm had no ability to selectively diminish protease transcriptional activity. Cartilage treated with this antioxidant showed significant increases in GAG loss to the medium, but diminished levels of chondrocyte apoptosis. Taken together, this work supports further investigation of the mechanisms of action of OP-1, IGF-1, and SODm in order to elucidate their possible therapeutic value, and demonstrates the usefulness of these complementary in vitro models of cartilage injury. / by Cameron A. Wheeler. / Ph.D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Biological Engineering Division, 2008. / Includes bibliographical references. / Parkinson's disease (PD) is a primarily motor disorder which affects at least half a million people in the US alone. Deep brain stimulation (DBS) is a neurosurgical intervention by which neural structures are stimulated electrically by an implanted pacemaker. It has become the treatment of choice for PD, when not adequately controlled by drug therapy. We introduced a novel robotic platform for the study of the effects of DBS on motor control in PD. Subjects performed discrete wrist movements with and without a force field. We found preliminary indication that motor learning may be taking place with stimulation, and demonstrated how robotic testing can augment existing clinical tools in evaluation of the disease. To study the effect of stimulation on movement frequency, we employed a rhythmic task that required movements of the elbow to remain within a closed shape on a phase plane. Three closed shapes required varying frequency/amplitude combinations of elbow movement. The task was performed with and without visual feedback. Analysis of data from the healthy control subjects revealed a non-monotonic relation between accuracy on the phase plane and movement speed. Further kinematic analyses, including movement intermittency and harmonicity, number and type of submovements (movement primitives) fit per movement cycle, and the effects of vision on intermittency were used to support the model we propose, whereby there exist two subtypes of rhythmic movement; small-amplitude, high-frequency movements are nearly maximally harmonic, and harness the elastic properties of the limb to achieve smoothness and accuracy, and large-amplitude, low-frequency movements share characteristics with a string of discrete movements, and make use of visual feedback to achieve smoothness and accuracy. / (cont.) Bradykinesia (slowness of movement) is one of the hallmarks of PD. We examined the effects of visual feedback on bradykinesia. PD patients off dopaminergic medication and healthy age-matched controls performed significantly faster movements when visual feedback was withdrawn. For the bradykinetic subjects, this increase in movement speed meant either a mitigation or an elimination of bradykinesia. Our results support a role of the basal ganglia in sensorimotor integration, and argue for the integration of nonvision exercises into patients' physical therapy regime. / by Shelly Levy-Tzedek. / Ph.D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Biological Engineering Division, 2006. / Includes bibliographical references (p. 153-165). / Cells communicate with other cells, and process cues from their environment, via signaling pathways, in which extracellular cues trigger a cascade of information flow, causing signaling molecules to become chemically, physically or locationally modified, gain new functional capabilities, and affect subsequent molecules in the cascade, culminating in a phenotypic cellular response. Mapping the influence connections among biomolecules in a signaling cascade aids in understanding of the underlying biological process and in development of therapeutics for diseases involving aberrant pathways, such as cancer and autoimmune disease. In this thesis, we present an approach for automatically reverse-engineering the structure of a signaling pathway, from high-throughput data. We apply Bayesian network structure inference to signaling protein measurements performed in thousands of single cells, using a machine called a flow cytorneter. Our de novo reconstruction of a T-cell signaling map was highly accurate, closely reproducing the known pathway structure, and accurately predicted novel pathway connections. The flow cytometry measurements include specific perturbations of signaling molecules, aiding in a causal interpretation of the Bayesian network graph structure. / (cont.) However, this machine can measure only -4-12 molecules per cell, too few for effective coverage of a signaling pathway. To address this problem, we employ a number of biologically motivated assumptions to extend our technique to scale up from the number of molecules measured to larger models, using measurements of overlapping variable subsets. We demonstrate this approach by scaling up to a model of 11 variables, using 15 overlapping 4-variable measurements. / by Karen Sachs. / Ph.D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Biological Engineering Division, 2007. / Includes bibliographical references. / Mechanical forces play an important role in cell morphology, orientation, migration, adhesion and can even induce apoptosis. The eukaryotic cell is equipped with a dynamic frame, known as the cytoskeleton, that provides the cell's structural integrity in order to sustain and react to such forces. Therefore, understanding the mechanical properties of the cytoskeleton is an important step towards building models describing cell behavior. Filamentous actin (F-actin), as one of the major constituents of the cytoskeleton, has been the target of extensive in vitro studies to determine its mechanical properties in bulk. However, there is still a lack in the understanding of how the molecular interactions between F-actin and the proteins that arrange these filaments into networks regulate the dynamic properties of the cytoskeleton Here we present a novel, single molecule assay to test the rupture force of a complex formed by an actin binding protein (ABP) linking two actin filaments. We readily demonstrate the adaptability of this assay by testing it with two different ABPs: filamin, a crosslinker, and a-actinin, a bundler. We measured rupture forces of 28-73 pN and 30-56 pN for filamin/actin and a-actinin/actin respectively, suggesting that the former is a slightly stronger interaction. Moreover, since no ABP unfolding events were observed at our force levels, our results suggest that ABP unbinding is a more relevant mechanism than unfolding for the temporal regulation of the mechanical properties of the actin cytoskeleton. In addition, we explore the micro-scale properties of F-actin networks reconstituted in vitro. / (cont.) Using imaging and microrheology techniques we characterized the effects of filament length and degree of crosslinking on the structural arrangement and mechanical properties of F-actin networks. We found that the mechanical properties of these networks are length-scale dependent. Also, when probed with active methods, the F-actin networks exhibited strain hardening followed by a gradual softening at forces -30 pN, in good agreement with the single molecule rupture force of 28-73 pN. Thus, with the combination of single molecule and network studies, we can expand the knowledge-base on the regulation and control of the cellular machinery starting from the molecular building blocks. / by Jorge M. Ferrer. / Ph.D.

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