Thesis (Ph. D.)--Massachusetts Institute of Technology, Biological Engineering Division, 2007. / Vita. / Includes bibliographical references. / The Escherichia coli AlkB protein is an exceptionally versatile DNA repair enzyme. Its expression is induced upon exposure to alkylating agents as part of the Ada-mediated adaptive response. This member of the ac-ketoglutarate/iron(II)-dependent dioxygenase family was originally discovered to reverse directly methylated lesions formed preferentially in single-stranded regions of DNA, such as 1-methyladenine and 3- methylcytosine. Repair proceeds via an oxidative demethylation pathway, in which the aberrant methyl group is hydroxylated and spontaneously lost as formaldehyde. Since these early studies, the list of lesions repaired by AlkB through this pathway has been extended to include 1-methylguanine, 3-methylthymine, 3-ethylcytosine, and 1-ethyladenine. Furthermore, the protein possesses a second, distinct chemical mechanism through which it can repair another class of lesions, the etheno-adducts formed by the reaction of DNA with metabolites of the carcinogen vinyl chloride or with breakdown products generated by lipid oxidation. In this case, direct repair proceeds through epoxidation of the etheno bond, creating an intermediate that hydrolyzes to a glycol form and finally releases the two-carbon bridge as glyoxal, restoring the unadducted adenine or cytosine. Thus, the AlkB protein bridges the repair of alkylative lesions with those induced by oxidative stress and embodies the multi-faceted protection required to preserve genomic stability and coding information despite the constant threats to which organisms are exposed. / (cont.) Herein, we exploit and characterize a pair of E. coli strains differing only in AlkB status to demonstrate the ability of AlkB to repair the etheno-lesions, the structural analog 1,N6-ethanoadenine (EA), and 3-methyluracil in vivo. Additionally, we establish the ability of the EA "repair product" to form interstrand cross-links in certain sequence contexts of duplex DNA. We also show that although the adaptive response proteins repair lesions generated by oxidative stress, oxidative agents do not induce expression of the response. Finally, we establish that certain hypothesized substrates for AlkB are not in fact repaired by the enzyme, nor are they repaired by another adaptive response protein, AidB. This work extends the current knowledge regarding the amazing ability of AlkB to protect cellular nucleic acids from damage arising from a diverse array of both endogenous and exogenous sources. / by Lauren Elizabeth Frick. / Ph.D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Biological Engineering Division, February 2008. / Includes bibliographical references (p. 213-231). / The combination of optical tweezers and single molecule fluorescence into an instrument capable of making combined, coincident measurements adds an observable dimension that allows for the examination of the localized effects of applied forces on biological systems. This technological advance had remained elusive due to the accelerated photobleaching of fluorophores in the presence of the high photon flux of the optical trap. This problem was circumvented by alternately modulating the trapping and fluorescence excitation laser beams, a technique named IOFF. Results show that our solution extends the longevity of Cy3 fluorophores by a factor of 20 without compromising the stiffness of the optical trap. This versatile arrangement can be extended to other fluorophores and was applied to unzip a 15 base pair region of dsDNA and to induce reversible conformational changes in a dsDNA hairpin labeled with a FRET pair. Next, this work developed an immobilization strategy and two single molecule assays for the CIpX ATPase, an enzyme capable of unfolding substrates that have been targeted for proteolytic degradation. In the first assay, which employs single molecule fluorescence, CIpX was found to unfold and translocate pre-engaged GFP substrates with a time constant of 22 s at saturating ATP concentrations, a rate that is 8-fold faster than bulk measurements clouded by binding and unbinding events. The second assay measured the strength of the ClpX-substrate interaction with optical tweezers. Results show that CIpX holds on to its substrates with forces on the order of 55 pN regardless of the nature and concentration of the nucleotide in solution. / (cont.) Finally, optical tweezers were used to characterize the rheological properties of methylcellulose and polarized cells, to quantify the mechanical properties of bacteriophage, and to measure the forces generated by a cellular actin spring. / by Ricardo R. Brau. / Ph.D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Biological Engineering Division, 2007. / Includes bibliographical references (leaves 112-118). / Recognition of polymicrobial infections is becoming important for understanding differential host responses to environmental exposures, vaccines, as well as therapeutics. Citrobacter rodentium is a well-characterized model of infectious colitis with particular usefulness for modeling human diarrheal disease or inflammatory bowel disease. Infection with Helicobacter hepaticus is subclinical and persistent in C57BL/6 mice, but causes disease in susceptible strains and immunodeficient mice. To test the hypothesis that subclinical persistent infection modulates the host response to diarrheal disease a polymicrobial mouse model utilizing H. hepaticus and C. rodentium was developed and characterized. Concurrent infection has been shown to modulate disease outcome through several mechanisms including: cross-reactivity between viral antigens; shifting T cell response from Th1 to Th2 by helminth infection; and induction of regulatory T cells that suppress host response. In this new model of polymicrobial infection, a new paradigm in which persistent infection prolonged the course of acute colitis associated with a deviation from Thl-biased disease to Th17 was observed. / (cont.) In addition, Foxp3+naturally-occurring regulatory T cells (nTre,) were markedly increased during active colitis. The accumulation of nTreg was sustained when mice were persistently infected with H. hepaticus, indicating on-going active colitis. Although persistent infection was able to modulate host response, protective immunity to a subsequent C. rodentium infection was not compromised. Persistent infection also modulated host response to soluble antigen by preventing induction of oral tolerance to single bolus, but not to continuous, high-dose antigen feeding. Using H. hepaticus infection of C57BL/6 mice, models to investigate the immunomodulatory potential of persistent infection on immunogenic responses of protective immunity to enteric infection, host response to polymicrobial enteric infection, as well as tolerogenic responses to soluble antigen were developed. These models establish baselines for further investigation into the influences of persistent infection on host immune responses. / by Megan Earley McBee. / Ph.D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Biological Engineering Division, 2007. / Leaf 187 blank. / Includes bibliographical references. / Gastric cancer is the second most common malignancy in the digestive system and the second leading cause of cancer-related death worldwide. Epidemiological data and experimental studies have identified several risk factors for gastric cancer, including Helicobacter pylori infection, low fruit and vegetable intake, N-nitrosoamine exposure, high salt diet, and smoking. Among these risk factors, H. pylori infection is the major cause of gastric cancer. Therefore, H. pylori has been classified as a type 1 (definite) carcinogen for gastric cancer by the World Health Organization (WHO) in 1994. H. pylori colonizes the human stomach and has been definitively linked to chronic gastritis. Infection in some: susceptible individuals results in serious gastric disease such as peptic ulcer or gastric cancer. The first aim of this thesis was to examine the role of different T cell subpopulations in H. pylori gastritis. Using a murine adoptive transfer model, adoptive transfer of wildtype (wt) effector T cells (TE) into H. pylori-infected lymphopenic Rag2-/- recipient mice resulted in H. pylori-associated corpus gastritis superimposed with non-specific gastroduodenitis. Cotransfer with TE and regulatory T cells (TR) from wt or IL10-/- mice reduced gastroduodenitis, but only wt TR cells reduced corpus gastritis. / (cont.) The second aim of this thesis was to evaluate the effect of vitamin C on H. pylori gastritis in vitamin C-deficient gulo-/- mice. It was found that a high vitamin C supplementation (3300 mg/L) in drinking water did not protect H. pylori gastritis, while a low vitamin C supplementation (33 mg/L) reduced the severity of H. pylori gastritis via an attenuated cellular immune response to H. pylori. The third aim of this thesis was to examine the role of DNA repair in H. pylori-associated gastric disease. We found that H. pylori-associated premalignant gastric atrophy was more severe in infected mice lacking DNA repair protein 3-alkyladenine DNA glycosylase or 06-methylguanine DNA methyltransferase in comparison to infected wt control mice. The forth aim of this thesis was to examine whether antimicrobial H. pylori eradication therapy could prevent gastric cancer development in INS-GAS mice, a model of gastric cancer. We found that antimicrobial H. pylori eradication therapy prevented the progression to gastric cancer in H. pylori-infected INS-GAS mice when treatment was instituted at an early stage of H. pylori infection. / (cont.) In conclusion, these studies provide further insight into the role of host immune responses in H. pylori pathogenesis. Additionally, information was garnered regarding the roles of vitamin C supplementation, DNA repair proteins, and H. pylori eradication therapy in H. pylori-associated gastric disease using genetically manipulated mice. / by Chung-Wei Lee. / Ph.D.

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.