Dissertations / Theses: 'Division of Biological Effects'

1

Kisiday, John D. (John David) 1970. "In vitro culture of a chondrocyte-seeded peptide hydrogel and the effects of dynamic compression." Thesis, Massachusetts Institute of Technology, 2003. http://hdl.handle.net/1721.1/29614.

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Thesis (Ph. D. in Bioengineering)--Massachusetts Institute of Technology, Biological Engineering Division, 2003.
Includes bibliographical references.
Emerging medical technologies for effective and lasting repair of articular cartilage include delivery of cells or cell-seeded scaffolds to a defect site to initiate de novo tissue regeneration. Biocompatible scaffolds assist in providing a template for cell distribution and extracellular matrix accumulation in a three-dimensional geometry. In these studies, a self-assembling peptide hydrogel is evaluated as a potential scaffold for cartilage repair using a model bovine cell source. A seeding technique is developed for 3-D encapsulation of chondrocytes in a peptide hydrogel. The chondrocyte-seeded peptide hydrogel was then evaluated cellular activities in vitro under standard culture conditions and also when subjected to dynamic compression. During 4 weeks of culture in vitro, chondrocytes seeded within the peptide hydrogel retained their morphology and developed a cartilage-like ECM rich in proteoglycans and type II collagen, indicative of a stable chondrocyte phenotype. Time dependent accumulation of this ECM was paralleled by increases in material stiffness, indicative of deposition of mechanically-functional neo-tissue. Culture of chondrocyte-seeded peptide hydrogels in ITS-supplemented medium was investigated as an alternative to high serum culture. Low serum (0.2%), ITS-supplemented medium was found to maintain high levels of cell division and extracellular matrix synthesis and accumulation, as seen in high serum culture. Furthermore, low serum, ITS medium induced minimal chondrocyte de-differentiation on the surface of the hydrogel. This is in contrast to high serum culture, where surface de-differentiation and subsequent proliferation led to a 5-10 cell thick layer that stained positive for type I collagen.
(cont.) The effects of dynamic compression of chondrocyte-seeded peptide hydrogels were evaluated over long-term culture. A non-continuous loading protocol was identified in which proteoglycan, but not protein, synthesis increased over static, free-swelling culture. Increases in GAG matrix accumulation were observed after at least 8 days of loading, while hydroxyproline accumulation was unaffected by dynamic compression. These data demonstrated dynamic compression differentially regulated the synthesis of proteoglycans. Analysis of GAG loss to the medium indicated peak proteoglycan catabolism occurred immediately after the initiation of loading. This phenomenon was further explored using a modified loading protocol that increased GAG loss to the medium. Peak GAG loss to the medium was 2-fold higher than previously observed, resulting in GAG accumulation values significantly less than controls. Hydroxyproline accumulation was minimally affected by loading, demonstrating that dynamic compression also differentially regulated the catabolism of proteoglycans. Proteoglycan catabolism was not predominantly due to physical disruption accumulated extracellular matrix or loss of newly-synthesized molecules. Instead, the presence of MMPs in the medium that coincided with GAG loss suggest a potential enzymatic mechanism. These results demonstrate the potential of a self-assembling peptide hydrogel as a scaffold for the synthesis and accumulation of a true cartilage-like extracellular matrix ...
John D. Kisiday.
Ph.D.in Bioengineering

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2

Szafranski, Jon D. (Jon David). "Cartilage mechanobiology : the effects of loading on the fine structure and function of chondroitin sulfate glycosaminoglycans." Thesis, Massachusetts Institute of Technology, 2005. http://hdl.handle.net/1721.1/33871.

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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.

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3

Wheeler, Cameron 1978. "Cartilage mechanobiology and transcriptional effects of combined mechanical compression and IGF-1 stimulation on bovine cartilage explants." Thesis, Massachusetts Institute of Technology, 2006. http://hdl.handle.net/1721.1/38613.

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Thesis (S.M.)--Massachusetts Institute of Technology, Biological Engineering Division, February 2007.
Includes bibliographical references.
Background: Investigators have focused on mechano-regulation of upstream signaling and responses at the level of gene transcription, protein translation and post-translational modifications. Intracellular pathways including those involving integrin signaling, mitogen activated protein kinases (MAPKs), and release of intracellular calcium have been confirmed in several laboratories. Studies with IGF-1: Insulin-like growth factor-I (IGF-1) is a potent anabolic factor capable of endocrine and paracrine/autocrine signaling. Previous studies have demonstrated that mechanical compression can regulate the action of IGF-1 on chondrocyte biosynthesis in intact tissue; when applied simultaneously, these stimuli act by distinct cell activation pathways. Our objectives were to elucidate the extent and kinetics of the chondrocyte transcriptional response to combined IGF-1 and static compression in cartilage explants. Discussion: Clustering analysis revealed five distinct groups. TIMP-3 and ADAMTS-5, MMP-l and IGF-2, and IGF-1 and Collagen II, were all robustly co-expressed under all conditions tested. In comparing gene expression levels to previously measured aggrecan biosynthesis levels, aggrecan synthesis is shown to be transcriptionally regulated by IGF- 1, whereas inhibition of aggrecan synthesis by compression is not transcriptionally regulated.
(cont.) Conclusion: Many genes measured are responsive the effects of IGF-1 under 0% compression and 50% compression. Clustering analysis revealed strong co-expressed gene pairings. IGF-1 stimulates aggrecan biosynthesis in a transcriptionally regulated manner, whereas compression inhibits aggrecan synthesis in a manner not regulated by transcriptional activity.
by Cameron A. Wheeler.
S.M.

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Villasenor, Jose Fernando. "HABITAT USE AND THE EFFECTS OF DISTURBANCE ON WINTERING BIRDS USING RIPARIAN HABITATS IN SONORA, MEXICO." The University of Montana, 2007. http://etd.lib.umt.edu/theses/available/etd-03282007-165836/.

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Riparian systems are important for breeding bird communities and are highly used as migratory corridors; however, their importance for wintering birds has not been assessed systematically. In order to assess the value of riparian areas for birds wintering in Sonora, data from 1,816 standard point counts were collected from 87 locations during January and February 2004-2006. A total of 253 species were detected across 14 vegetation types, including nine categories of riparian vegetation. The mean number of species and individuals detected per count was significantly higher in riparian vegetation than in non-riparian vegetation for migratory species, but not for residents. Riparian bird communities are different from those in non-riparian habitats, and contribute 22% of the regional avifauna's species.
Anthropogenic disturbance has imposed significant changes in riparian habitats, and is known to have negative effects on biological communities. To assess the effects of human induced disturbance on wintering bird communities, I recorded community composition, relative abundance of species, and three indicators of bird condition in relatively undisturbed and highly disturbed sites at three river systems in Sonora. There is, in general, little effect of disturbance on the composition of wintering communities, with less than 20% of the most common species having significant differences in their abundances between relatively undisturbed and highly disturbed sites. Condition indicators were similar in the two disturbance levels, but the mean heterophil/lymphocyte ratio in the blood of sampled birds showed increased levels of physiological stress in disturbed sites. A more experimental approach is needed to determine the specific cause of the stress expression in leucocytes.
Modification of natural flooding regimes has modified riparian areas, as has been the case in the Colorado River Delta. I present a summary of the changes experienced by riparian systems and some of the measures implemented for riparian restoration in the southwestern United States, and then I compare the scenario with that in central Sonora, where some of the same stressors exist on riparian systems, but where traditional management practices have also mitigated some of the negative consequences of flow control along mid-sized river systems.

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Anderson, Michelle Louise. "The edge effect: lateral habitat ecology of an alluvial river flood plain." The University of Montana, 2008. http://etd.lib.umt.edu/theses/available/etd-10012008-134442/.

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We describe the ecology of off-channel or "lateral" habitats as key attributes of the Shifting Habitat Mosaic (SHM) of a river flood plain at the Nyack Research Natural Area in northwestern Montana. Our working hypothesis was that lateral habitats are important to the cycling of energy and materials within the SHM and contribute greatly to the productivity and biodiversity of the floodplain ecosystem. We produce a quantitative description of temperature variation across aquatic and terrestrial habitats at Nyack. Thermal patterns across lateral habitats indicate vast differences exist between lateral habitats in habitat suitability for aquatic organisms over an annual cycle. Existing thermal regimes favor life history diversification. We further document the impacts of flood disturbances on organic matter accumulation by aquatic primary producers in off-channel environments. We were able to show that the annual flood pulse disturbance was the major force controlling periphyton community biomass, nutrient status, and species composition, with secondary control by surface and groundwater mediated water chemistry fluctuations during lower flow periods. We end with a study relating biodiversity pattern and process to lateral habitat heterogeneity. Data supported our prediction that densities and diversity of organisms and food webs of the flood plain would be greatly increased if off-channel habitats as well as main channel habitats were included. Collectively, lateral habitats uniquely support 50% of the total documented aquatic biodiversity of the entire floodplain. We conclude that lateral habitats contribute significantly to the biocomplexity of alluvial floodplain ecosystems.

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6

Sachs, Karen Ph D. Massachusetts Institute of Technology. "Bayesian network models of biological signaling pathways." Thesis, Massachusetts Institute of Technology, 2006. http://hdl.handle.net/1721.1/38865.

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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.

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7

Shiva, V. A. "Scalable computational architecture for integrating biological pathway models." Thesis, Massachusetts Institute of Technology, 2007. http://hdl.handle.net/1721.1/42384.

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Thesis (Ph. D.)--Massachusetts Institute of Technology, Biological Engineering Division, 2007.
MIT Institute Archives copy: DVD inserted in pocket on p. [3] of cover on v. 1.
"c2007"--p. ii.
Includes bibliographical references (v. 2, leaves 292-302).
A grand challenge of systems biology is to model the cell. The cell is an integrated network of cellular functions. Each cellular function, such as immune response, cell division, metabolism or apoptosis, is defined by an interconnected ensemble of biological pathways. Modeling the cell or even one cellular function requires a computational architecture that integrates multiple biological pathway models in a scalable manner while ensuring minimal effort to maintain the resulting integrated model. Scalable is defined as the ease in which more and more biological pathway models can be integrated. Current architectures for integrating biological pathway models are primarily monolithic and involve combining each biological pathway model's software source code to build one large monolithic model that executes on a single computer. Such architectures are not scalable for modeling complex cellular functions or the whole cell. We present Cytosolve, a new computational architecture that integrates a distributed ensemble of biological pathway models and computes solutions in a parallel manner while offering ease of maintenance of the integrated model. The individual biological pathway models can be represented in SBML, CellML or in any number of formats. The EGFR model of Kholodenko with known solutions is used to compare the Cytosolve solution and computational times with a known monolithic approach. A new integrative model of the interferon (IFN) response to virus infection is developed using Cytosolve. Each model within the integrated model, spans different time scales, is created by different authors from four countries and three continents across different disciplines, is written in different software codes, and is built on different hardware platforms.
(cont.) A new quantitative methodology and formalism is then derived for evaluating different types of monolithic and distributed architectures for integrating biological pathway models. As more biological pathway models develop in a disparate and decentralized manner, the Cytosolve architecture offers a unique platform to build and test complex models of cellular function, and eventually the whole cell.
by V.A. Shiva Ayyadurai.
Ph.D.

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8

Margolin, Yelena 1977. "Analysis of sequence-selective guanine oxidation by biological agents." Thesis, Massachusetts Institute of Technology, 2007. http://hdl.handle.net/1721.1/42381.

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Thesis (Ph. D.)--Massachusetts Institute of Technology, Biological Engineering Division, February 2008.
Vita.
Includes bibliographical references.
Oxidatively damaged DNA has been strongly associated with cancer, chronic degenerative diseases and aging. Guanine is the most frequently oxidized base in the DNA, and generation of a guanine radical cation (G'") as an intermediate in the oxidation reaction leads to migration of a resulting cationic hole through the DNA n-stack until it is trapped at the lowest-energy sites. These sites reside at runs of guanines, such as 5'-GG-3' sequences, and are characterized by the lowest sequence-specific ionization potentials (IPs). The charge transfer mechanism suggests that hotspots of oxidative DNA damage induced by electron transfer reagents can be predicted based on the primary DNA sequence. However, preliminary data indicated that nitrosoperoxycarbonate (ONOOCO2"), a mediator of chronic inflammation and a one-electron oxidant, displayed unusual guanine oxidation properties that were the focus of present work. As a first step in our study, we determined relative levels of guanine oxidation, induced by ONOOCO2 in all possible three-base sequence contexts (XGY) within double-stranded oligonucleotides. These levels were compared to the relative oxidation induced within the same guanines by photoactivated riboflavin, a one-electron reagent. We found that, in agreement with previous studies, photoactivated riboflavin was selective for guanines of lowest IPs located within 5'-GG-3' sequences. In contrast, ONOOCO2" preferentially reacted with guanines located within 5'-GC-3' sequences characterized by the highest IPs. This demonstrated that that sequence-specific IP was not a determinant of guanine reactivity with ONOOCO2". Sequence selectivities for both reagents were double-strand specific. Selectivity of ONOOCO2 for 5'-GC-3' sites was also observed in human genomic DNA after ligation-mediated PCR analysis.
(cont.) Relative yields of different guanine lesions produced by both ONOOCO2" and riboflavin varied 4- to 5-fold across all sequence contexts. To assess the role of solvent exposure in mediating guanine oxidation by ONOOCO2", relative reactivities of mismatched guanines with ONOOCO2" were measured. The majority of the mismatches displayed an increased reactivity with ONOOCO2 as compared to the fully matched G-C base-pairs. The extent of reactivity enhancement was sequence context-dependent, and the greatest levels of enhancement were observed for the conformationally flexible guanine- guanine (G-G) mismatches and for guanines located across from a synthetic abasic site. To test the hypothesis that the negative charge of an oxidant influences its reactivity with guanines in DNA, sequence-selective guanine oxidation by a negatively charged reagent, Fe+2-EDTA, was assessed and compared to guanine oxidation produced by a neutral oxidant, y-radiation. Because both of these agents cause high levels of deoxyribose oxidation, a general method to quantify sequence-specific nucleobase oxidation in the presence of direct strand breaks was developed. This method exploited activity of exonuclease III (Exo III), a 3' to 5' exonuclease, and utilized phosphorothioate-modified synthetic oligonucleotides that were resistant to Exo III activity. This method was employed to determine sequence-selective guanine oxidation by Fe+2-EDTA complex and y-radiation and to show that both agents produced identical guanine oxidation pattems and were equally reactive with all guanines, irrespective of their sequence-specific IPs or sequence context.
(cont.) This showed that negative charge was not a determinant of Fe+2-EDTA-mediated guanine oxidation. Finally, the role of oxidant binding on nucleobase damage was assessed by studying sequence-selective oxidation produced by DNA-bound Fe+2 ions in the presence of H202. We found that the major oxidation targets were thymines located within 5'-TGG-3' motifs, demonstrating that while guanines were a required element for coordination of Fe+2 to DNA, they were not oxidized. Our results suggest that factors other than sequence-specific IPs can act as major determinants of sequence-selective guanine oxidation, and that current models of guanine oxidation and charge transfer in DNA cannot be used to adequately predict the location and identity of mutagenic lesions in the genome.
by Yelena Margolin.
Ph.D.

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9

Canton, Bartholomew (Bartholomew John). "Engineering the interface between cellular chassis and synthetic biological systems." Thesis, Massachusetts Institute of Technology, 2008. http://hdl.handle.net/1721.1/44918.

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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 (p. 165-176).
The aim of my thesis is to help enable the engineering of biological systems that behave in a predictable manner. Well-established techniques exist to engineer systems that behave as expected. Here, I apply such techniques to two aspects of the engineering of biological systems. First, I address the design and construction of standard biological devices in a manner that facilitates reuse in higher-order systems. I describe the design and construction of an exemplar device, an engineered cell-cell communication receiver using standard biological parts (refined genetic objects designed to support physical and functional composition). I adopt a conventional framework for describing the behavior of engineered devices and use the adopted framework to design and interpret experiments that describe the behavior of the receiver. The output of the device is the activity of a promoter reported in units of Polymerases Per Second (PoPS), a common signal carrier. Second, I begin to address the coupling that exists between engineered biological systems and the host cell, or chassis. I propose that the coupling between engineered biological systems and the cellular chassis might be reduced if fewer resources were shared between the system and the chassis. I describe the construction of cellular chassis expressing both T7 RNA polymerases (RNAP) and orthogonal ribosomes that are unused by the chassis but are available for use by an engineered system. I implement a network in which the orthogonal ribosomal RNA and the gene encoding T7 RNAP are transcribed by T7 RNAP. In turn, the orthogonal ribosomes translate the T7 RNAP message. In addition, the T7 RNAP and orthogonal ribosomes express a repressor that inhibits transcription of both the T7 RNAP and orthogonal ribosomes.
(cont.) As a result, the orthogonal RNAP and ribosomes are auto-generating and self-regulating. The provision of resources unused by the cellular chassis and dedicated to an engineered biological system forms the beginnings of a biological virtual machine.
by Bartholomew Canton.
Ph.D.

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10

Frick, Lauren Elizabeth. "The versatile E. coli adaptive response protein AlkB mitigates toxicity and mutagenicity of etheno-, ethano-, and methyl-modified bases in vivo." Thesis, Massachusetts Institute of Technology, 2007. http://hdl.handle.net/1721.1/42382.

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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.

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11

Brau, Ricardo R. (Ricardo Rafael) 1979. "Exploring the mechanome with optical tweezers and single molecule fluorescence." Thesis, Massachusetts Institute of Technology, 2007. http://hdl.handle.net/1721.1/43795.

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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.

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McBee, Megan Earley. "Immunomodulation by subclinical persistent infection with Helicobacter hepaticus." Thesis, Massachusetts Institute of Technology, 2007. http://hdl.handle.net/1721.1/39916.

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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.

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13

Lee, Chung-Wei Ph D. Massachusetts Institute of Technology. "Pathogenesis of the carcinogenic bacterium, Helicobacter pylori." Thesis, Massachusetts Institute of Technology, 2007. http://hdl.handle.net/1721.1/39909.

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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.

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France, Danielle Cook. "Structure and mechanics of the spasmoneme, a biological spring within the protozoan Vorticella convallaria." Thesis, Massachusetts Institute of Technology, 2007. http://hdl.handle.net/1721.1/39904.

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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.

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15

Garcia-Webb, Michael G. (Michael Gregory). "An apparatus for high throughput muscle cell experimentation." Thesis, Massachusetts Institute of Technology, 2006. http://hdl.handle.net/1721.1/38240.

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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.

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16

Sams, Alexandria V. (Alexandria Victoria). "Development and characterization of an in vitro culture system as a physiological model for chronic Hepatitis B infection." Thesis, Massachusetts Institute of Technology, 2007. http://hdl.handle.net/1721.1/39915.

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Thesis (Ph. D.)--Massachusetts Institute of Technology, Biological Engineering Division, 2007.
Includes bibliographical references (p. 152-164).
Human Hepatitis B virus (HBV) is the prototype member of the family Hepadnaviridae that consists of enveloped, partially double stranded DNA viruses that specifically target hepatocytes for viral replication. Although a vaccine has been available for more than 20 years chronic HBV infection afflicts 350-400 million worldwide. It is estimated that 0.5-1.2 million people die each year from HBV-attributable cases of chronic hepatitis, cirrhosis, and hepatocellular carcinoma. Significant disadvantages exist among currently available therapeutics (e.g. IFNca, lamivudine, adefovir, etc.) that include limited efficacy and the promotion of drug-resistant viral strains. These therapeutics are the research products of the HBV molecular biology that can be manipulated in the laboratory setting. Future antiviral drug therapy is dependent upon the development of better cell culture systems that will allow the study of the complete viral life cycle. The use of primary human and primate hepatocytes is restricted by multiple experimental limitations including a rapid loss of susceptibility to infection in culture, lot-to-lot variability inherent in primary cell culture, and the necessity of treatment with chemical agents such as DMSO for reproducible infection.
(cont.) Permissive cell lines are capable of supporting viral replication upon transfection with the HBV genome. These cell lines have helped to elucidate the later events in the viral life cycle. However, there is less understanding of the early stages that include virus attachment, internalization, uncoating, nuclear transport, and genome repair. Our group has developed an in vitro system that recreates many of the features of a perfused capillary bed structure. Various metrics (e.g. biochemical production, tissue morphology, liver-enriched mRNA expression, and drug metabolism) confirm that this system maintains a well-differentiated liver phenotype. Using DHBV as a surrogate model, this study has attempted to demonstrate that hepatocytes maintained in a more sophisticated culture system retain susceptibility to infection. This study has endeavored to establish the perfused three-dimensional culture system as potential tool to study early events of the viral life cycle. This research lays the foundation for the future development of a human HBV infection model in which early stages of the viral life cycle can be studied and therapeutic targets identified.
by Alexandria V. Sams.
Ph.D.

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17

Kosuri, Sriram. "Simulation, models, and refactoring of bacteriophage T7 gene expression." Thesis, Massachusetts Institute of Technology, 2006. http://hdl.handle.net/1721.1/39912.

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Thesis (Sc. D.)--Massachusetts Institute of Technology, Biological Engineering Division, February 2007.
Includes bibliographical references (leaves 108-124).
Our understanding of why biological systems are designed in a particular way would benefit from biophysically-realistic models that can make accurate predictions on the time-evolution of molecular events given arbitrary arrangements of genetic components. This thesis is focused on constructing such models for gene expression during bacteriophage T7 infection. T7 gene expression is a particularly well suited model system because knowledge of how the phage functions is thought to be relatively complete. My work focuses on two questions in particular. First, can we address deficiencies in past simulations and measurements of bacteriophage T7 to improve models of gene expression? Second, can we design and build refactored surrogates of T7 that are easier to understand and model? To address deficiencies in past simulations and measurements, I developed a new single-molecule, base-pair-resolved gene expression simulator named Tabasco that can faithfully represent mechanisms thought to govern phage gene expression. I used Tabasco to construct a model of T7 gene expression that encodes our mechanistic understanding. The model displayed significant discrepancies from new system-wide measurements of absolute T7 mRNA levels during infection.
(cont.) I fit transcript-specific degradation rates to match the measured RNA levels and as a result corrected discrepancies in protein synthesis rates that confounded previous models. I also developed and used a fitting procedure to the data that let us evaluate assumptions related to promoter strengths, mRNA degradation, and polymerase interactions. To construct surrogates of T7 that are easier to understand and model, I began the process of refactoring the T7 genome to construct an organism that is a more direct representation of the models that we build. In other words, instead of making our models evermore detailed to explain wild-type T7, we started to construct new phage that are more direct representations of our models. The goal of our original design, T7. 1, was to physically define, separate, and enable unique manipulation of primary genetic elements. To test our initial design, we replaced the left 11,515 bp of the wild-type genome with 12,179 bp of engineered DNA. The resulting chimeric genome encodes a viable bacteriophage that appears to maintain key features of the original while being simpler to model and easier to manipulate. I also present a second generation design, T7.2, that extends the original goals of T7.1 by constructing a more direct physical representation of the T7 model.
by Sriram Kosuri.
Sc.D.

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18

Borenshtein, Diana. "Development, characterization and transcriptional profiling of a mouse model of fatal infectious diarrhea and colitis." Thesis, Massachusetts Institute of Technology, 2007. http://hdl.handle.net/1721.1/39843.

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Thesis (Ph. D.)--Massachusetts Institute of Technology, Biological Engineering Division, 2007.
This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections.
Includes bibliographical references (p. 195-208).
Citrobacter rodentium is a naturally occurring murine bacterial pathogen which is used to model human diarrheagenic E. coli (EPEC and EHEC) infections in mice. C. rodentium causes colonic hyperplasia and a variable degree of colitis and mortality in the majority of inbred and outbred lines of mice. Differences in C. rodentium-induced disease are determined by the genetic background of the host. Here, C. rodentium infection in resistant outbred Swiss Webster (SW) mice was compared with infection in the cognate inbred FVB strain for the first time. In contrast to subclinical infection in SW mice, adult FVB mice developed overt disease with significant weight loss, severe colitis, and over 75% mortality. Fluid therapy intervention completely prevented mortality in FVB mice, and expression of pro-inflammatory and immunomodulatory genes in the colon was similar in both lines of mice, suggesting that mortality in C. rodentiuminfected FVB mice is due to hypovolemia resulting from severe dehydration. To identify host factors responsible for the development of mortality, gene expression in the distal colon of FVB and SW mice was investigated using a whole mouse genome Affymetrix array.
(cont.) Transcripts represented by 1,547 probe sets (3.4%) were differentially expressed between FVB and SW mice prior to infection and at 4 and 9 days post-inoculation. Data analysis suggested that intestinal ion disturbances rather than immune-related processes are responsible for susceptibility in C. rodentium-infected FVB mice. Marked impairment in intestinal ion homeostasis predicted by microarray analysis was confirmed by quantitative RT-PCR and serum electrolyte measurements that showed hypochloremia and hyponatremia in susceptible FVB mice. C. rodentium infection was next characterized in additional inbred strains of Swiss origin. SWR and SJL mice developed minimal morbidity and no mortality in response to the pathogen, demonstrating resistance to disease. Furthermore, C3H mice developed severe diarrhea and gene expression changes comparable to those in infected FVB mice, suggesting common pathogenic mechanisms in susceptible strains. In conclusion, C. rodentium infection in FVB mice is a useful model for fatal infectious diarrhea. These studies contribute to our understanding of C. rodentium pathogenesis and identify possible candidates for susceptibility to fatal enteric bacterial infection.
by Diana Borenshtein.
Ph.D.

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19

Yap, Jonathan Woon Teck. "Dendritic cell maturation and activation via RNA/DNA danger signals : co-delivery of protein antigen with siRNA or CpG DNA." Thesis, Massachusetts Institute of Technology, 2005. http://hdl.handle.net/1721.1/38449.

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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.

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20

Fitzgerald, Jonathan Basil. "Chondrocyte gene expression and intracellular signaling pathways in cartilage mechanotransduction." Thesis, Massachusetts Institute of Technology, 2005. http://hdl.handle.net/1721.1/33869.

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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.

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21

Sun, Hsiao-Lan Patty. "Mechanisms of toxicity and carcinogenicity of three alkylanilines." Thesis, Massachusetts Institute of Technology, 2006. http://hdl.handle.net/1721.1/37958.

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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.

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22

Camp, James (James Patrick) 1977. "Development of simple 3D-printed scaffolds for liver tissue engineering." Thesis, Massachusetts Institute of Technology, 2002. http://hdl.handle.net/1721.1/16864.

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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

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23

Lee, Peter S. M. Massachusetts Institute of Technology. "Using optical tweezers, single molecule fluorescence and the ZIF268 protein-DNA system to probe mechanotransduction mechanisms." Thesis, Massachusetts Institute of Technology, 2005. http://hdl.handle.net/1721.1/34490.

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Thesis (S.M.)--Massachusetts Institute of Technology, Biological Engineering Division, 2006.
Includes bibliographical references (p. 42-43).
Optical tweezers instruments use laser radiation pressure to trap microscopic dielectric beads. With the appropriate chemistry, such a bead can be attached to a single molecule as a handle, permitting the application of force on the single molecule. Measuring the force applied in real-time is dependent on detecting the bead's displacement from the trapping laser beam axis. Back-focal-plane detection provides a way of measuring the displacement, in two-dimensions, at nanometer or better resolution. The first part of this work will describe the design of a simple and inexpensive position sensing module customized for optical tweezers applications. Single molecule fluorescence is another powerful technique used to obtain microscopic details in biological systems. This technique can detect the arrival of a single molecule into a small volume of space or detect the conformational changes of a single molecule. Combining optical tweezers with single-molecule fluorescence so that one can apply forces on a single molecule while monitoring its effects via single molecule fluorescence provides an even more powerful experimental platform to perform such microscopic studies. Due to the enhanced photobleaching of fluorophores caused by the trapping laser, this combined technology has only been demonstrated under optimized conditions.
(cont.) The second part of this work will describe a straightforward and noninvasive method of eliminating this problem. The study of mechanotransduction in biological systems is critical to understanding the coupling between mechanical forces and biochemical reactions. Due to the recent advances in single molecule technology, it is now possible to probe such mechanisms at the single molecule level. The third and final part of this work will describe a basic mechanotransduction experiment using the well-studied ZIF268 protein-DNA system. An experimental assay and method of analysis will be outlined.
by Peter Lee.
S.M.

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24

Dong, Min 1968. "Nucleobase deamination as a biomarker of inflammatory processes." Thesis, Massachusetts Institute of Technology, 2005. http://hdl.handle.net/1721.1/28763.

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Thesis (Ph. D.)--Massachusetts Institute of Technology, Biological Engineering Division, 2005.
Includes bibliographical references.
(cont.) kinetic studies. Moderate increases ([approximately]30%) in nucleobase deamination products were observed in the SJL mice bearing the RcsX tumor, but the biological meaning of these increases awaits studies of DNA repair kinetics. dO was not detected in any study at levels above 5 per 10⁸ nt, leading to the prediction that it will not be present at significant levels in inflamed tissues in humans. As a complement to the LC-MS method for the quantification of nucleobase deamination products, enzymatic probes were also developed for oxidative and nitrosative DNA lesions. These probes would not only allow differential quantification of the two types of DNA damage, but would also allow the lesions to be mapped in any DNA sequence by coupling their activity with the technique of ligation-mediated PCR. As an extension of the biomarker study, the effects of ONOO⁻ dose and dose-rate on the DNA damage and mutations induced in the supF gene were investigated. The observations suggest that both the dose and dose-rate at which a genetic target is exposed to ONOO⁻ substantially influence the damage and mutational response and these parameters will need to be considered in assessing the potential effects of ONOO⁻ in vivo. Finally, an extended study using the analytical method developed in this thesis yielded results in E. coli consistent with a new paradigm: perturbations of nucleobase metabolism may lead to incorporation of the purine precursors hypoxanthine (I) and xanthine (X) into DNA. This can be regarded as another endogenous process causing DNA damage that may lead to human diseases such as cancer ...
The objective of this thesis project was to develop nucleobase deamination products as biomarkers of inflammation and to study the role of these DNA lesions in the pathophysiology of inflammation-induced carcinogenesis. The basis of this research is the epidemiological evidence that chronic inflammation is associated with an increased risk of cancer, yet the link between the inflammatory process and the development of cancer has eluded definition. Biomarker development began with the establishment of a sensitive liquid chromatography-mass spectrometry (LC-MS) method to quantify four of the nucleobase deamination products: 2'-deoxyxanthosine (dX) and 2'-deoxyoxanosine (dO) from the deamination of dG; 2'-deoxyuridine (dU) from dC; and 2'-deoxyinosine (dl) from dA. The analytical method was then validated and tested with both in vitro and in vivo studies involving quantification of nucleobase deamination products in isolated plasmid DNA and human lymphoblastoid cells exposed to nitric oxide (NO)Ì at controlled physiological concentrations. Finally, the formation of nucleobase deamination products was analyzed in SJL/RcsX mice, an established mouse model of NO Ìover production. This set of studies revealed several important features of the nitrosative chemistry of NO Ìderivatives. The in vitro formation of dX, dl and dU was found to occur at nearly identical rates (k = 1.2 x 10⁵ M⁻¹s⁻¹). Low levels of nucleobase deamination products were formed in cells exposed to NO,Ì which suggests that cellular factors significantly influence the nitrosative chemistry. However, cellular glutathione (GSH) was found to play a smaller role than expected, considering the effects of GSH on steady-state concentrations of N₂0₃ in in vitro
by Min Dong.
Ph.D.

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25

Vuong, Yihvan 1981. "Development of a biocompatible method of implanting multi-electrode arrays for stable chronic deep neuronal recording." Thesis, Massachusetts Institute of Technology, 2004. http://hdl.handle.net/1721.1/28864.

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Thesis (M. Eng.)--Massachusetts Institute of Technology, Biological Engineering Division, 2004.
Includes bibliographical references (leaf 43).
(cont.) electrodes.
Flexible multi-electrode arrays represent an evolving technology for chronic deep neuronal recording. We focus on developing a biocompatible method of implanting a novel 12-electrode device for long-term recording (weeks to months) in a deep target responsible for object identification, the inferotemporal cortex (IT). Numerous electrodes embedded in a bioerodible polymer matrix composed of 50/50 poly(D,L-lactide-co-glycolide) created an electrode bundle that was sufficiently stiff for insertion into brain tissue, but was expected to become more flexible after polymer degradation. Titanium electrode ports used to house these multi-electrode arrays were successfully designed, constructed, and implanted on a non-human primate as well. These ports were tested for mechanical durability over a course of 6-8 weeks. To evaluate the recording properties of the electrodes, many parameters of the electrode bundle tip were explored and evaluated, including gold-plating, tip cutting (flat cut, angle cut, or individual wire cut), and splaying. Gold-plating lowered electrode impedance and improved signal quality on originally high impedance electrodes. The angle cut and individually cut electrodes were superior to the flat cut ones; they recorded from 6-7 active channels at varying depths during advancement. Splaying, the removal of polymer some distance from the array tip to separate the bundle into individual electrodes, also improved the recording properties in acute experiments. Pending chronic experiments will further reveal 1) the true degradation rate of the array and thus the flexibility restored, 2) the electrode tip characteristics following polymer erosion, and 3) the long-term recording quality and stability of all the
by Yihvan Vuong.
M.Eng.

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26

Gruhl, Amanda Natalie. "Metakaryotic biology : novel genomic organization in human stem-like cells of fetal-juvenile development and carcinogenesis." Thesis, Massachusetts Institute of Technology, 2008. http://hdl.handle.net/1721.1/44864.

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Thesis (Ph. D.)--Massachusetts Institute of Technology, Biological Engineering Division, 2008.
Includes bibliographical references (leaves 66-75).
Eight distinct nuclear shapes, or morphologies, have been discovered in human proto-organs and tumors, including bell-shaped nuclei with stem-like properties. These bell-shaped, or "metakaryotic," nuclei are abundant in fetal tissues and neoplasias, but rare in normal adult somatic tissues. Metakaryotic nuclei employ an unusual process for division in which DNA synthesis, partial genomic condensation, and separation of the two nuclei in a cup-from-cup fashion occur concurrently, as shown by Feulgen densitometry and single-stranded DNA assays by Dr. Elena Gostjeva. This is clearly different from the sequential steps of S-phase DNA synthesis, chromatin condensation, chromosomal separation, and genomic segregation that occur in mitotic eukaryotic cells. In order to discover how a genome apparently devoid of chromosomes might be organized, this thesis focused on recognizable DNA sequences common to all chromosomes: centromeres and telomeres. Fluorescence In Situ Hybridization (FISH) with pan-centromeric and pan-telomeric probes was applied to samples of human tissue. (A collaborating lab used centromeric and telomeric antibodies to confirm results.) An optimized FISH protocol was developed specifically for metakaryotic nuclei and tested in both human cell lines and eukaryotic cells as experimental controls. Staining of metakaryotic nuclei resulted in approximately 23 centromeric regions in each, unlike the expected number of 46 regions seen in eukaryotic nuclei. Many of these staining regions contained paired centromere signals, or doublets. This suggested a genomic organization of homologous chromosomes paired at their centromere regions. If this were the case, one would expect 46 telomeric signals per nuclei, if telomeres were also homologously paired.
(cont.) Unexpectedly, an average of 23 telomeric regions were found in many, if not all, bell-shaped metakaryotic nuclei. This, along with the observation of a condensed double ring around the mouth of the bell-shaped nuclei, suggested the possibility of a genome organized as paired, continuous genomic circles. Studies of telomere joining in metakaryotic nuclei by Dr. Per Olaf Ekstrom have provided further evidence for the paired genomic circle model. The results in this thesis are an original contribution to the field of stem cell physiology, a starting point for further investigation of DNA organization, synthesis, and repair in these metakaryotic cells, and hopefully will lead to a greater understanding of human development, growth, and cancer.
by Amanda Natalie Gruhl.
Ph.D.

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27

Ganz, Michal. "Investigation of growth factors and cytokines that suppress adult stem cell asymmetric cell kinetics." Thesis, Massachusetts Institute of Technology, 2005. http://hdl.handle.net/1721.1/33874.

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Thesis (S.M.)--Massachusetts Institute of Technology, Biological Engineering Division, 2005.
Includes bibliographical references (leaves 40-43).
Adult stem cells are potentially useful in many biomedical applications that can save lives and increase the quality of a patient's life, such as tissue engineering, cell replacement, and gene therapy. However, these applications are limited because of the difficulty in isolating and expanding pure populations of adult stem cells (ASCs). A major barrier to ASC expansion in vitro is their property of asymmetric cell kinetics. Our lab has developed a method, Suppression of Asymmetric Cell Kinetics (SACK), to expand ASCs in vitro by shifting their cell kinetics program from asymmetric to symmetric. We have found that guanine nucleotide precursors can be used to convert the kinetics of adult stem cells from asymmetric to symmetric, which promotes their exponential expansion. Previously, we have used the SACK method to derive hepatic and cholangiocyte stem cell strains from adult rat livers in vitro. These cell strains provide an assay to evaluate whether growth factors and cytokines previously implicated in proliferation of progenitor cells act by converting the kinetics of the stem cells in the population from asymmetric to symmetric, and thus identify new SACK agents. We are evaluating three agents, Wnt, IGF- 1, and Sonic hedgehog (Shh).
(cont.) Wnt has been found to cause self-renewal and proliferation of hematopoietic stem cells (HSCs) in vitro. IGF- 1 also plays a role in hematopoietic progenitor self-renewal in vivo as well as in tissue maturation. Shh has been implicated in the proliferation of primitive neural cells as well as in cellular proliferation during invertebrate development. Thus far, we have found that Wnt peptide shifts the cell kinetics from asymmetric to symmetric and may reduce the generation time, whereas IGF-1 appears only to affect generation time. Studies involving Shh are currently underway. We are also currently investigating whether Wnt acts additively or synergistically with guanine nucleotide precursors to shift cell kinetic symmetry. Discovering new SACK agents will allow us to obtain purer populations of ASCs that can be used to study properties unique to stem cells. Furthermore, the observation that Wnt shifts the kinetics of adult rat hepatic stem cells from asymmetric to symmetric implicates the involvement of similar cell kinetics symmetry mechanisms in the proliferation effect of Wnt on murine and human HSCs.
by Michal Ganz.
S.M.

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28

Locasale, Jason W. "Design principles of mammalian signaling networks : emergent properties at modular and global scales." Thesis, Massachusetts Institute of Technology, 2008. http://hdl.handle.net/1721.1/44862.

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Thesis (Ph. D.)--Massachusetts Institute of Technology, Biological Engineering Division, 2008.
Includes bibliographical references (leaves 244-249).
This thesis utilizes modeling approaches rooted in statistical physics and physical chemistry to investigate several aspects of cellular signal transduction at both the modular and global levels. Design principles of biological networks and cell signaling processes pertinent to disease progression emerge from these studies. It is my hope that knowledge of these principles may provide new mechanistic insights and conceptual frameworks for thinking about therapeutic intervention into diseases such as cancer and diabetes that arise from aberrant signaling. Areas of interest have emphasized the role of scaffold proteins in protein kinase cascades, modeling relevant biophysical processes related to T cell activation, design principles of signal transduction focusing on multisite phosphorylation, quantifying the notion of signal duration and the time scale dependence of signal detection, and entropy based models of network architecture inferred from proteomics data. These problems are detailed below. The assembly of multiple signaling proteins into a complex by a scaffold protein guides many cellular decisions. Despite recent advances, the overarching principles that govern scaffold function are not well understood. We carried out a computational study using kinetic Monte Carlo simulations to understand how spatial localization of kinases on a scaffold may regulate signaling under different physiological condition. Our studies identify regulatory properties of scaffold proteins that allow them to both amplify and attenuate incoming signals in different biological contexts. In a further, supplementary study, simulations also indicate that a major effect that scaffolds exert on the dynamics of cell signaling is to control how the activation of protein kinases is distributed over time[2].
(cont.) Scaffolds can influence the timing of kinase activation by allowing for kinases to become activated over a broad range of times, thus allowing for signaling across a broad spectrum of time scales. T cells orchestrate the adaptive immune response and are central players in maintenance of functioning immune system. Recent studies have reported that T cells can integrate signals between interrupted encounters with Antigen Presenting Cells (APCs) in such a way that the process of signal integration exhibits a form of memory. We carried out a computational study using a simple mathematical model of T cell activation to investigate the ramifications of interrupted T cell-APC contacts on signal integration. We considered several mechanisms of how signal integration at these time scales may be achieved. In another study, we investigated the role of spatially localizing signaling components of the T cell signaling pathway into a structure known as the immunological synapse. We constructed a minimal mathematical model that offers a mechanism for how antigen quality can regulate signaling dynamics in the immunological synapse These studies involving the analysis of signaling dynamics led us to investigate how differences in signal duration might be detected. Signal duration (e.g. the time scales over which an active signaling intermediate persists) is a key regulator of biological decisions in myriad contexts such as cell growth, proliferation, and developmental lineage commitments. Accompanying differences in signal duration are numerous downstream biological processes that require multiple steps of biochemical regulation. We present an analysis that investigates how simple biochemical motifs that involve multiple stages of regulation can be constructed to differentially process signals that persist at different time scales[3].
(cont.) Topological features of these networks that allow for different frequency dependent signal processing properties are identified. One role of multisite phosphorylation in cell signaling is also investigated. The utilization of multiple phosphorylation sites in regulating a biological response is ubiquitous in cell signaling. If each site contributes an additional, equivalent binding site, then one consequence of an increase in the number of phosphorylations may be to increase the probability that, upon disassociation, a ligand immediately rebinds to its receptor. How such effects may influence cell signaling systems is not well understood. A self-consistent integral equation formalism for ligand rebinding, in conjunction with Monte Carlo simulations, was employed to further investigate the effects of multiple, equivalent binding sites on shaping biological responses. Finally, this thesis also seeks to investigate cell signaling at a global scale. Advances in Mass Spectrometry based phosphoproteomics have allowed for the real-time quantitative monitoring of entire proteomes as signals propagate through complex networks in response to external signals. The trajectories of as many as 222 phosphorylated tyrosine sites can be simultaneously and reproducibly monitored at multiple time points. We develop and apply a method using the principle of maximum entropy to infer a model of network connectivity of these phosphorylation sites. The model predicts a core structure of signaling nodes, affinity dependent topological features of the network, and connectivity of signaling nodes that were hitherto unassociated with the canonical growth factor signaling network. Our combined results illustrate many complexities in the broad array of control properties that emerge from the physical effects that constrain signal propagation on complex biological networks.
(cont.) It is the hope of this work that these studies bring coherence to seemingly paradoxical observations and suggest that cells have evolved design rules that enable biochemical motifs to regulate widely disparate cellular functions.
by Jason W. Locasale.
Ph.D.

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29

Ferrer, Jorge M. 1976. "Mapping the actin and actin binding proteins interactions : from micromechanics to single molecule force spectroscopy." Thesis, Massachusetts Institute of Technology, 2007. http://hdl.handle.net/1721.1/40950.

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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.

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30

Bever, Caitlin Anne. "Selecting high-confidence predictions from ordinary differential equation models of biological networks." Thesis, Massachusetts Institute of Technology, 2008. http://hdl.handle.net/1721.1/45212.

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Thesis (Ph. D.)--Massachusetts Institute of Technology, Biological Engineering Division, 2008.
Includes bibliographical references (p. 139-153).
Many cellular processes are governed by large and highly-complex networks of chemical interactions and are therefore difficult to intuit. Computational modeling provides a means of encapsulating information about these interactions and can serve as a platform for gaining understanding of the biology and making predictions about cellular response to perturbation. In particular, there has been considerable interest in ordinary differential equation (ODE) models, which have several attractive features: ODEs can describe molecular interactions with mechanistic detail, it is relatively straightforward to implement perturbations, and, in theory, they can predict the concentration and activity of every species as a function of time. However, both the topology and parameters in such models are subject to considerable uncertainty. We explore the ramifications of these sources of uncertainty for making accurate predictions and develop methods of selecting high confidence predictions from uncertain models. In particular, we promote a shift in emphasis from model selection to prediction selection, and use consensus among model ensembles to identify the predictions most likely to be accurate. By constructing decision trees, this consensus can also be used to partition the space of potential perturbations into regions of high and low confidence. We apply our methods to the Fas signaling pathway in apoptosis to satisfy two goals: first, to design a therapeutic cocktail to reduce cell death in the presence of high levels of stimulus, and second, to design experiments that may lead to a better understanding of the biological network.
by Caitlin Anne Bever.
Ph.D.

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31

Robbins, Jennifer L. "The role of mismatch repair in mediating cellular sensitivity to cisplatin : the Escherichia coli methyl-directed repair paradigm." Thesis, Massachusetts Institute of Technology, 2006. http://hdl.handle.net/1721.1/34155.

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Thesis (Ph. D.)--Massachusetts Institute of Technology, Biological Engineering Division, 2006.
Includes bibliographical references (v. 2, leaves 195-258).
The anticancer drug cisplatin is in widespread use but its mechanism of action is only poorly understood. Moreover, human cancers acquire resistance to the drug, which limits its clinical utility. A paradox in the field is how loss of mismatch DNA repair leads to clinical resistance to this widely used drug. The phenomenon of cisplatin tolerance in mismatch repair deficient cells was initially discovered in E. coli, where methylation deficient dam mutants show high sensitivity to cisplatin and dam mutants with an additional mutation in either of the mismatch repair genes mutS or mutL show near wildtype levels of resistance. A prevalent explanation for this observation is the abortive repair model, which proposes that in dam mutants, where the strand discrimination signal is lost, mismatch repair attempts futile cycles of repair opposite cisplatin-DNA adducts. Previous findings have supported this model to the extent that MutS, the E. coli mismatch recognition protein, specifically recognizes DNA modified with cisplatin. However it has recently been shown that MutS binding to cisplatin adducts may contribute to toxicity by instead preventing the recombinational repair of a cisplatin-modified substrate, and we have previously shown that recombination is an essential mechanism for tolerating cisplatin damage.
(cont.) In the present study, we examined the global transcriptional responses of wildtype, dam, dam mutS, and mutS mutant E. coli after treatment with a toxic dose of cisplatin. We also determined any dose-response at the transcriptional level of several SOS response genes and other genes involved in DNA repair by real time RT-PCR. Furthermore, we performed single-cell electrophoresis in order to determine the effect of mismatch repair on the level of double-strand break formation in cisplatin-treated cells. Our results show that Dam-deficient strains exhibit unique gene regulation that may be due to mismatch-repair induced DNA damage in the absence of adenine methylation. In addition, cisplatin treatment induces double-strand break formation and the SOS response in a dose-dependent manner, and both break formation and the SOS response are greatest in the hypersensitive dam mutant strain. The higher level of cisplatin-induced double-strand breaks in the dam mutant may be dependent on functional mismatch repair.
by Jennifer L. Robbins.
Ph.D.

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32

Green, Jordan Jamieson. "Enhanced polymeric nanoparticles for gene delivery." Thesis, Massachusetts Institute of Technology, 2007. http://hdl.handle.net/1721.1/44299.

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

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33

Rothman, Craig Jeremy. "Tissue-specific classification of alternatively spliced human exons." Thesis, Massachusetts Institute of Technology, 2007. http://hdl.handle.net/1721.1/39920.

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Thesis (M. Eng.)--Massachusetts Institute of Technology, Biological Engineering Division, 2007.
Includes bibliographical references (p. 53-57).
Alternative splicing is involved in numerous cellular functions and is often disrupted and involved in disease. Previous research has identified methods to distinguish alternative conserved exons (ACEs) in human and mouse. However, the cellular machinery, the spliceosome, does not use comparative genomics to decide when to include and when to exclude an exon. Human RefSeq exons obtained from the University of California Santa Cruz (UCSC) genome browser were analyzed for tissue-specific skipping. Expressed sequence tags (ESTs) were aligned to exons and their tissue of origin and histology were identified. ACEs were also identified as a subset of the skipped exons. About 18% of the exons were identified as tissue-specifically skipped in one of sixteen different tissues at four stringency levels. The different datasets were analyzed for both general features such as exon and intron length, splice site strength, base composition, conservation, modularity, and susceptibility to nonsense-mediated mRNA decay caused by skipping. Cis-element motifs that might bind protein factors that affect splicing were identified using overrepresentation analysis and conserved occurrence rate between human and mouse.
(cont.) Tissue-specific skipped exons were then classified with both a decision-tree based classifier (Random ForestsTM) and a support vector machine. Classification results were better for tissue-specific skipped exons vs. constitutive exons than for tissue-specific skipped exons vs. exons skipped in other tissues.
by Craig Jeremy Rothman.
M.Eng.

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Fernández, Cecilia A. 1969. "Functional and structural uncoupling of the angiogenic and enzymatic inhibitory activity of TIMPs : loop 6 of TIMP-2 is a novel inhibitor of angiogenesis." Thesis, Massachusetts Institute of Technology, 2004. http://hdl.handle.net/1721.1/31110.

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Thesis (Ph. D. in Applied Biosciences)--Massachusetts Institute of Technology, Biological Engineering Division, 2004.
Includes bibliographical references (leaves 119-130).
Tissue inhibitors of metalloproteinases (TIMPs) regulate tumor growth, progression and angiogenesis in a variety of experimental cancer models and in human malignancies. However, numerous studies have revealed important differences between TIMP family members in their ability to inhibit angiogenic processes in vitro and angiogenesis in vivo despite their universal ability to inhibit matrix metalloproteinase (MMP) activity. To address these differences, structure-function studies were conducted to identify and characterize the anti-angiogenic domains of TIMP-2, the endogenous MMP inhibitor that uniquely inhibits capillary endothelial cell (EC) proliferation and angiogenesis in vivo. Only the carboxy-terminal domain of TIMP-2 (T2C) and not the MMP-inhibitory N-terminal domain (T2N), inhibited capillary EC proliferation. Although both T2N and T2C inhibited embryonic angiogenesis, only T2C potently inhibited mitogen-stimulated angiogenesis. These findings demonstrate that TIMP-2 possesses two distinct types of anti-angiogenic activities which can be uncoupled from each other. The anti-proliferative activity of T2C was further mapped to the 24-amino acid peptide, Loop 6, which proved to be a potent inhibitor of both embryonic and nitogen-stimulated angiogenesis in vivo. Initial studies into the mechanism(s) by which Loop 6 inhibits angiogenesis revealed that the anti-proliferative effects of Loop 6 are due, at least in part, to the inhibition of cell cycle progression and not to the induction of apoptosis. This inhibition was associated with increased levels of cell cycle inhibitor p27. Although Loop 6 did not compete with bFGF for binding to its receptor,
(cont.) five potential cell surface complexes were observed in crosslinking studies of capillary EC treated with ¹²⁵I-labeled T2C or Loop 6. Finally, given the high degree of homology between TIMP-2 and TIMP-4, we hypothesized that TIMP-4 might share anti-proliferative and MMP inhibition- independent anti-angiogenic activities with TIMP-2. Our results demonstrate that although TIMP-4 inhibits capillary EC migration, it does not inhibit capillary EC proliferation. Furthermore, TIMP-4 did not result in significant inhibition of embryonic angiogenesis in the CAM. These results suggest that TIMP-2 is unique among TIMP family members in its ability to inhibit angiogenesis via two distinct pathways. One of these activities, housed within Loop 6, results in the potent inhibition of angiogenesis in vivo.
by Cecilia A. Fernández.
Ph.D.in Applied Biosciences

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35

Thomson, Ty M. (Ty Matthew). "Models and analysis of yeast mating response : tools for model building, from documentation to time-dependent stimulation." Thesis, Massachusetts Institute of Technology, 2008. http://hdl.handle.net/1721.1/45206.

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Thesis (Ph. D.)--Massachusetts Institute of Technology, Biological Engineering Division, 2008.
Includes bibliographical references (p. 309-336).
Molecular signaling systems allow cells to sense and respond to environmental stimuli. Quantitative modeling can be a valuable tool for evaluating and extending our understanding of signaling systems. In particular, studies of the mating pheromone response system in yeast (Saccharomyces cerevisiae) have revealed many protein families and regulatory motifs also found in higher eukaryotes. This thesis develops several computational and experimental approaches that facilitate characterization of cellular signaling systems, and tests these approaches using yeast mating response as a model. Limitations in the current approach to building models of molecular systems were addressed first. For example, published computational models are often difficult to evaluate and extend because researchers rarely make available the information and assumptions generated throughout model building. I developed tools that facilitate model construction, evaluation, and extension. I used these tools to develop the YeastPheromoneModel (YPM) information repository, in which construction of an exhaustive model of the yeast mating system is documented (http://www.YeastPheromoneModel.org). Next, motivated by an ability to rapidly make many derivative models from the YPM repository and by carefully measured abundances of mating system proteins, I analyzed a model of the mating system mitogen activated protein kinase cascade. I found that varying the abundance of the scaffold protein Ste5, but not the abundances of other proteins, is expected to result in a quantitative tradeoff between total system output and dynamic range. Thus, the abundance of scaffold proteins in signaling systems may generally be under selective pressure to support specific quantitative system behavior.
(cont.) Finally, because traditional methods for characterizing signaling systems can be slow and tedious, I postulated that time-dependent stimulation of signaling systems might increase the richness and value of data derived from individual experiments. To do this, I devised a custom microfluidic device to expose yeast cells to pheromone in a time-dependent manner. I also developed computational approaches to investigate the use of time-dependent stimulation to characterize receptor and G protein response dynamics. I found that, at least for the receptor/G protein portion of the mating system, time-dependent stimulation does not appear to offer significant gains for constraining kinetic parameters relative to traditional step-response experiments.
by Ty M. Thomson.
Ph.D.

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36

Farkas, Dóra 1976. "Development of a collagen gel sandwich hepatocyte bioreactor for detecting hepatotoxicity of drugs and chemicals." Thesis, Massachusetts Institute of Technology, 2004. http://hdl.handle.net/1721.1/18040.

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Thesis (Ph. D.)--Massachusetts Institute of Technology, Biological Engineering Division, 2004.
Includes bibliographical references (leaves 124-140).
Understanding the hepatotoxicity of drugs and chemicals is essential for progress in academic research, medical science and in the development of new pharmaceuticals. Studying hepatotoxicity in vitro is a challenging task because hepatocytes, the metabolically active cells of the liver, are very difficult to maintain in culture. After just 24 hours, the cells detach from the plate and die, and even if they survive they usually do not express the metabolic functions which they have in vivo. It has been observed by others that culturing hepatocytes between two layers of collagen type I maintains in vivo-like morphology and also many drug metabolizing enzymes for weeks. In spite of the research examining drug metabolism in collagen sandwiches, there are very few studies evaluating this system for investigating hepatotoxicity. We cultured primary rat hepatocytes in the collagen sandwich configuration and our goal was to optimize this system for long-term studies and to examine toxicity of a variety of hepatotoxins. By measuring secretions of urea and albumin, and P4501A activity, we determined the optimal cell density to be 50,000 cells/cm2. We also evaluated the need for epidermal growth factor (EGF) in our cultures, by comparing urea and albumin secretions in cultures grown with and without EGF. The cultures without EGF had significantly less secretion of both urea and albumin just two days after plating. Therefore, we decided to include EGF in the medium. The toxins we examined were aflatoxin B1, acetaminophen, carbon tetrachloride, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), methyl methane sulfonate (MMS), cadmium, vinyl acetate and dimethylformamide (DMF). The cells were sensitive to aflatoxin B1, MMS, MNNG and cadmium. However, they were
(cont.) immune to acetaminophen, carbon tetrachloride, vinyl acetate and DMF. Our Western Blots showed that CYP1A, 2B and 3A were maintained in the culture for a week, but CYP2E1 was lost gradually over time. CYP2E1 is also the primary metabolic enzyme for acetaminophen, carbon tetrachloride and DMF. Thus, it is possible that the lack of toxicity is due to the loss of the enzyme responsible for the metabolism of these compounds. Immunity to vinyl acetate suggests that carboxylesterase is also lost in culture, since this enzyme is the one which converts vinyl acetate to acetaldehyde. The metabolism of acetaminophen was also examined with liquid chromatography and mass spectrometry. Liquid chromatography showed that acetaminophen is metabolized primarily to the sulfate and glucuronide metabolites. In order to investigate whether the glutathione adduct was formed, we synthesized the adduct and determined its retention time with liquid chromatography and its fragmentation pattern with mass spectrometry. We isolated the fraction with the same retention time from the medium of acetaminophen-treated cells, and showed that it contains a peak with the same mass to charge ratio and fragmentation pattern as the glutathione adduct. We also examined the conditioned medium from the hepatocytes to investigate the secreted protein profile, which could potentially be used to find toxicity biomarkers. We were able to remove most of the albumin from the medium using an immuno-affinity column containing anti-albumin antibodies bound to protein A-agarose beads ...
by Dóra Farkas.
Ph.D.

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37

Yin, David 1973. "The applications of comb polymer to the study of liver cell adhesion and signaling." Thesis, Massachusetts Institute of Technology, 2004. http://hdl.handle.net/1721.1/18044.

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Thesis (M. Eng.)--Massachusetts Institute of Technology, Biological Engineering Division, 2004.
Includes bibliographical references (p. 70-73).
Comb polymer, which consists of a hydrophobic poly(methyl methacrylate) (PMMA) backbone with hydrophilic hydroxy-poly(ethylene oxide) (HPOEM) side chains, is a tool that has many possible applications for the study of liver cell adhesion and signaling. This polymer has the unique properties of being cell resistant and chemically versatile such that various cell ligands can be coupled to its side chains. These properties allow adhesion through specific cell receptors to be studied without the effect of background adhesion to adsorbed proteins. By taking advantage of the ability to target specific receptors the comb polymer could be used as a powerful sorting tool. Sorting could be accomplished by finding cell type specific adhesion ligands. Several possible such ligands were screened. A ligand containing the tripeptide sequence RGD was found to elicit a strong cell adhesion response. However, this ligand is adherent to many cell types of the liver and would not be suitable for sorting purposes. Other cell type specific ligands tested showed little to no affinity for liver cell adhesion. Additionally, the comb was utilized to study α₅β₁ integrin-specific hepatocyte adhesion and the effect of Epidermal Growth Factor on adhesion. α₅β₁ integrin adhesion was mediated using a novel branched peptide, SynKRGD. This peptide consists of a linear peptide sequence containing RGDSP and the synergy site sequence PHSRN connected by the sequence GGKGGG. By utilizing the amine side group of Lysine a GGC branch was added. The terminal cysteine was used to conjugate SynKRGD to comb polymer surfaces using N-(p-Maleimidophenyl) isocyanate (PMPI) chemistry. EGF has a great potential to benefit the field of tissue engineering due to its influence on cell
(cont.) proliferation, migration, and differentiation. EGF is also known to have a de-adhesive effect in some cell types. Hepatocytes were studied on comb surfaces of variable SynKRGD densities with and without the presence of EGF in the media. Distinct morphological differences were observed for hepatocytes on substrates of varying adhesivity with and without the presence of EGF. EGF was found to have a de-adhesive effect on α₅β₁ integrin adhesion in hepatocytes. This effect became more pronounced as substrate adhesiveness increased.
by David Yin.
M.Eng.

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38

Kim, Ji-Eun 1974. "Regulation of tumor necrosis factor-alpha induced apoptosis via posttranslational modifications in a human colon adenocarcinoma cell line." Thesis, Massachusetts Institute of Technology, 2004. http://hdl.handle.net/1721.1/28865.

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Thesis (Ph. D.)--Massachusetts Institute of Technology, Biological Engineering Division, 2004.
Includes bibliographical references.
(cont.) phosphoproteomics technology, IMAC/LC/MS/MS, [approximately] 200 phosphosites were identified from HT-29 cells, some of which were detected only from insulin-treated cells. Our phosphoproteomics approach also enabled us to detect alteration of both known and unknown phosphorylation states of apoptosis-related proteins at two time points during early apoptosis induced by tumor necrosis factor-α
Apoptosis, a physiologically regulated cell death, plays critical roles in development and immune system by maintaining tissue homeostasis. The thesis project investigates regulations of apoptosis in a human colon adenocarcinoma cell line, HT-29, exposed to diverse cellular stimuli, focusing on a specific protein as well as global level of proteins. The first part of the thesis demonstrated S-nitrosation of procaspase-9. S-nitrosation is a novel protein modification to regulate protein-protein interaction or protein activity. This modification has been implied to inactivate caspases. We could visualize S-nitrosation of an initiator caspase, procaspase-9, by enriching low-abundant procaspase-9 with immunoprecipitation and stabilizing S-nitroso-cysteine with biotin labeling. Nitric oxide synthase inhibitors and tumor necrosis factor-α (TNF-α) reduced the S-nitrosation level of procaspase-9, suggesting that S-nitrosation may be regulated by a nitric oxide synthase and denitrosation is likely a mechanism of apoptosis. The second part of the thesis is to examine survival effects of insulin on cells undergoing TNF-α-induced apoptosis. Insulin decreased the TNF-α-induced cleavage of key apoptotic mediators, caspases, and their substrates as well as apoptosis, in part, depending on phosphatidylinositol-3 kinase (PI-3K)/Akt pathway. One of protective mechanisms by insulin is likely to decrease the TNF-α-induced dissociation of a potent inhibitor of caspases, X-chromosome linked inhibitor of apoptosis protein (XIAP), from procaspase-9 via PI-3K/Akt pathway. Lack of phosphoproteomics data in HT-29 cells led the third part of the thesis to focus on investigating global level regulation of phosphoproteins during apoptosis. With a
by Ji-Eun Kim.
Ph.D.

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39

Behr, Jonathan Robert. "Novel tools for sequence and epitope analysis of glycosaminoglycans." Thesis, Massachusetts Institute of Technology, 2007. http://hdl.handle.net/1721.1/42383.

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

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40

Hwa, Albert J. "Microvessel structure formation in a 3D perfused co-culture of rat hepatocytes and liver endothelial cells." Thesis, Massachusetts Institute of Technology, 2006. http://hdl.handle.net/1721.1/34154.

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

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41

Hendricks, Carrie A. (Carrie Anne) 1975. "Mitotic homologous recombination at engineered repeats in S. cerevisiae and in novel transgenic mice." Thesis, Massachusetts Institute of Technology, 2003. http://hdl.handle.net/1721.1/29615.

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

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42

Prabhakar, Vikas. "Therapeutics discovery via glycotechnology : the development of protein engineered enzymes for the study of galactosaminoglycan neuromedicine." Thesis, Massachusetts Institute of Technology, 2006. http://hdl.handle.net/1721.1/38917.

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Abstract:

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

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43

Sudo, Hiroko 1977. "Point mutations in normal lungs of smokers and non-smokers." Thesis, Massachusetts Institute of Technology, 2004. http://hdl.handle.net/1721.1/36321.

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Abstract:

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

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44

Kwan, Chi-Pong 1973. "Glycosaminoglycan-protein interactions : the fibroblast growth factor paradigm." Thesis, Massachusetts Institute of Technology, 2002. http://hdl.handle.net/1721.1/8088.

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Thesis (Ph. D.)--Massachusetts Institute of Technology, Biological Engineering Division, 2002.
Includes bibliographical references (leaves 193-218).
Specific interactions between heparan sulfate glycosaminoglycans (HSGAGs) and proteins are central to a wide range of biological processes such as anticoagulation, angiogenesis and growth factor activation. The specificity involved in the HSGAG-protein interactions stems from the structural heterogeneity of HSGAGs, which are highly acidic biopolymers associated on the cell surface and in the extracellular matrix. It is believed that structural specificity in the HSGAG-protein interactions determines the biological functions mediated by HSGAG-binding proteins such as basic fibroblast growth factor (FGF2). A number of models have been proposed to account for the mode of FGF-FGFR interactions and the role of HSGAGs in modulating FGF2 signaling. It was hypothesized that one role played by HSGAGs was to stabilize FGF2 oligomers in a "side-by-side" or cis fashion for presentation to fibroblast growth factor receptor (FGFR). In this thesis research, we systematically examined different proposed modes of FGF2 dimerization and showed that extensive oligomerization of a FGF2 mutant protein could be achieved by oxidatively crosslinking. Heparin, a highly sulfated form of HSGAGs, was demonstrated to increase the extent of oligomerization. Therefore, the results reported here were consistent with the hypothesis that HSGAGs promoted FGF2 oligomerization in a "side-by-side" mode. The functional significance of a FGF2 dimer was tested using a genetically engineered dimeric FGF2 (dFGF2). Biochemical and biophysical properties of dFGF2, such as protein folding, heparin affinity and receptor
(cont.) binding, were assayed. dFGF2 was found to exhibit higher activities in stimulating cell proliferation and cell survival in vitro compared with the monomeric wildtype. An in vivo rat cornea pocket model further corroborated the in vitro findings. The functional role of HSGAGs derived from the cell surface was studied here. It was found that distinct HSGAG fragments released by heparinase treatment were capable of modulating FGF2-stimulated cell proliferation depending on the expression of FGFR isoforms. This founding is consistent with the proposal that structural specificity of distinct HSGAG fragments dictated the interaction of HSGAGs with FGF and FGFR. The role of heparinase-generated HSGAG fragments in inhibiting cell proliferation was investigated. B16 melanoma cells treated with heparinase III were found to exhibit biochemical and morphological hallmarks of apoptosis. Conditioned medium derived from heparinase-treated cells was shown to be effective in suppressing cell growth. Gene array experiments and caspase activity assays further suggested that apoptotic cell death was mediated through a caspase 8-, death receptor-dependent pathway. Thus, the present study lends further credence to the proposal that cell surface HSGAGs plays a critical role in orchestrating cell phenotype. This thesis work provides the framework for understanding the molecular mechanism of growth factor activation and the structure-function relationship of HSGAG-mediated cell signaling. Results from this study may potentially be useful for therapeutic protein engineering and carbohydrate-based drug discovery.
by Chi-Pong Kwan.
Ph.D.

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45

Fan, Vivian H. (Vivian Hanbing). "Polymer-tethered epidermal growth factor as an inductive biomaterial surface for connective tissue progenitors." Thesis, Massachusetts Institute of Technology, 2006. http://hdl.handle.net/1721.1/37959.

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Thesis (Ph. D.)--Massachusetts Institute of Technology, Biological Engineering Division, September 2006.
"July 2006."
Includes bibliographical references (leaves 123-137).
Connective tissue progenitors (CTP) can act as a pluripotent source of reparative cells during injury and therefore have great potential in regenerative medicine and tissue engineering. However, the response of CTP to most growth factors and cytokines is unknown. Many envisioned applications of CTP, such as treating large defects in bone, involve in vivo implantation of CTP attached to a scaffold, a process that creates an acute inflammatory environment that may be hostile to CTP survival. This project entails the design of a two-component polymeric implant system to aid in the healing process of bony defects by influencing cell behaviors at the implant site through the covalent modification of the implant surface with selected ligands. We investigate cellular responses of CTP on a biomaterial surface covalently modified with epidermal growth factor (EGF) and find that surface-tethered EGF (tEGF) promotes both cell spreading and survival more strongly than saturating concentrations of soluble EGF. By sustaining MEK-ERK signaling, tEGF increases the contact of CTP with an otherwise moderately adhesive synthetic polymer and confers resistance to apoptosis induced by the proinflammatory cytokine, FasL.
(cont.) We confirm that these signaling, spreading, and apoptotic responses are conserved across three sources of CTP: an hTERT-immortalized human mesenchymal stem cell (MSC) line, primary porcine bone-marrow CTP, and primary human bone-marrow-derived CTP. We conclude that tEGF may offer a protective advantage to CTP in vivo during acute inflammatory reactions to tissue engineering scaffolds. The tEGF-modified polymers described here could be used together with structural materials to construct CTP scaffolds for the treatment of hard-tissue lesions, such as large bony defects.
by Vivian H. Fan.
Ph.D.

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46

Gervais, Thomas. "Mass transfer and structural analysis of microfluidic sensors." Thesis, Massachusetts Institute of Technology, 2005. http://hdl.handle.net/1721.1/34489.

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Thesis (Ph. D.)--Massachusetts Institute of Technology, Biological Engineering Division, February 2006.
Includes bibliographical references (leaves 181-191).
Surface-based sensors take advantage of the natural high surface-to-volume ratios in microfluidic devices, low reagent consumption and high potential for integration in more complex micro total analysis systems (microTAS or pTAS). This thesis studies the fundamental limits of on-chip integrated microfluidic sensors. More specifically, it focuses on detection methods involving surface interaction in channels with thicknesses on the order of a few microns or less. Through mass transfer analysis, we demonstrate that, for thin enough channels, sample detection is limited by the convective transport of analytes, and neither by diffusion nor reaction. The results provided extend the validity of transport models to include transport in the absence of mass transfer boundary layer. All existing analytic solutions to the Graetz problem are described and compiled. The analysis, complemented by finite element simulations, successfully predicts experimental observations made for on-chip immunoassays in micron-thick fluidic channels. Subsequently, our study of on chip detection systems is carried on with emphasis on resonating cantilever sensors. In order to interpret the output signal from these devices, we develop a dynamic cantilever model to link spatially and temporally dependent mass adsorption with resonance frequency change.
(cont.) The mass adsorption is then directly related to the sensors' operating conditions via the mass transfer models previously developed. We then develop a 2D finite-element model capable of predicting the devices response and of extracting bimolecular rate constants. Finally, since hydraulic resistance severely increases as channels get shallower, we provide a structural analysis of polymer-based microsystems. Through scaling and numerical simulations we demonstrate the effect of channel deformation on the flow conditions inside the device and vice versa. Finally, channel deformation is experimentally quantified using optical methods and compared with the models developed. Throughout this thesis, the approach to physical modeling has been to use mathematical and numerical analysis as predictive tools in the design of integrated lab-on-a-chip systems. Whenever possible, scaling and analytic solutions are developed, since they provide a direct relationship between experimental observations, geometry and the multiple dependent variables in the system, and can be readily used as design criteria by the experimentalist.
by Thomas Gervais.
Ph.D.

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47

Townsend, Seth A. (Seth Alan). "Development of novel diagnostics and therapeutics for amyotrophic lateral sclerosis." Thesis, Massachusetts Institute of Technology, 2008. http://hdl.handle.net/1721.1/45948.

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Thesis (Ph. D.)--Massachusetts Institute of Technology, Biological Engineering Division, 2008.
Includes bibliographical references (p. 224-236).
Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease with diagnostics and treatments that are ineffective at stopping the progression. This thesis examines new ways of both diagnosing and treating ALS, including 1) a gadolinium tetanus toxin C fragment (Gd-TTC) biomarker for axonal retrograde transport, 2) TTC-conjugated biodegradable nanoparticles, and 3) poly(glycerol-co-sebacate) acrylate (PGSA) and 3D scaffolds for human embryonic stem cell (hESC) and neuronal encapsulation.A Gd-TTC conjugate was developed and characterized that was shown to be highly visible under MRI and preserved the functionality of the native TTC protein in vitro. Live animal MRI imaging and immuno fluorescent staining of the spinal cord showed that the conjugate was transported to the central nervous system (CNS) and localized in motor neurons. H&E staining and biodistribution studies showed that GdTTC was well tolerated and bio available. Quantification of MRI and staining images showed that Gd-TTC was retrograde transported and that that this rate decreased during the disease progression of ALS in a transgenic mouse model, suggesting that Gd-TTC could be used as a biomarker for neurodegenerative diseases.TTC-conjugated nanoparticles were developed by synthesizing PLGA-PEG-biotin and using biotin binding proteins (avidin, streptavidin, and neutravidin) to specifically conjugate TTC to the nanoparticle surface. TTC nanoparticles were shown to selectively target neurons and not other cell types in vitro.
(cont.) Subsequent in vivo experiments showed that nanoparticles were well tolerated and that TTC was co-localized with neurons unilaterally, suggesting that TTC-conjugated nanoparticles may be a useful drug delivery system. Porous PGSA scaffolds were prepared and characterized by porosity, swelling, mass loss, toxicity and mechanical properties, and subsequently used to encapsulated hESC and neuroblastoma cells in vitro. Neuroblastoma cells proliferated and formed matrix fibrils, and fluorescent staining of undifferentiated hESCs showed the presence of all three germ layers. In vivo experiments showed that porous PGSA scaffolds were well-tolerated and promoted vascular ingrowths.
by Seth A. Townsend.
Ph.D.

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48

Chan, Joyce M. Eng Massachusetts Institute of Technology. "A central composite design to investigate antibody fragment production by Pichia pastoris." Thesis, Massachusetts Institute of Technology, 2005. http://hdl.handle.net/1721.1/33872.

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Thesis (M. Eng.)--Massachusetts Institute of Technology, Biological Engineering Division, 2005.
Includes bibliographical references (p. 77-80).
This study aims to investigate the relationships between growth parameters (agitation, glycerol concentration, salt concentration) and responses (biomass, growth rate, protein expression), by a 3-factor-3-level central composite factorial design. This experimental design involved running shake flask culture at 15 different experimental conditions with duplicates. Optical density (OD600), dry cell weight (DCW), and BCA Protein Assays were done on each experiment. Mathematical models in terms of these parameters' effects and their interactions were proposed for each of the responses. The significance of each effect and interaction, as well as the goodness-of-fit of mathematical models to data were examined by analysis of variance. It was found that biomass (with R²Adj=0.951) is a strong function of glycerol concentration (higher glycerol concentration leads to higher biomass), but it varies much less with agitation, and it is completely independent of salt concentration. Growth rate (R²Adj=0.901), however, varies strongly with agitation and salt concentration, but much more weakly with glycerol concentration. Protein production has a low R²Adj value of 0.746, implying that higher-order terms, e.g. x₁² and x₂², should be tested for significance in the model.
(cont.) Collected data were fitted to the proposed models by response surface regression, after which surface and contour plots of responses were generated to identify trends in them. High agitation (300 rpm in shaker) gave rise to both highest biomass and growth rate. In addition, biomass at high glycerol concentration (3% v/v) was almost twice as much as biomass at low glycerol concentration (1% v/v) at high agitation rate (19 g/L compared to 11 g/L). At the same agitation rate, growth rate shows the largest increase of 20.5% with increasing salt concentration from 0.7% to 2.1%. Protein production reached maximum of 7.3 mg/mL at medium agitation rate (250 rpm), high salt and glycerol concentrations.
by Joyce Chan.
M.Eng.

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49

Huang, Hua Ming Paul. "Quantitative analysis of the EGFRvIII mutant receptor signaling networks in Glioblastoma." Thesis, Massachusetts Institute of Technology, 2008. http://hdl.handle.net/1721.1/45382.

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Thesis (Ph. D.)--Massachusetts Institute of Technology, Biological Engineering Division, 2008.
"September 2008."
Includes bibliographical references and index.
Glioblastoma multiforme (GBM) is the most aggressive adult brain tumor and remains incurable despite multimodal intensive treatment regimens. EGFRvIII is a truncated extracellular mutant of the EGF receptor (EGFR) that is commonly found in GBMs and confers tumorigenic behavior. Although much work has been done over the past decade to elucidate pathways involved in EGFRvIII receptor signaling, the global map of the signaling networks that it activates remains incomplete, making it difficult to assess downstream components involved in EGFRvill-mediated transformation. To gain a molecular understanding of the mechanisms by which EGFRvIII acts, we have employed a mass spectrometry-based phosphoproteomic approach to quantitatively map cellular signaling events activated by this receptor. Using this approach, we have determined the major downstream pathways activated as a function of titrated EGFRvIII receptor levels. This analysis highlighted several aspects of EGFRvIII tumor biology, including crosstalk between EGFRvIII and other receptor tyrosine kinases. Specifically, we have identified the c-Met receptor as a co-target in the treatment of EGFRvIII positive GBM cells, and have shown that an EGFR and c-Met combination inhibitor strategy may be applicable in overcoming the poor efficacy of EGFR kinase inhibitor monotherapy in GBM patients. We then went on to investigate the mechanisms by which signaling networks are regulated in response to site-specific tyrosine mutations on EGFRvIII. This analysis has revealed a receptor compensation mechanism that is capable of restoring network architecture, upon the loss of a major tyrosine phosphorylation site on EGFRvIII.
(cont.) This is, to our knowledge, the first demonstration of signal compensation at the level of receptor phosphorylation and highlights an unexpected level of complexity within the signaling network. Our data also indicates that EGFRvIII fine-tunes the activity of the Erk pathway; some Erk activity is required for growth but excessive pathway activation results in cell death. We believe that the sensitivity to modulation of the Erk pathway may be exploited as a potential means of therapy for EGFRvIII positive tumors. Taken together, our study highlights the utility of quantitative phosphoproteomic analysis as a tool to gain molecular insights in cancer biology and a means for drug target discovery.
by Hua Ming Paul Huang.
Ph.D.

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50

Rakestraw, James A. "A directed evolution approach to engineering recombinant protein production in S. cerevisiae." Thesis, Massachusetts Institute of Technology, 2006. http://hdl.handle.net/1721.1/38242.

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Thesis (Ph. D.)--Massachusetts Institute of Technology, Biological Engineering Division, 2006.
Vita.
Includes bibliographical references.
The continued success of protein therapeutics has put a strain on industry's ability to meet the large demand. Creating a more productive expression host for the manufacture of these proteins is a potential solution. Although heterologous proteins are frequently made in organisms as disparate as E. coli and bovines, the single-celled organism S. cerevisiae has emerged as a well-qualified candidate due to its approachable genetic and fermentation attributes as well as its ability to stably fold disulfide bonded and multi domain proteins. Because S. cerevisiae screens for enhanced protein secretion have traditionally utilized low-throughput and often plate-based methods, a high-throughput, liquid phase assay could offer a real advantage in secretory selection. In this thesis, yeast surface display is investigated as a potential proxy for heterologous protein secretion. Although ultimately unsuitable as a screening proxy, the surface display experiments did show a novel method of improving protein secretion by co-expressing a more stably folded protein with the protein of interest. In these studies the secretion of an scFv-Aga2p fusion was stimulated 1 0-fold by the concomitant surface expression of BPTI.
(cont.) BPTI surface expression also stimulated the secretion of secreted scFv three-fold suggesting a niche for protein coexpression as well as secretion by way of Aga2p fusions. A new screening method was developed that involves the capture of secreted protein on the surface of the cell where it can be labeled and sorted by FACS. This new method was verified to achieve thirty-five fold enrichment per pass for a three-fold enhanced protein secretor making it easily suitable for screening. The new screening methodology, the Cell Surface Secretion Assay (CeSSA), was also modeled and verified with time course data that enabled optimization of sort parameters and predicted sort outcomes based on user-derived selection parameters. The CeSSA was used to screen a library of mutant yeast alpha mating factor leader sequences for improved secretion of the scFv 4m5.3. The improved leaders imparted up to a twenty-fold improvement in scFv secretion per cell and up to thirty-fold improvement after expression tuning. These engineered leader sequences also conferred improved secretion on other scFv's and proteins including whole IgG. Moreover, the leader sequence mutants give indications of where the important residues in secretory leaders lie and the aberrations in protein traffic that result in reduced secretion.
by James A. Rakestraw.
Ph.D.

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Dissertations / Theses on the topic 'Division of Biological Effects'

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