Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biological Engineering, 2009. / Cataloged from PDF version of thesis. / Includes bibliographical references (p. 106-116). / Heparin-binding epidermal growth factor-like growth factor (HB-EGF) exhibits activity as a juxtacrine, paracrine, and autocrine ligand for the epidermal growth factor receptor (EGFR), and possesses the ability to bind heparan sulfate proteoglycans (HSPGs). The interaction of HB-EGF with HSPGs has been previously studied only with the soluble (autocrine/paracrine) form of the protein (sHB-EGF), produced after proteolytic cleavage of the transmembrane form (proHB-EGF) from the cell surface. It was hypothesized that HSPGs interact with proHB-EGF in ways that could alter behavior of the transmembrane form of this ligand and consequent processes. Using an engineered form of proHB-EGF that allowed for independent tracking of the extracellular domain and the C-terminal tail, proHB-EGF was observed primarily at sites of cell-cell contact. However, a dramatic change in this localization was observed upon the addition of exogenous heparin, heparan sulfate, heparinase III or mutation of the heparin-binding domain of proHB-EGF, suggesting that an interaction with HSPGs is responsible for localizing proHB-EGF to sites of cell-cell contact. Further studies in wild-type CHO-Ki cells and heparan sulfate deficient CHOpgsD-677 cells demonstrated that a trans interaction between proHB-EGF and HSPGs on neighboring cells was responsible for this localization. Additionally, this interaction inhibited proteolytic processing of the ligand, as heparin and mutation of the heparin-binding domain increased the amount of sHB-EGF accumulated in the media. / by Robin N. Prince. / Ph.D.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, September, 2020 / Cataloged from the official PDF version of thesis. / Includes bibliographical references. / Viruses are the most abundant and diverse life form on Earth. With over 10³¹ viral particles in existence, viruses fundamentally shape global biogeochemistry and ecology. Most viruses infect bacteria, archaea, and other microbes, and the threat of infection continually challenges microbes' survival. As a consequence of this expansive war, bacteria and archaea have acquired a potent arsenal of molecular defense systems in order to survive. Known defense systems, such as CRISPR, have given rise to transformative technologies including genome editing. However, defense systems as a whole remain underexplored. Continued investigation of these systems and the warfare between microbes and viruses may lead not only to a better understanding of basic microbiology and evolution, but also to new technologies and therapeutic applications. In this thesis, we investigate the collective arsenal of molecular defense systems that bacteria and archaea use to fight viral infections. First, we focus on known defense systems and use protein engineering to increase the specificity and targeting range of CRISPR enzymes for human genome editing. Second, by computational mining and experimental reconstitution, we discover 29 novel defense gene cassettes that are collectively present in one third of all sequenced bacterial and archaeal genomes. These systems incorporate enzymatic activities not previously implicated in antiviral defense, including RNA editing and retron satellite DNA synthesis. In addition, we predict a diverse set of other putative defense genes that remain to be characterized. These results highlight an immense array of molecular functions that bacteria and archaea employ against viruses. / by Linyi Gao. / Ph. D. / Ph.D. Massachusetts Institute of Technology, Department of Biological Engineering

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, May, 2020 / Cataloged from student-submitted PDF version of thesis. "March 2020." Date of graduation, May 2020. / Includes bibliographical references (pages 140-146). / Expansion microscopy (ExM) enables 3-D, nanoscale-precise imaging of biological specimens by isotropic swelling of hydrogel-embedded, chemically processed tissue. Such capability raises the question of whether nanoscale mapping of biomolecules could be performed in an entire organism, which would allow super-resolution-mediated in situ analyses, such as digital quantification of biomolecules and mapping of synaptic contacts, to be performed within the context of an entire nervous system. The nematode Caenorhabditis elegans could be a suitable model for such organism-wide analyses, due to its tractable physical size, deterministic cell lineage, ease of genetic control, and well-established literature. However, C. elegans is enclosed in a chemically impermeable and mechanically tough cuticle, which could hinder the deployment of ExM. In this thesis, we present a strategy, expansion of C. elegans (ExCel), to expand fixed, cuticle-enclosed intact animals of C. elegans. ExCel enables simultaneous readout of fluorescent proteins, RNAs, DNA locations, and anatomical structures at resolutions of ~65-75 nm (3.3-3.8x linear expansion). We also developed epitope-preserving ExCel, which enables imaging of endogenous proteins stained by antibodies, and iterative ExCel, which enables imaging of fluorescent proteins at a ~25-nm resolution (20x linear expansion). We demonstrate the utility of the ExCel toolbox for multiplexed imaging of multiple molecular types, for mapping synaptic proteins, for identifying previously unreported proteins at cell junctions, and for gene expression analysis in multiple individual neurons of the same animal. In addition to ExCel, we discuss two other ExM-related technologies, including tetragel, which is a highly homogeneous hydrogel network that improves the nanoscale isotropy of biological ultrastructure expanded by ExM, and stochastic arrangement of reporters in clusters (STARC), which is a strategy for recording neuronal activity at a subneurite-level resolution, in densely labeled neuronal populations. Taken together, the work presented in this thesis extends the capabilities of ExM, and lays the foundation for a comprehensive, functionally and structurally informed analysis of an entire organism, which could reveal new insights in neuroscience, organismal development, and systems biology. / by Chih-Chieh (Jay) Yu. / Ph. D. / Ph. D. Massachusetts Institute of Technology, Department of Biological Engineering

Astrocytes are among the most functionally diverse population of cells in the central nervous system (CNS) as they are essential to many important neurological functions including maintaining brain homeostasis, regulating the blood brain barrier, and preventing build-up of toxic substances within the brain, for example. Astrocyte importance to brain physiology and pathology has inspired a host of studies focused on understanding astrocyte behavior primarily from a biological and chemical perspective. However, a clear understanding of astrocyte dysfunction and their link to disease has been hampered by a lack of knowledge of astrocyte behavior from a biomechanical perspective. Furthermore, astrocytes (and all cells) can sense and respond to their external biomechanical environment via the extracellular matrix and various other biomechanical cues. One such biomechanical cue, substrate stiffness changes within the brain under certain pathologies, which subsequently leads to changes in the biomechanical behavior of the cell. For example, increased tissue stiffness is a hallmark of brain tumors that subsequently alters astrocyte biomechanical behavior. Therefore, to gain a better understanding of this process we cultured astrocytes on stiffnesses that mimicked that of the normal brain, meningioma, and glioma and investigated astrocyte biomechanical behavior by measuring cell-substrate tractions and cell-cell intercellular stresses utilizing traction force microscopy and monolayer stress microscopy, respectively. Our findings showed an increase in traction forces, average normal intercellular stress, maximum shear intercellular stress, and strain energy proportional to increased substrate stiffness. A substrate stiffness of 4 kPa showed 2.1 fold increase in rms tractions, 1.8 fold increase in maximum shear stress, 2.6 fold increase in average normal stress, and 1.6 fold increase in strain energy. While 11 kPa showed a 4.6 fold increase in rms tractions, 6.6 fold increase in maximum shear stress, 5.2 fold increase in average normal stress, and 2.3 fold increase in strain energy. Cell velocity, on the other hand, showed a decreasing trend with increasing stiffness. This study demonstrates for the first time that astrocytes can bear intercellular stresses and that astrocyte intercellular stresses and traction can be modified using substrate stiffness. We believe this study will be of great importance to brain pathology, specifically as it relates to treatment methods for brain tumors.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, June 2017 / "May 2017." Cataloged from PDF version of thesis. / Includes bibliographical references (pages 103-111). / Biological hydrogels exhibit complex properties that cannot be recapitulated by current synthetic materials. Examples include mucus, which acts as a barrier against toxins and pathogens while simultaneously hosting trillions of microbes within the gut; cartilage which resists repetitive compressive forces while maintaining highly lubricated layers for efficient movement; and nuclear pore matrices which act as selective barriers in the transport of proteins and nucleic acids. An underlying theme that gives biological hydrogels their unique mechanical and biological functions is the presence of long polymeric molecules. These polymers are typically comprised of repeating subunits that are essential for correct polymer function, such as the phenylalanine-glycine (FG) repeats in nucleoporin proteins of nuclear pore complexes (NPCs) and the proline-threonine-serine (PTS) domains in mucin polymers found in mucus. / Although these polymeric subunits are well-identified, to date their structural complexity has limited our understanding of how they contribute to the overall hydrogel function. In this thesis, we focus on two main biological hydrogels: the self-assembled matrix of the nuclear pore complex that controls the passage of molecules between the nucleus and the cytoplasm, and mucus, which protects against invading pathogens and toxins. As both hydrogels consist of functionally redundant polymers and associated factors, understanding the relationship between polymer sequence and hydrogel function is a significant technical challenge. To simplify the problem, we design structurally reduced peptides and polymers with targeted individual biological features such as amino acid identity, spatial localization of charge, and glycosylation identity. We then study the effect of one or a combination of these properties on the overall hydrogel function. / Using this technique, we first demonstrate that peptide charge type and amino acid placement are important features for regulating selective transport through NPCs. For mucins, we identify single glycans that are sufficient to recapitulate the biofilm inhibition properties of mucin, and present novel evidence that mucins modulate horizontal gene transfer rates for opportunistic and commensal bacteria. / by Wesley George Chen. / Ph. D. / Ph.D. Massachusetts Institute of Technology, Department of Biological Engineering

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, May, 2020 / Cataloged from PDF version of thesis. / Includes bibliographical references (pages 203-216). / Reductionist biology of the 20th century rooted pure culture methods and antibiotics as pillars of humankind's interaction with microbiology, igniting a revolution in medicine and biotechnology. The revolution was not without cost. By overlooking complex biological interactions, it introduced new problems--from the sharp rise in immune disorders to the antibiotic resistance crisis--that 21st century tools must address. While 'omics methods have fundamentally expanded our understanding of biological complexity, we lack a generalized method for measuring how the parts of a complex system, such as the individual strains of a microbial community, interact with each other. In this thesis, I present kChip, a new platform for constructing massively parallel combinatorial arrays of these parts in order to measure their interactions directly. I describe how kChip has been used to reveal patterns in microbial community assembly, unearth minimal microbial combinations with desirable functions, and screen for compounds that potentiate antibiotic activity. I demonstrate how kChip can advance the development of new technologies like microbial consortia and combinatorial drug therapies. / by Jared Scott Kehe. / Ph. D. / Ph.D. Massachusetts Institute of Technology, Department of Biological Engineering

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

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

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

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