Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2015. / Cataloged from PDF version of thesis. / Includes bibliographical references. / Human exposure to dangerous genotoxins is unavoidable, as DNA damaging agents are ubiquitous both in our environment and within our cells. The diversity in lesions induced by these agents led to the evolution of several DNA repair pathways that suppress the mutagenic and toxic effects of DNA damage. Ironically, many cancer chemotherapeutics are themselves genotoxic due to their ability to target tumor cells that are more vulnerable to DNA damage. Therefore, knowledge about DNA damage and repair is relevant to cancer prevention, susceptibility and treatment. Despite its fundamental importance, measurement of DNA damage and repair in people is far from routine, primarily due to technological obstacles. Recently, we have developed the CometChip, a novel platform for measuring DNA damage that has its basis in the well-accepted comet assay. To advance the CometChip for broader utility in screens and population studies, we further improved the platform to provide greater sensitivity, reproducibility and throughput. We then characterized the conditions of the CometChip to analyze different DNA repair pathways. We showed that the CometChip is effective for evaluating repair kinetics and for detecting deficiencies of base excision repair, nucleotide excision repair, mismatch repair, and non-homologous end-joining. The ability to assess multiple repair pathways opens door to many applications. In terms of basic research, we applied the CometChip technology to perform cross-pathway analysis of two repair proteins, Xrccl and DNA-PKcs. Results confirm known activities as well as reveal new cross-pathway functions of these proteins. In terms of clinical relevance, we used the CometChip to evaluate small molecule inhibitors of DNA repair proteins, which are potential chemotherapeutics. In terms of public health, we exploited the CometChip to learn about differences in DNA repair among individuals. We discovered that there are subpopulations of people who share similar BER characteristics and there are shared factors between BER and NER among individuals. Taken together, the CometChip platform represents a significant technological advance. From integrated analysis of repair proteins, to evaluation of clinically relevant chemicals, to studies of inter-individual variations, the CometChip has demonstrated value in uncovering new information about DNA repair in humans. / by Jing Ge. / Ph. D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biological Engineering, 2011. / Cataloged from PDF version of thesis. / Includes bibliographical references (p. 125-144). / The volume of protein structure data has grown rapidly over the past 30 years, leaving a wake of facts that still require explanation. We endeavored to answer a few open questions on the structure-function relationship of intriguing mechanochemical protein systems. To this end this thesis work contains five studies that offer novel insights into molecular biomechanical systems that may guide future basic research or applications development. The first study concerns the biophysics of cadherin-mediated cell sorting observed in developing solid tissue. We investigated the evolutionary dynamics of the cadherin superfamily of cell-cell adhesion proteins to infer a structural basis for their paradoxical mixture of pairwise binding specificity and promiscuity. Our analysis predicts a small set of specificity-determining residues located within the protomer-protomer binding interface. The putative specificity-determinants form a design space with potential for engineering novel cell-cell adhesive interactions. The second study addresses the open question of how to automatically identify regions within a protein that engage in allosteric communication. To identify allostery we developed and tested two computational tools that operate on protein conformational dynamics data. These tools are useful for generating testable hypotheses about proteins with multiple functional sites for the design of non-competitive protein inhibitors. The third study asks, "What is the consequence of allosteric cooperation between the tandem binding sites in a class of proteins that bundle filamentous actin (F-actin)?" Through simulation we demonstrate that cooperative F-actin bundling tends to strengthen bundles by driving the formation of cross-links between neighboring filaments while depleting F-actin binding sites that are occupied but not cross-linked. We hence propose that allostery may be a natural feature of ABPs with tandem F-actin binding sites if nature indeed selects for sturdy F-actin bundles. The final two studies examine the impact of two structural perturbations to Factin on its mechanics. Using structure-based computer modeling we develop a simple explanation for the mechanism by which the structure of actin's polymorphic subdomain 2 mediates 4-fold changes in F-actin's flexibility. We further demonstrate that two calponin homology domains stabilize F-actin by binding in a configuration that tends to relax the stress concentration at actin-actin interfaces. / by Philip W. Bransford. / Ph.D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biological Engineering, February 2013 / Cataloged from PDF version of thesis. / Includes bibliographical references. / There is increasing recognition that aberrant protein modifications play an important role in the pathophysiology of inflammation and oxidative stress in cells. We recently discovered that N⁶-formylation of lysine is an abundant endogenous modification of histone and chromatin proteins. The high abundance of N⁶-formyllysine in histone proteins and its chemical similarity to the biologically important N⁶- acetyllysine has raised questions about its mechanisms of formation and biological consequences. Using novel ultrasensitive and specific liquid chromatography-coupled tandem mass spectrometry methods (LC-MS/MS) to quantify N⁶-formyllysine lesions in proteins, we aimed to investigate the sources as well as the fate of this abundant endogenous protein modification. We present evidence that endogenous formaldehyde is a major source of N⁶-formyllysine and that this adduct is widespread among proteins in all cellular compartments. We observed in vitro as well as in vivo that formaldehyde exposure leads to a dose-dependent increase in N⁶-formyllysine protein adducts, with the use of isotopically-labeled formaldehyde to dissect endogenous from exogenous formaldehyde as sources of the adduct. Further, other isotope labeling studies revealed that lysine demethylation in histone proteins is not a source of N⁶-formyllysine. With regard to N⁶-formyllysine persistence in cells, our investigation of histone deacetylases revealed that despite chemical similarity of N⁶-formyllysine to N⁶-acetyllysine, the former is refractory to removal by histone deacetylases, which suggests that they will persist throughout the life of individual histone proteins. If not repaired, lysine formylation could accumulate to significant levels. The resemblance of N⁶-formyllysine to N⁶-acetyllysine, together with recent studies that mapped its location on many conserved lysine acetylation and methylation sites along histone proteins, support the idea that this abundant protein modification could interfere with normal regulation of gene expression, potentially leading to an epigenetic mechanism of disruption of cell function. / by Bahar Edrissi. / Ph.D.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2018. / Page 165 blank. Cataloged from PDF version of thesis. / Includes bibliographical references (pages 151-164). / Experiments in biology are often combinatorial in nature and require analysis of large multi-dimensional spaces, but the scales of these experiments are limited by logistical complexity, cost, and reagent consumption. By miniaturizing experiments across nanoliter-scale emulsions that can be processed at large scales, droplet microfluidic platforms are poised to attack these challenges. Here we describe a droplet microfluidic platform for combinatorial experiments that automates the assembly of reagent combinations, with order-of-magnitude improvements over conventional liquid handling. Moreover, our design is accessible, requiring only standard lab equipment such as micropipettes, and improves the chemical compatibility of droplet microfluidic platforms for small molecules. We applied our platform to two experimental problems: combinatorial drug screening and microbial ecology. First, we used our platform to enable screening of pairwise combinations of a panel of antibiotics and 4,000+ investigational and approved drugs to overcome intrinsic antibiotic resistance in the model Gram-negative bacterial pathogen E. coli. This screen processed 4+ million droplet-level assays by hand in just 10 days to discover more than 10 combinations of antibiotics and non-antibiotic drugs for further study. We then applied our platform to microbial ecology, where the interactions between microbes in communities can dictate functions important for both basic science and biotechnology. As a proof of concept, we used our platform to survey 960 pairwise interactions of microbes isolated from soil, and deconstruct higher-order interactions in a 4-strain community. Altogether, we expect that our platform can be used to efficiently attack combinatorial problems across molecular and cellular biology. / by Anthony Benjamin Kulesa. / Ph. D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biological Engineering, 2013. / Cataloged from PDF version of thesis. / Includes bibliographical references (p. 73-83). / Environmental bacteria display tremendous genetic diversity, but we are still learning how this diversity arises and relates to their wide range of habitats. Investigating how bacteria adapt helps us understand their contributions to environmental processes and informs forward engineering of bacteria for industrial applications. Experimental evolution is a powerful approach, with microbes especially, but it has mostly been applied to model organisms and metabolic functions. In the work here, we investigated the possibility, degree, and variability of adaptation of an environmental Vibrio strain by applying a little-used selection method appropriate to a relevant condition, salinity. We successfully isolated mutants with higher salt tolerance by selecting on salt gradient plates. Resequencing the genomes of the evolved strains revealed unprecedented hypermutation in three of nine parallel lineages. These mutator lines arose independently, and each of them accumulated more than 1500 single-base mutations. By comparison, there are only 302 single-base differences between the ancestor strain and another strain isolated in the wild. Hypermutation was associated with a deletion resulting from improper prophage excision. Members of this family of prophages are found in other proteobacteria, including well-studied human pathogens, from very different environments. Mutators are known to arise spontaneously in wild and clinical bacteria, but the extent of their adaptive contribution is unknown. We have preliminary evidence that this mechanism of evolution could be relevant in the environment, where horizontal gene transfer and mobile elements play known, significant roles in bacterial evolution. / by Sean Aidan Clarke. / Ph.D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biological Engineering, 2009. / Cataloged from PDF version of thesis. / Includes bibliographical references. / One of the primary goals of cancer chemotherapy is the design of antitumor agents that achieve selective targeting of tumor cells while minimizing toxicity to normal tissues. We have synthesized a series of DNA damaging agents that are designed to disrupt selectively the DNA repair and signaling pathways in tumor cells. My dissertation focuses on the mechanistic studies of the rationally designed genotoxicants E2-7aX and 2Pl, which target cancer cells that express the estrogen receptor (ER). The studies on the biological activity of E2-7ca in ovarian cancer cells reveal that the compound induces levels of cytotoxicity comparable to those of cisplatin, which is the current front-line therapeutic for ovarian cancer. E2-7ca induces crosslink formation in the ovarian cell line SKOV-3, which potentially leads to the S-phase arrest observed in these cells. Further, the arrest is persistent even after drug removal, indicating that the persistence might be one of the reasons for the enhanced toxicity of E2-7ca compared to the control compound chlorambucil. E2-7ca induces cell death in SKOV-3 cells through autophagy, as opposed to the classical cell death pathway of apoptosis. Mechanistic studies on the role of ER in E2-7a toxicity in SKOV-3 cells show that the absence of the ER (facilitated by the knockdown of ER protein through RNA interference in SKOV-3 cells) makes the cells less sensitive to the drug. In addition, the ER(-) population displays a lower level of drug- DNA adducts than its ER(+) counterpart, as detected by Accelerator Mass Spectrometry (AMS). The combination of these pieces of evidence strongly suggests that as per its intended mechanism of action, E2-7a. mediates its effects through the involvement of the ER, and appears to be a promising candidate as an antitumor agent in the clinical setting. In addition, the toxicity and DNA damage caused by the 2-phenylindole compounds 2PI and 2PI(OH) were studied in ovarian cancer cell lines; these studies underscore the differences in the mechanism of action of the two compounds. Both the compounds demonstrate promise as potent anti-tumor agents, and the selectivity of these compounds in different cellular contexts (ER (+)/(-) or p53 (+)/(-)), once established, could help strengthen the basis of their therapeutic efficacy. / by Sreeja Gopal. / Ph.D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biological Engineering, 2010. / Cataloged from PDF version of thesis. / Includes bibliographical references (p. 105-112). / Improved methods are needed for routine, inexpensive monitoring of biomarkers that could facilitate earlier detection and characterization of complex diseases like cancer. Development of new assay formats based on microfluidic, label-free platforms enable radical reductions in assay complexity and reagent requirements with the potential for such applications. Suspended microchannel resonators (SMRs) are highly sensitive, batch-fabricated microcantilevers with embedded microchannels that can measure mass with femtogram precision. Biomolecules such as proteins and nucleic acids are denser than water, and their presence can thus be quantified by their buoyant mass, or increase in mass relative to the solution they displace. This thesis presents two approaches to conducting label-free, buoyant-mass immunoassays with SMRs with potential for clinical applications. The sensor surface can be functionalized to bind targets directly, or individually weighed polystyrene beads can be used as mobile supports. As in other label-free detection methods, biomolecular measurements in complex media such as serum are challenging due to high background signals from non-specific binding. We demonstrate that carboxybetaine-derived polymers developed to adsorb directly onto SMR SiO2 surfaces act as ultra-low fouling and functionalizable surface coatings. Coupled with a reference microcantilever, this approach enables detection of activated leukocyte cell adhesion molecule (ALCAM), a model cancer biomarker, in undiluted serum with a limit of detection of 10 ng/mL. Decoupling the complexity of surface modifications from the sensor precludes the need for specialized reagents. Monodisperse, micron-scale polystyrene beads are widely available and can be used as mobile supports, with the mean mass of a bead population quantifying target binding onto bead surfaces. Inherent mass variability in the bead population is masked by matching solution density to bead density. We demonstrate that by weighing hundreds of beads in 30 min, mean mass can be estimated with a resolution of 100 attograms. A proof-of-principle assay is demonstrated that quantifies IgG binding onto functionalized beads at 5.20 femtograms per bead. / by Marcio Goldani von Muhlen. / Ph.D.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2018. / Cataloged from PDF version of thesis. / Includes bibliographical references (pages 221-242). / Macaques are the most commonly used non-human primate in cognitive neuroscience research due to similarities between the macaque and human brain. Cephalic recording chambers (CRCs) are often surgically implanted to obtain neuronal recordings. CRCs represent a persistent source of microbial contamination, which can occasionally progress to clinical sequelae of meningitis and brain abscesses. In this thesis, we first examined aerobic and anaerobic bacterial species colonizing CRCs using both traditional culture-dependent methods and 16S microbiota culture-independent methods. We evaluated the most prevalent species, and compared CRC bacterial communities to skin, oral and fecal bacterial communities. Our results indicated that CRC bacterial communities are predominantly composed of anaerobic flora and are relatively unique between individual macaques. Additionally CRC bacterial communities are more similar to skin and oral bacterial communities than fecal bacterial communities, indicating that fecal contamination of CRCs is a less likely source of contamination. Aerobic culture and sensitivity data from samples collected in 2011 identified Staphylococcus aureus, Enterococcus faecalis and Proteus spp. as the most prevalent species isolated, and that E.faecalis isolates displayed marked resistance to multiple antimicrobial classes. Routine CRC sanitization procedures were revised in September 2014 to prohibit antimicrobial use within CRCs, and we evaluated how E.faecalis lineages persisted and evolved between 2011 and 2017. We identified a shift in sequence type (ST) from ST4 and ST55, predominating in 2011, to ST48 predominating in macaques implanted after 2013. ST48 lineages were less resistant to antimicrobials and stronger biofilm producers as compared to ST4 and ST55 lineages. We concluded that loss of selective pressure from antimicrobial use within CRCs permitted ST48 to emerge as the predominant lineage due to its strong biofilm-forming abilities. Finally, we evaluated alternative E.faecalis biofilm treatment strategies. We isolated lytic bacteriophages with activity against ST55 E.faecalis and evaluated the use of phages and antimicrobial peptides LL-37 and PR-39 against E. faecalis biofilm, alone, and in combination with antimicrobials. Our results identified that bacteriophages successfully decreased biofilm produced by ST55 and ST4 E. faecalis isolates and should be evaluated further for treatment of animal and human enterococcal-associated biofilm infections. / by Mia Tova Rock Lieberman. / Ph. D.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2015. / This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections. / Cataloged from student-submitted PDF version of thesis. / Includes bibliographical references. / Malaria remains a major burden to global public health. Antimalarial drugs are a mainstay in efforts to control and eventually eradicate this disease. However, increasing drug resistance threatens to reverse recent gains in malaria control, making the discovery of new antimalarials critical. Antimalarial discovery is especially challenging due to the unique biology of malaria parasites, the scarcity of tools for identifying new drug targets, and the poorly understood mechanisms of action of existing antimalarials. Therefore, this work describes the development of two chemical biology tools to address unmet needs in antimalarial drug discovery. A particular challenge in antimalarial development is a shortage of validated parasite drug targets. Potent antimalarials with demonstrated clinical efficacy, like the aminoquinolines and artemisinins, represent a promising basis for rational drug development. Unfortunately, the molecular targets of these drugs have not been identified. While both are thought to interact with parasite heme, linking in vitro heme binding with drug potency remains challenging because labile heme is difficult to quantify in live cells. This work presents a novel genetically-encoded heme biosensor and describes its application to quantify labile heme in live malaria parasites and test mechanisms of antimalarial action. Another challenge is posed by the widespread malaria parasite Plasmodium vivax, which, unlike P. falciparum, cannot be propagated in vitro, hindering research into parasite biology and drug target identification. P. vivax preferentially invades reticulocytes, which are impractical to obtain in continuous supply. The basis for this invasion tropism remains incompletely understood, mainly because current tools cannot directly link molecular binding events to invasion outcomes. This work presents novel methods for immobilizing synthetic receptors on the red blood cell surface. These receptors are used in proof-of-concept experiments to investigate requirements for efficient invasion via a well-characterized P. falciparum invasion pathway, suggesting this method can be used to elucidate molecular mechanisms underlying parasite invasion tropisms. Future receptor designs could promote the invasion of P. vivax into mature red blood cells and potentially facilitate practical in vitro culture. Taken together, these tools present new opportunities for drug discovery to aid efforts in malaria control and eventual eradication. / by James R. Abshire. / Ph. D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biological Engineering, February 2011. / Cataloged from PDF version of thesis. / Includes bibliographical references (p. 119-128). / Angiogenesis is crucial during many physiological processes, and is influenced by various biochemical and biomechanical factors. Models have proven useful in understanding the mechanisms of angiogenesis and the characteristics of the capillaries formed as part of the process. We have developed a 3D hybrid, agent-field model where individual cells are modeled as sprout-forming agents in a matrix field. Cell independence, cell-cell communication and stochastic cell response are integral parts of the model. The model simulations incorporate probabilities of an individual cell to transition into one of four states - quiescence, proliferation, migration and apoptosis. We demonstrate that several features such as continuous sprouts, cell clustering and branching that are observed in microfluidic experiments conducted under controlled conditions using few angiogenic factors can be reproduced by this model. We also identify the transition probabilities that result in specific sprout characteristics such as the length and number of continuous sprouts. We have used microfluidics to study cell migration and capillary morphogenesis. The experiments were conducted under different concentrations of VEGF and Ang I. We demonstrated that capillaries with distinct characteristics can be grown under different media conditions and that characteristics can be altered by changing these conditions. A two-channel microfluidic device fabricated in PDMS was used for all experiments. The rationale underlying the design of the experiments was twofold: the first goal was to generate reproducible and physiologically relevant results in a microfluidic device, and the second goal was to quantify the capillary characteristics and use them to estimate the transition parameters of the model. We developed stable, well-maintained sprouts by using human microvascular endothelial cells in 2.5 mg/ml dense collagen I gel and by using media supplemented with 40 ng/ml VEGF and 500 ng/ml Ang 1 for two days. It has been shown in many studies that VEGF acts as an angiogenic factor and Ang 1 acts as stabilizing factor. Here we showed that their roles are maintained in the 3D microenvironment, and the sprout characteristics obtained by using this baseline condition could be altered by changing the concentrations of these two growth factors in a systematic way. Sprout and cell characteristics obtained in the experiments and simulations were analyzed by adapting Decision Tree Analysis. This methodology provides us with a useful tool for discerning the impact of different growth factors on the process of cell migration or proliferation as they alter general sprout morphology. The imprints obtained via experiments and simulations were compared; by choosing appropriate values of the transition probabilities, the model generates capillary characteristics similar to those seen in experiments (R2 ~ 0.82- 0.99). Thus, this model can be used to cluster sprout morphology as a function of various influencing factors and, within bounds, predict if a certain growth factor will affect migration or proliferation as it impacts sprout morphology. This was demonstrated in the case of anti-angiogenic agent, PF4. We showed that at high concentration of PF4 (- 1000 ng/ ml), the transition to migration is more profoundly affected while at low concentrations of - 10 ng/ ml, PF4 does not have much of an effect on either migration or proliferation. / by Anusuya Das. / Ph.D.