Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2016. / Cataloged from PDF version of thesis. / Includes bibliographical references (pages 105-123). / In vitro multi-cellular 3D cultures can provide tremendous insight into pathologies arising from dysregulation of extracellular communication by recapitulating key features of the microenvironment, such as ECM-cytokine interactions, spatially regulated cell-cell interactions, and matrix biophysical cues. Relevant 3D in vitro systems have been limited by the difficulty to assay the local microenvironment that cells directly experience, and by the lack of modularity of naturally-derived hydrogels such as Matrigel and collagen gels. Here, we exploit the modularity of synthetic hydrogels to overcome these limitations by designing a novel 3D culture system that can be locally and accurately interrogated. We begin by investigating the underlying principles directing morphogenesis in 3D synthetic PEG hydrogel cultures by screening the relevant parameter space - specifically, the intertwined relationship between adhesion and matrix degradation, the effect of cell-cell contacts, and cell-mediated matrix deposition. We use vasculogenesis of human iPSC-derived endothelial cells as a case study. Understanding vasculogenesis is a crucial requirement for engineering tissue models exceeding 100 gm due to nutrient diffusion limitations. Here we define microenvironment conditions that are permisive of the formation of 3D interconnected structures of iPSC endothelial cells in PEG hydrogels. Lastly, we adapted the synthetic hydrogels to enable local interrogation of the 3D microenvironment -a hitherto unavailable feature. Here we report a novel hydrogel system that can be used to both encapsulate complex co-cultures, and recover intact cells and local cytokines from the 3D matrix by repurposing a mutant of sortase A (SrtA). We demonstrate SrtA-mediated gel dissolution preserves important cell morphological features and can be used to measure recovered cytokines much more accurately compared to other proteolytic degradation methods. We observe discrepancies between the cytokine concentrations inside the gel and those in the culture medium. These local measurements reveal behaviors in response to an inflammatory stimulus that cannot be captured by monitoring only the cytokine concentrations in the medium. This system allows for better understanding of protein communication networks in relevant complex 3D multicellular cultures and can aid in identifying and testing targets in drug discovery and development. / by Jorge L. Valdez Macias. / Ph. D.

Thesis (S.M.)--Massachusetts Institute of Technology, Dept. of Biological Engineering, February 2012. / Cataloged from PDF version of thesis. / Includes bibliographical references (p. 46-48). / Agents that damage our DNA are omnipresent in our environment and inside our cells themselves. Left unrepaired, DNA damage can lead to premature aging, neurodegeneration and cancer. Humans have thus evolved intricate and widespread mechanisms to repair and manage this damage. These mechanisms-called the DNA damage response-often involve cell cycle arrest. Cell cycle arrest gives the cells precious extra time to utilize its diverse set of repair pathways. Among these is the homologous recombination pathway, which repairs DNA double-strand breaks. When the damage is deemed irreparable, a cell can choose to die: this allows for the maintenance of genomic integrity of the organism. Humans share 99.9% of the same genetic information. The remaining 0.1% is responsible for all genetic variations between individuals. This includes differences in disease susceptibility. In this study, we examined the inter-individual differences in the DNA damage response. To do so, we used a panel of twenty-four B lymphoblastoid cell lines derived from twenty-four healthy individuals of diverse ancestries. This panel had already been shown to display a broad range of sensitivity to several DNA damaging agents. We focused our attention on the alkylating agents temozolomide and methylnitronitrosoguanidine (MNNG). While MNNG has been extensively studied as a model DNA damaging drug, temozolomide is used in the clinic today to treat astrocytoma and glioblastomas. The two drugs are often referred to as functional analogues. We wanted to see if the cell lines' relative sensitivities to both drugs would be similar, which would support the analogy made between the drugs, or different, which would refute it. Furthermore, we measured the amounts of sister chromatid exchanges (SCEs) induced by temozolomide treatment to determine if the sensitivity measured by growth inhibition post-treatment was correlated with the amount of temozolomide-induced SCEs. For the cell lines tested, we found that the MNNG-induced sensitivity was similar to that induced by temozolomide. We also found a cell line in which temozolomide induced a large growth inhibition, all the while inducing no detectable SCEs. / by Meriem Sefta. / S.M.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biological Engineering, 2012. / Cataloged from PDF version of thesis. / Includes bibliographical references (p. 175-195). / Proteorhodopsin (PR) phototrophy plays an important role in the marine ecosystem, harvesting energy from sunlight for a diverse community of hetertrophic organisms. The simple proteorhodopsin photosystem (PRPS) composed of six to seven genes is sufficient for producing a functional light-driven proton pump, capable of powering cellular processes. This thesis describes the functional characterization of a subcloned PRPS previously identified from a large insert metagenomic library (Martinez et al., 2007). Incorporation of the PRPS into a strain of Pseudomonas putida resulted in a light-dependent increase in viable cell yield of cultures grown in low carbon media. The light-dependent effect demonstrates a dependence on carbon, reducing at increasing carbon concentrations until no differential effect is observed. A survey of six PR-containing vectors from metagenomic libraries revealed PR transcription in two hosts, P. putida and Pseudoalteromonas atlantica, and of the three additional vectors with PRPS tested, two demonstrated the same qualitative light-dependent yield increase. This work illustrates the utility of a simple rhodopsin photosystem for supplementing the cellular energy system of a heterologous host, paving the way for future engineering applications in photoheterotrophy. / by Justin David Buck. / Ph.D.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2018 / Cataloged from PDF version of thesis. / Includes bibliographical references. / DNA is the blueprint of life, and the high fidelity transmission of genetic information from parent cells to progeny is essential for an organism's viability. However, our genomes are constantly being damaged by reactive molecules generated from cellular metabolic processes or introduced from the environment. The resulting DNA damage can alter the information encoded in DNA, and can interfere with the accurate transmission of genetic information when cells divide. The accumulation of cells with highly damaged or altered DNA within an organism can cause diseases, such as growth defects, aging and cancer. Fortunately, cells possess the capability to repair damaged DNA. Since DNA repair mechanisms can reverse the deleterious effects of DNA damage, they are important in disease prevention, and in particular play an important role in preventing cancer. DNA repair factors are also important targets for cancer therapies. / Tumor cells frequently harbor defects in DNA repair, rendering them vulnerable to DNA damage. Many cancer therapies exploit this vulnerability by treatment with DNA damaging agents. However, tumor cells can have differential DNA repair capacities based on the expression levels of various DNA repair genes. Thus, some cancer cells are variable in their response to chemotherapy and radiation. It is well established that inhibiting DNA repair can increase the efficacy of treatment. Therefore, it is critical to develop a better understanding of the network of genes that regulate DNA repair mechanisms both to understand susceptibility to cancer, and also in order to improve the outcomes of cancer therapy. DNA repair is a complex process that requires the coordination of many proteins to respond to specific classes of DNA damage. Many of the major proteins that directly participate in DNA repair pathways are well characterized. / However, recent research has indicated that the core DNA repair factors make up only a small fraction of the proteins that respond to DNA damage, suggesting that a large number of novel DNA repair factors have yet to be discovered and characterized. In this work, we leveraged the CometChip, a high-throughput DNA damage quantification assay, to screen thousands of genes for their ability to modulate DNA repair, by knocking them down with shRNAs. We first designed hardware for the CometChip to make it compatible with high-throughput robotics so as to reduce the amount of manual labor needed to execute our screen. We then exploited the ability of our assay to measure DNA damage at an unparalleled rate to screen an shRNA library targeting 2564 oncology-associated genes. We performed gene network analysis on the top candidate genes and found LATS2 to be a novel DNA repair factor. Further investigation revealed that LATS2 is a modulator of the homologous recombination repair pathway. / In addition, we merged our screen data with that from an assay that queries proteins for their ability to bind to DNA double strand breaks. Our results showed that we were able to identify known DNA repair factors via the intersection of the two datasets, and we pinpointed at least one other novel DNA repair gene for further investigation. Taken together, this work represents an advancement in the ability to discover novel DNA repair factors by large-scale parallel measurement of physical DNA damage in cells. Our technology enables high-throughput screening for DNA damage and repair factors faster than ever before, allowing for extensive studies of DNA damage and opening doors to the discovery of new genes and molecules that affect DNA repair. / by Ian Jun Jie Tay. / Ph. D. / Ph.D. Massachusetts Institute of Technology, Department of Biological Engineering

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2019 / Cataloged from PDF version of thesis. / Includes bibliographical references. / Modern molecular biology, biochemical, and chemical techniques have made it possible to identify individual natural products that possess pharmacological activity from medicinal plants. While approximately 50% of all new FDA-approved small molecule therapeutics in the past 40 years were natural products or natural product analogs, challenges including limited natural resources and the difficulty of solving the total synthesis or semi-synthesis of natural products has limited our ability to harness the full diversity of chemical structures provided by nature to treat human diseases. One solution to these challenges is the elucidation of plant specialized metabolite biosynthetic pathways. Identifying and characterizing the enzymes involved in specialized metabolite biosynthesis will provide insight into the evolution of enzymes performing interesting chemistries and provide new tools for the enzymatic production of therapeutically promising natural products. The goal of this dissertation is to explore the aspects of both medicinal chemistry and the elucidation of biosynthetic pathways that can contribute to the development of novel therapeutics. First, we analyzed the structure-activity relationship of analogs of the the flavonoid icariin and identified analogs with improved potency in inhibiting human phosphodiesterase-5. We subsequently identified and characterized a novel flavonoid prenyltransferase and O-methyltransferase from the medicinal herb Epimedium sagittatum that is known to produce many bioactive prenylated and methylated flavonoids. / by Yasmin-Pei Chau. / Ph. D. / Ph.D. Massachusetts Institute of Technology, Department of Biological Engineering

Production of dairy and associated products is a source of millions of gallons of wastewater every year. Water used in cleaning feeding stalls as well as the liquid component of the animal waste are two of the major volumetric components of this wastewater. This water is nutrient rich, often limiting the viability as a land applied fertilizer. However, these same nutrients could be used as an inexpensive feedstock for the cultivation of algae, which can then be used to produce downstream products including animal feed and aquaculture. As part of this study, algal biomass was cultivated on dairy wastewater from the Utah State University Caine Dairy. A Rotating Algal Biofilm Reactor (RABR) system was used to grow the biomass. The RABR is a biofilm technology designed and developed at Utah State University and has been applied to the treatment of municipal wastewater. In this study, the RABR was adapted for use in a dairy wastewater stream. The RABR was operated at temperatures ranging from 7-27 °C, and organic carbon levels in the wastewater ranged from 300-1200 mg/L of Total Organic Carbon (TOC). Areal algal biofilm growth rates were calculated, and statistical analysis showed that both increasing temperature and levels of organic carbon contributed to an increase in biomass growth and an increase in nutrient removal. Equations were then developed using a linearization method and corresponding constants and equations were generated that can be used to evaluate algal biomass productivity and nutrient removal rates in future experiments and designs for dairy wastewater.

This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections. / Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2019 / Cataloged from student-submitted PDF version of thesis. / Includes bibliographical references. / A billion years of evolution has crafted a diverse set of proteins capable of complex and varied functionalities. Within recent decades, scientists have applied both rational design and directed evolution to accelerate development of high-value proteins, including biotherapeutics. While computational modelling increasingly facilitates protein design, empirically screening large collections of protein variants remains an essential component of protein engineering. This process requires generating protein variation, partitioning variants with a selection pressure, and identifying highly functional proteins. This thesis presents computational tools for initial protein library design, leverages high-throughput sequencing for phage display screenings, and reports biotemplating of an inorganic phase-change material onto the filamentous M13 phage surface. / Designing ensembles of protein variants involves optimizing library size and quality with constraints on screening capacity, cost, and experimental complexity. Incorporating degenerate codons during oligonucleotide synthesis enables residue-specific protein randomization with a known amino acid scope. However, this widely adopted method often generates uneven variant abundances that diverge exponentially with additional randomized residues. The first section of this work presents tools for the design and assessment of degenerate-codon-based protein libraries. This framework facilitates incorporating an arbitrarily large number of randomized sites, non-standard genetic codes, and non-equimolar nucleotide mixtures. In addition to library size and coverage calculations, whole-population diversity metrics and abundance quantiles are reported. / An evolutionary solver to optimize non-equimolar base compositions to match amino acid profiles, as well as mutational profiling for spike-in oligonucleotides is also presented. The second section of this thesis develops an experimental and data analysis pipeline for integrating high-throughput DNA sequencing with M13 phage display biopanning. Deeply sequencing naïve M13 peptide libraries elucidated censorship patterns for both p3 and p8 coat protein fusions. Streptavidin panning recapitulated the HPQ binding motif after a single panning round, and additional biopannings pursued M13 p8 variants that interact with both gold films and carbon nanotubes. Furthermore, this thesis explores the effect of M13 p8 surface charge on the biotemplating of an inorganic phase-change material. An ambient temperature synthesis for modulating the atomic composition of germanium-tin-oxide nanomaterials is reported. / by Griffin James Clausen. / Ph. D. / Ph.D. Massachusetts Institute of Technology, Department of Biological Engineering

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2019 / Cataloged from PDF version of thesis. / Includes bibliographical references. / Bone marrow stromal cells (BMSCs), a subset of which are considered mesenchymal stem cells (MSCs), have been used in over 600 clinical trials for indications ranging anywhere from autism to liver cirrhosis to diabetes. They have cited enthusiasm in the cell therapy community not only for their demonstrated differentiation potential toward several lineages, but also due to the anti-inflammatory and immunomodulatory effects of their secretome. However, the necessary in vitro expansion of BMSCs renders cell populations functionally diverse, and understanding of what drives heterogeneity onset - as well as which distinct phenotypes elicit therapeutic responses of interest - remains an open challenge. This lack of characterization confounds studies focused on basic cell behavior as well as translational applications, and U.S. Food & Drug Administration approval for BMSC therapies has yet to be achieved for any of the several dozen indications explored to date. / This thesis describes our work toward understanding the extent, mechanisms, and possible mitigation strategies regarding heterogeneity in BMSC cultures, by exploring the biophysical and transcriptomic profiles of single cells. We report our findings that cell generation most succinctly dictates the combined biophysical properties studied and that at the transcriptome level four distinct functional phenotypes exist. We further explore mechanisms by which heterogeneity emerges, demonstrating that cellular senescence and asynchronous proliferation kinetics leads to a distribution of biophysical properties and that at fixed time points cells are somewhere along a gene expression cascade trajectory from one functional state to another. / We also report our discovery of novel surface marker candidates for enrichment of specific phenotypes and demonstrate that these discrete subpopulations differentially express genes implicated in the distinct, yet established therapeutic applications of immunosuppression, neurogeneration, and wound healing. These findings were enabled by our technological advancements that include complex time lapse imaging protocols, innovative assays for probing of label-free cell behavior, establishment of best practices for generating single BMSC transcriptome libraries, and robust analytical pipelines for time lapse imaging and single-cell RNA sequencing datasets. Collectively, these tools and analyses provide a strong foundation toward leveraging the discrete functional roles of this diverse collection of cells for both well-designed basic research studies and improved therapeutic efficacy. / by Deena Antoinette Rennerfeldt. / Ph. D. / Ph.D. Massachusetts Institute of Technology, Department of Biological Engineering

Promiscuous mutant EcoRI endonucleases produce lethal to sub-lethal effects because they cleave E. coli DNA despite the presence of the EcoRI methylase. Three promiscuous mutant forms, Ala138Thr, Glu192Lys and His114Tyr, have been characterized with respect to their binding affinities and first-order cleavage rate constants towards the three classes of DNA sites: specific, miscognate (EcoRI*) and nonspecific. We have made the unanticipated and counterintuitive observations that the mutant endonucleases that exhibit relaxed specificity in vivo nevertheless bind more tightly than the wild-type enzyme to the specific recognition sequence in vitro and show even greater preference for binding to the cognate GAATTC site over miscognate sites. Binding preference for EcoRI* over nonspecific DNA is also improved. The mutant enzymes cleave the cognate site GAATTC at a normal rate, but cleave EcoRI* sites faster than does the wild-type enzyme. Thus, the mutant enzymes use two mechanisms to partially bypass the multiple fail-safe mechanisms that protect against cleavage of genomic DNA in cells carrying the wild-type EcoRI restriction-modification system: (a) Binding to EcoRI* sites is more probable than for wild-type enzyme because nonspecific DNA is less effective as a competitive inhibitor; (b) The combination of increased affinity and faster cleavage at EcoRI* sites makes double-strand cleavage of these sites a more probable outcome than it is for the wild-type enzyme. The crystal structure of the A138T "promiscuous" mutant enzyme in complex with specific DNA shows reveals no changes in protein contacts to the bases of the GAATTC site relative to the wild-type complex; however, there are changes in water-mediated contacts between the enzyme and flanking bases, and changes in protein-phosphate contacts. These observations lead us to hypothesize that the improved specific DNA binding of the A138T enzyme relative to the wild-type enzyme is not attributable to a single new protein DNA contact, but rather the distributed effect of the improved complementarity between the mutant and the flanking bases, as well as the optimization of several phosphate contacts. The aggregate of biochemical data presented in this thesis leads us to propose a model where the A138T miscognate DNA binding ensemble (for a subset of miscognate sites) is partitioned more towards complexes that are on the path to the transition state than wild-type miscognate DNA binding ensembles; the mutant accomplishes this by forming 'specific-like' phosphate contacts to these sites, which in turn stabilize the DNA distortion that is critical for efficient cleavage. Given the proximity of amino acid 138 to the residues which make contacts to the DNA phosphates in the specific complex, we hypothesize that the A138T mutation has an effect on the structure and/or dynamics of the "arm" protein segment (contains residues contacting the DNA backbone) such that it is more adaptable, resulting in formation of functional phosphate contacts to a broader range of DNA substrates.

Chromosome instability (CIN), a continuous change in the structure or number of chromosomes, is proposed to be a key mechanism driving the genomic changes associated with tumorigenesis. One major cause of CIN in cells is chromosome segregational defects occurring during mitosis. Two such examples are anaphase bridges and multipolar spindles, which are common in most cancer cells and many tumor tissues. Anaphase bridges are chromatin bridges in between separating chromosome masses during anaphase, which may result in gene amplification or loss when breaking. We have found that cigarette smoke condensate (CSC) induced anaphase bridges in cultured primary human cells, which in a short time led to genomic imbalances. The frequency of the induced bridges within the entire population decreased with time, independent of the p53-mediated apoptotic pathway. We also showed that CSC induced DNA double-stranded breaks (DSBs) in cultured cells as well as purified DNA. The reactive oxygen species (ROS) scavenger, 2 deoxyguanosine 5-monophosphate (dGMP) prevented CSC-induced DSBs, anaphase bridge formation and genomic imbalances. Therefore, we propose that CSC induces bridges and genomic imbalances via DNA DSBs. Further analysis in live oral cancer cells shows that cells with anaphase bridges mostly survive and these bridges frequently result in micronuclei formation, indicating that anaphase bridges actively contribute to CIN. Multipolar spindles (MPS) are aberrant mitotic structures when cells divide with greater than two spindle poles, which may result in uneven chromosome segregation. Multipolarity is strongly linked to centrosomal amplification, the mechanism of which remains controversial. We have examined the origin and fate of cells with MPS in real time. In both human embryonic kidney and oral cancer cells, the vast majority of multipolar cells originated from multinucleated cells. The frequency of cytokinesis failure was similar to the frequency of MPS, and each observed bipolar division that ended in a cytokinesis failure led to MPS formation in the subsequent mitosis. While grossly abnormal, these cells are still capable of dividing, often giving rise to a mixed progeny of multinucleated and mononucleated cells. These observations support the model that failure of cytokinesis may be the most common mechanism by which cells form MPS.