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

Thesis (M.Eng.)--Massachusetts Institute of Technology, Biological Engineering Division, 2003. / Includes bibliographical references (leaves 126-134). / by Tomoko Iida. / Thesis (M.Eng.)--Massachusetts Institute of Technology, Biological Engineering Division, 2003.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2019 / Cataloged from PDF version of thesis. / Includes bibliographical references (pages 143-163). / Three-dimensional in vitro tissue and organ cultures have immense promise as models of human pathophysiology and stand to make a significant impact on the process of drug discovery and development. Many existing model systems do not capture the relevant complexity of the native tissue environment, relying on poorly characterized natural extracellular matrices (ECMs) for growth and development. These models are notably limited by the lack of vasculature, a key functional component of most human tissues, enabling oxygen and nutrient exchange, as well as facilitating paracrine signaling with surrounding epithelial cells. Fully-defined and tunable synthetic ECMs that support the generation of vascular network structures in dense tissue environments represent a path towards overcoming the limitations of existing model systems. / This thesis focuses on the development and characterization of polymeric biomaterials that can be used to enhance in vitro tissue models through engineering the cell-material interface to guide a particular biological response. A major application focus of this research is to engineer biomaterial tools that would enable vascularization of dense epithelial tissue in vitro. We developed and characterized a poly(ethylene glycol)-based microbead angiogenesis scaffold with tunable physical and biochemical properties, identifying a critical ligand concentration regime on the microbead surface that promotes integrin-mediated endothelial cell attachment and invasion into both a synthetic ECM as well as a tissue aggregate of hepatocarcinoma cells. / Furthermore, we investigated a novel hybrid PEG-polypeptide polymer, poly([gamma]-propargyl- L-glutamate) (PPLG) as a hydrogel substrate that can enhance endothelial cell attachment and spreading through modulation of the macromer structure and hydrophobicity properties. This work demonstrates how rational biomaterial design through chemical and structural modifications to polymer scaffolds can control cell fate within an in vitro tissue culture system. / by Marianna Sofman. / Ph. D. / Ph.D. Massachusetts Institute of Technology, Department of Biological Engineering

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, September, 2019 / Cataloged from the PDF version of thesis. / Includes bibliographical references. / Many methods for increasing the optical transparency of non-living brain tissue have come into widespread use because of their utility in enabling better anatomical brain imaging. In the first part of this thesis, we explore whether this is also possible for living brain tissue. We report a general principle for doing so, namely the reduction of refractive index mismatch between cellular membranes and the extracellular space, using high refractive index biocompatible reagents that have high molecular weights, so that they can be used at low concentrations. We implement this via multiple reagents that satisfied these criteria, including the iodinated radiocontrast agent iodixanol, high molecular weight polyethylene glycol (PEG), high molecular weight Dextran, and PEG-ylated Silicon nanoparticles. We achieve ~2x increases in the brightness of cells expressing red fluorescent proteins in vivo in mice, as measured by conventional one-photon epifluorescence imaging, using concentrations of reagents that increased the refractive index of the extracellular space by just 0.01. Lastly, We show that Dextran does not have a statistically significant effect on neural physiology or neural network properties. We expect such strategies to not only facilitate live imaging of the brains of mice and other mammals, but open up a new class of strategies for changing the electromagnetic properties of living systems. We conclude this thesis with two nanotechnologies that may be leveraged for making higher performance reagents for increasing the optical transparency of living brain tissue. (1) A method for the synthesis of high-yield and high-monodispersity nanoparticles of a variety of materials with tailored surface ligands, using common benchtop equipment. This method may be useful for developing nanoparticles with better biosafety, efficacy and performance. (2) A method for the delivery of hydrophobic NVNDs to neural cell membranes using PEG-ylated liposomes. These PEG-ylated liposomes may be used for delivery of hydrophyllic nanoparticles to neural soma and achieve maximal transparency. / by Ishan Gupta. / Ph. D. / Ph.D. Massachusetts Institute of Technology, Department of Biological Engineering