Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, February, 2021 / Cataloged from the official PDF version of thesis. / Includes bibliographical references (pages 185-197). / Forward genetic screens are powerful tools for the unbiased discovery and functional characterization of specific genetic elements associated with a phenotype of interest. By perturbing thousands of genes simultaneously and selecting for a desired phenotype, genetic features can be systematically mapped to phenotypic changes. Recently, CRISPR-Cas9 has emerged as a powerful genetic perturbation technology, opening up new opportunities for forward genetic screens. In this thesis, I present work to advance this approach and demonstrate its application in a range of contexts relevant to human health. We first established a detailed CRISPR-Cas9 screening protocol that outlines experimental design considerations. We then applied this methodology to develop a CRISPR toolkit for screening and characterizing long non-coding RNAs in the human genome, many of which remain uncharacterized. We identified the EMICERI locus as a regulator of four neighboring genes, one of which conferred resistance to a melanoma therapeutic. We next sought to use CRISPR activation screening to gain insight into the cellular processes that govern tumor resistance to immunotherapy. We identified four candidate genes in our screen, which we validated in diverse cancer cell types and explored through mechanistic studies, leading to the discovery of novel immunotherapy resistance pathways. Finally, we developed a pooled transcription factor (TF) screening platform that provides a generalizable approach for studying cellular programming. We created a comprehensive human TF library and applied it to identify TFs that can drive differentiation of embryonic stem cells toward neural cell fates. We discovered that one TF, RFX4, leads to differentiation of neural progenitors that produced inhibitory neurons, providing an efficient method for generating this important cell type. During the COVID-19 pandemic, we paused the screening work and developed a streamlined SARS-CoV-2 detection assay, STOPCovid, suited for low-complexity settings. STOPCovid combines viral RNA concentration with isothermal amplification and CRISPR-mediated detection. STOPCovid achieved a sensitivity and specificity of 93.1% and 98.5%, respectively, on patient samples. Together, our applications of forward genetic screens address diverse problems in human health and broadly demonstrate the potential of this approach for systematically interrogating genetic elements. / by Julia Joung. / Ph. D. / Ph.D. Massachusetts Institute of Technology, Department of Biological Engineering

Understanding how marine biodiversity is distributed, and what drives these patterns, relies on good descriptions of marine ecosystems. This information should inform the protection of biodiversity and guide its management. Relationships between marine landscapes and biodiversity therefore need to be described at scales that are useful to regional management. Simultaneous sampling of marine biodiversity and the seafloor is challenging, so baseline ecosystem descriptions are often mismatched in their abiotic and biotic components. Cameras can sample the seafloor and its associated biodiversity concurrently, with good coverage and at low cost. These are important considerations for sustainable monitoring to inform conservation management in resource-limited regions. Terrestrial landscape characterisations cannot simply be translated to the ocean because interpreting remote ocean terrain assessments in a manner relevant to ecological analysis is complicated by depth, circulation, light attenuation, and other oceanographic variables. The integration of some of these concepts into rapid marine biodiversity assessments therefore needs ground-truthing where they are applied in new regions, to advance sustainability in long-term marine monitoring. This thesis investigated the relationship between landscape composition and benthic marine biodiversity in False Bay, South Africa using novel methods that extended biodiversity sampling across more depths and habitats than any single, previous survey of the bay. This study's approach piloted sampling and interpreting the marine landscape and biodiversity over matching spatial and temporal scales. The coverage, repeatability and ecosystem-level scale applied to this study make it a useful basis to develop monitoring protocols that are appropriate to conservation management at relevant regional scales. New insights for the region include a) a new description of the seafloor using classifications that explain the variation in epibenthic megafauna and ichthyofauna communities, b) a quantitative account of the epibenthic megafauna on the eastern reefs where species diversity was highest, and c) the synthesis of seafloor descriptions with the epibenthic megafauna and ichthyofauna to describe nine regions of False Bay, relative to two previous descriptions of "grounds". Photographs and multibeam bathymetry characterised the seafloor on eight transects across the bay and were ground-truthed by grab samples repeated at representative sites. Two new classifications were applied to describe the seafloor. Horizontal seafloor heterogeneity was highest in the east, and reef was distributed along the eastern and western margins. The Collaborative and Automated Tools for Analysis of Marine Imagery (CATAMI) scheme captured accurate broad-scale descriptions of the physical landscape when applied to photographs. Grabs are still needed to provide fine-scale particle size data on soft sediments where most invertebrate diversity is likely infauna. However, CATAMI abstracts fine-scale sediment variation into simpler groupings more useful for rapid ecosystem assessment. Photographic sampling is repeatable, which is useful for long-term ecosystem monitoring. Photographs taken using a jump camera rig assessed the epibenthic megafauna across habitats and along depth gradients. Rényi diversity showed that species diversity increased in shallow waters up to 40 m, reaching a peak between 30 and 40 m, before decreasing with increasing depth. Species diversity was highest in the east, where seafloor heterogeneity was also highest. This result is interesting because eastern False Bay falls mostly outside the current marine protected area (MPA) network and has been relatively under-represented in previous surveys. The jump camera documents ecosystem-level biodiversity patterns and processes, and the random point count method in Coral Point Count (CPCe) is useful to assess community composition and cover on reefs. The relative abundance and distribution of ichthyofauna were assessed using baited remote underwater video systems (BRUVs). Fifty-seven fish species from 30 families were recorded between 4 and 84 m. Rényi diversity showed that species richness was similar for reef and sand overall, but the Shannon-Wiener diversity index (H') was significantly higher on reef sites than on sand sites (t = 1.972, p < 0.0001). Species richness for the whole bay was similar in winter and summer, which indicates that the same species are likely present year-round; however, the Shannon-Wiener diversity index was significantly higher in winter (t = 1.973, p < 0.013) and evenness was greater in winter at the level of the site. These findings highlight the difficulty in protecting sufficient sand habitat to encompass the patchy distribution of sand-associated species and highlight seasonal differences in optimal visibility for future camera monitoring surveys by conservation management. There are clear patterns in the marine biodiversity of False Bay, at various scales, that can be detected using novel methods for the region. The study's approach to classifying both the landscape and its associated biodiversity creates a framework for future ecosystem threat assessment that can be applied elsewhere, especially along the South African coastline.

The dynamic Benguela Upwelling System is one of four major upwelling regions in the world and is subdivided into two sub-systems, the northern and southern Benguela. This current study was conducted within the southern Benguela, which lies between 27°S and 35°S (Orange River Mouth to East London) and is characterized on the west coast by seasonal, wind-driven, coastal upwelling. The study targeted three picophytoplankton groups, Synechococcus, Prochlorococcus and picoeukaryotes, which are the three most abundant < 2 µm size class phytoplankton. Flow cytometry was employed to enumerate picophytoplankton abundances, using their pigments and cell sizes to identify the different groups. The aim of the study was to investigate the role of picophytoplankton in the southern Benguela coastal upwelling ecosystem. These aims were met by: i) determining the spatiotemporal variability of each of the three picophytoplankton groups over a period of 8 sampling cruises at 44 stations across four latitudinal lines in the study region, ii) determining short-term changes in carbon and nitrogen biomass of picophytoplankton and their growth rates over a 10-day period, using abundance estimates from a station off St. Helena Bay, and iii) estimating mortality of microbial communities in a laboratory study using samples collected from a coastal upwelling environment. Results showed no strong seasonality in picophytoplankton abundances but evidence of latitudinal and zonal effects. Investigations over the short term showed that populations of picophytoplankton in the southern Benguela change on the same timescale of ~3 days as the larger phytoplankton during an upwelling event. Determining mortality rates using a dilution experiment presented some challenges. Instead of increased growth rates, the study showed decreased growth rates as predator numbers decreased. These shortcomings were investigated in a second experiment, which both excluded large predators (<200µm) and also ran a parallel experiment excluding smaller predators (10-200 µm). The last of these experiments resulted in increased growth rates as predator numbers decreased. The complexity of the southern Benguela system, with its pulsed, high productivity and large concentrations of nutrients, traditionally is known to show variability through effects on the biology of large phytoplankton. However, picophytoplankton also were variable in the study area, resulting from bottom up effects of the environment, confounded by biotic factors such as predation, parasitism and competition

Mussels living in coastal environments are often exposed to natural inorganic particles and hence may be well adapted to dealing with high sediment loads. The mechanisms by which they deal with particle loads do, however, cause stress and alter metabolic processes. An increasingly common anthropogenic addition to particle loads in the ocean are microplastic particles. Numerous recent experiments have addressed the impacts of microplastics on metabolic performance, but few of these have used natural reference particles to control for the concurrent effects of particle load itself. This study aims to compare the effects of microplastic and of natural particle exposure on the mussel Mytilus galloprovincialis, an invasive species which has become the dominant mussel in the mid- to low-shore of the south and west coasts of South Africa, but is absent from areas prone to sand inundation. These effects will be compared to those on the native mussel Choromytilus meridionalis, which resides on the low shore, and unlike M. galloprovincialis often occurs in areas prone to sand inundation. Respiration rates, byssus production, clearance rate, body condition (BCI) and survival of mussels exposed to four concentrations of two particle types, polyvinyl chloride (PVC) and red clay were measured. A significant concentration effect was found in the respiration rates of Mytilus galloprovincialis, while C. meridionalis respiration rates were largely unaffected by both particle type and particle concentration. The byssus numbers of M. galloprovincialis were significantly reduced by microplastic exposure, whilst no particle type effects were found in C. meridionalis. Clearance rates of C. meridionalis, on the other hand, were significantly affected by particle concentration, while no effects were found on M. galloprovincialis. The BCI of C. meridionalis was also found to be affected by particle concentrations, while M. galloprovincialis was unaffected. All C. meridionalisindividualssurvived the experiment, while 29 M. galloprovincialis died. Mortality of M. galloprovincialis exposed to the two particle types was not significantly different, although more mortality was suffered in PVC treatments than in red clay treatments. The results reveal that there was indeed a difference in the response of M. galloprovincialis to the different particle types, and that the two species did exhibit different strategies to both particle type, and concentration. Experimental studies of this nature are imperative in order to disentangle microplastic effects from those of particles in general, and to develop a better understanding of potential impacts of plastic debris on marine ecosystems.

This study examines the navigation of structural pressures, limitations and conservation policies by the community of Hobeni Village in the practice of traditional rituals within the context of nature conservation. It considers how ceremonies such as uKuqatywa komntwana, Intonjane, Ulwaluko, ukunikezelwa ko-Mkhontho, and Umcimbi/ Umgidi play a vital role in the construction of personhood among small groups of men, women and teenagers identifying as Xhosa people in the Hobeni Village in the Eastern Cape. The qualitative research drew insights from indigenous knowledge already known to the researcher, current research, participatory observation and semistructured interviews conducted on twenty-four members of the community. The ethnographic study found that systemic methods of nature conservation Vis a Vis symbolic oppression, sit in tension with the rituals performed by members of the AmaXhosa at Hobeni village. It argues that for Hobeni residents, accessing the natural resources placed under conservation is a vital aspect of their identity formation, which is impacted by conservation. The research found that current conservation practices pose a threat not only to AmaXhosa practices of identity formation and sense of belonging but also to the maintenance of their culture. The connection of the Hobeni people with nature is limited by conservation methods that force them to adapt their traditional practices that attempt to find congruence with their belief systems, but that strain the relationship between these villagers and their ancestors. In the search for alternative methods to preserve natural resources and maintain the culture of Hobeni village, this dissertation calls for the establishment of a different approach to conservation that is context-specific and community-centred. A transformative approach to conservation could advance environmental justice without compelling the community to negatively negotiate, as is currently the case, their cultural practices or erode their entanglement with nature. The contribution of this study lies in challenging the narrative or ideologically laden discourses that perceive people as a threat to nature and the environment. This dissertation concludes that people possess diverse knowledge systems and resources that enable them to coexist and conserve nature in their surroundings or living environment.

South African mosasaur remains consist of a frontal with articulating portions of the parietal and postorbitofrontals (SAM-PK-5265); two dentary fragments (SAM-PK-5265) assigned to ‘Tylosaurus capensis' by Broom in 1912 (SAM-PK-5265); an undescribed muzzle unit and associated isolated teeth (CGP/1/2265) from Pondoland and a more recently discovered isolated partial vertebra from St Lucia. Some research has been done on the material, but there is still uncertainty concerning their relationships and taxonomy. This research aims to provide a taxonomic assessment of all the SA mosasaurid material to better understand their phylogenetic relationships and to place them in the context of mosasaurs from other parts of Africa and globally. In addition, isotopic analyses, micro-computed tomography (micro-CT), mineralised tissue histology and scanning electron microscopy (SEM) are applied to the SA mosasaur remains to decipher various aspects of their palaeobiology. This study identifies three mosasaur taxa from SA: Mosasaurus sp., cf. M. hoffmannii., cf. Taniwhasaurus, and cf. Prognathodon. The isolated vertebra is assigned to Mosasaurus sp., cf. M. hoffmannii. The frontal and dentary fragments (SAM-PK-5265) originally described as Tylosaurus appears to be a mix of two taxa: One of the dentary fragments possesses replacement teeth with enamel ornamentation that resembles, Ta. mikasaensis, but is tentatively assigned to cf. Taniwhasaurus based on tooth recurvature. The other dentary fragment and a frontal with articulated elements are suggested to belong to the same individual as the muzzle unit for which the suggested assignment is cf. Prognathodon. Strontium analysis of tooth enamel dated the cf. Prognathodon material to the end of the Maastrichtian (87Sr/86Sr = 0.707817; age = 66.85Ma). The cf. Taniwhasaurus dentary fragment is likely Santonian-aged, as originally indicated in 1901. SEM of enamel from cf. Prognathodon reveals a complex array of prismless enamel types and pervasive aggregations of fossilised bacteria in the underlying dentine. The δ18OPO4 derived body temperature estimate (Tb) of the cf. Prognathodon (Tb = 33.21°C) compares well with previously reported Tb for mosasaurs and may indicate that the mosasaur was capable of maintaining a Tb higher than that of the surrounding seawater.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2019 / Cataloged from PDF version of thesis. / Includes bibliographical references (pages 186-197). / Hands-on demonstrations greatly enhance the teaching of STEM concepts and foster engagement and exploration in the sciences. While numerous chemistry and physics classroom demonstrations exist, few biology demonstrations are practical and accessible due to the challenges and concerns of growing living cells in classrooms. Here I introduce a platform to develop hands-on molecular and synthetic biology educational activities based on easy-to-use, shelf-stable, freeze-dried, cell-free (FD-CF) reactions, which are simply activated by water. By using fluorescent proteins as a visual output, I created a variety of engaging modules using this platform that can teach the central dogma of biology, how certain cellular functions work, and other basic molecular biology topics that are otherwise difficult to easily teach in a hands-on manner. By expanding the platform to other non-visual outputs (such as smell or touch), as well as further incorporating components, such as RNA switches, I also developed modules that can teach more advanced biology topics, such as biochemistry, biomaterials, and synthetic biology, as well as basic laboratory skills such as pipetting, experimental design, and the scientific method. Pilot testing of a prototype kit based on these elements were tested in classrooms across the country and initial results suggest that the activities are accessible, easy to use, educational, and engaging for high school students. Overall, the platform introduces low-cost, user-friendly, and hands-on activities that can be used in classrooms to improve the quality of biology education and open the door for student-driven, independent explorations in the life sciences. / by Ally Huang. / 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 (pages 154-176). / Lack of medication adherence is a worldwide problem. As many as 50-70% of patients have trouble following treatment recommendations. Whereas adherence is driven by many factors including the socioeconomic status of a patient and the quality of the health care team, drug regimen complexity also affects treatment outcomes. For example, adherence decreases as the number of pills per dose and the number of doses per day increases. For diseases where potent medications are available, depot formulations provide sustained drug release to simplify dosing. For diseases lacking potent compounds for treatment, there remains an unmet need for depot systems that could transform medication adherence. Tuberculosis (TB) is one such disease with a high pill burden, where poor patient adherence to the treatment regimen is a major cause of treatment failure and contributes to the emergence of drug-resistant TB strains. / For example, an average 60-kg patient with TB needs to take 3.3 g of antibiotics per day, which is a dose that exceeds the largest swallowable capsule and current depot systems. According to the World Health Organization (WHO), 10 million people developed TB in 2017 with a global economic burden amounting to $12 billion annually. This thesis presents a solution to the challenge of prolonged dosing for regimens such as TB that require multigram drug dosing. First, a gastric resident system (GRS) compatible with transesophageal administration was designed using biocompatible materials. The GRS consists of a series of drug pills on a coiled superelastic nitinol wire; the ends are protected with a retainer and tubing. Safe administration, gastric retention for 1 month, and retrieval of the GRS were demonstrated in a swine model. Next, sustained release formulations for 6 TB antibiotics were formulated into drug-polymer pills, and first-order drug release kinetics were achieved in vitro. / Then, the GRS was demonstrated to be capable of safely encapsulating and releasing 10 grams of an antibiotic over the period of weeks in a swine model. Lastly, end-user assessment was evaluated with a field questionnaire in India and an economic model to estimate the impact of the GRS on the health care system. There are multiple applications of the GRS in the field of infectious diseases, as well as for other indications where multigram depots could impart meaningful benefits to patients, helping maximize adherence to their medication. / "Funding and Resources: -- Bill and Melinda Gates Foundation -- National Institutes of Health -- National Science Foundation Graduate Research Fellowship -- MIT Tata Center and leadership team for believing in and guiding our project" / by Malvika Verma. / 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. / Fireflies (Lampyridae) and certain other families of beetles including the American railroad worms (Phengodidae), Asian starworms (Rhagophthalmidae), and American click-beetles (Elateridae), produce light in a process known as bioluminescence. The bioluminescent systems of beetles, natively used for the purposes of mating communication and/or an aposematic warning signal, are now well understood and have been widely applied in biotechnology and biomedical research. There have been considerable advancements in the engineering of the luciferin substrate, and the luciferase enzyme, for beneficial characteristics such as altered emission wavelength, improved thermostability, and improved catalytic parameters, but despite this substantial effort focused on the biotechnological applications of beetle bioluminescence, major questions remain regarding its natural biochemistry and evolutionary origins. / Four major questions that were unanswered at the beginning of this PhD study were: (1) Do fireflies possess a storage form of their luciferin? (2) What is the evolutionary relationship of bioluminescence amongst the bioluminescent beetles families, and has this trait independently evolved multiple times? (3) How is firefly luciferin biosynthesized? And (4) Are there accessory genes from the bioluminescent beetles which act in bioluminescent metabolism, and might these genes be useful for biotechnological applications? Here I describe the discovery and characterization of the presumed storage form of luciferin in fireflies, sulfoluciferin, and the enzyme which produces it, luciferin-sulfotransferase. / Furthermore, I describe the sequencing, assembly, and characterization of the genome of the North American "Big Dipper" firefly Photinus pyralis, along with the Japanese "heike" firefly Aquatica lateralis genome, and the genome of the Puerto Rican bioluminescent click beetle or "cucubano" Ignelater luminosus. Genomic comparisons amongst these three species support the hypothesis that firefly and click beetle luciferase evolved independently, suggesting an independent evolutionary origin of the bioluminescent systems between these fireflies and click beetles. I also describe stable isotope tracing experiments in live fireflies, establishing that adult and larval fireflies likely do not de novo biosynthesize firefly luciferin, and may instead rely on a "recycling" pathway to re-synthesize luciferin from the luminescence product oxyluciferin. Lastly, I discuss the future directions resulting from this thesis, and the yet unanswered questions. / by Timothy Robert Fallon. / 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 (pages 139-148). / Synthetic mRNA is an emerging therapeutic modality for gene and cell therapy. Unlike their synthetic DNA counterparts, synthetic mRNA has an increased safety profile due to its transient gene expression and ability to express outside of the nucleus. Furthermore, it can be more easily delivered to cells via entry only into the cytoplasm. While synthetic biology as a field has existed for over two decades, the main area of research and development has focused on DNA interfaces, building on the mechanisms of transcription factors with small molecule interfaces to create multi-input/multi-output genetic circuitry. Until recently, the field had not developed sufficient synthetic circuit control devices at the translational level due to 1) lack of perceived need and 2) deficiency of available natural systems for adaptation. In this thesis, I present the construction of a diverse synthetic biology toolbox for RNA-only synthetic biology. The creation of new synthetic biology frameworks can be broken down into three modules: Build, Control, and Apply. In the Build phase, I demonstrate how the current toolbox of mRNA binding and recognition proteins can be utilized to form diverse and orthogonal gene regulatory networks. In Control, I construct regulatory networks capable of responding to exogenous signals and utilize advanced circuit design to motivate dynamic control for novel behaviors. When I transition to Apply, I illustrate that these next-generation circuits can be layered into biologically active modalities that are therapeutically relevant. Taken as a whole, the work presented here represents a merging of the fields of synthetic biology and mRNA therapeutics, and serves as a foundational proof-of-principle for future efforts to expand synthetic biology across novel modalities. / by Jacob Robert Becraft. / Ph. D. / Ph.D. Massachusetts Institute of Technology, Department of Biological Engineering