Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2019 / Cataloged from PDF version of thesis. / Includes bibliographical references (pages 147-168). / Cell-cell communication between malignant, immune, and stromal cells influences many aspects of in vivo tumor biology, including tumorigenesis, tumor progression, and therapeutic resistance. As a result, targeting receptor-ligand interactions, for instance with immune check-point inhibitors, can provide significant benefit for patients. However, our knowledge of this complex network of cell-cell interactions in a tumor microenvironment is still incomplete, and there is a need for systematic approaches to study cell-cell communication. This thesis presents computational approaches for characterizing cell-cell communication networks in three different experimental studies. In the first study, we modeled metastatic triple negative breast cancer in the liver using a microphysiological system and identified inflammatory cytokines secreted by the microenvironment that result in the proliferation of dormant metastases. In the second study, we used single-cell RNA sequencing (scRNA-seq) to quantify receptor-ligand interactions in six syngeneic mouse tumor models. To identify specific receptor-ligand interactions that predict tumor growth rate and immune infiltration, we used receptor-ligand interactions as features in regression models. For the third study, we extended our scRNA-seq approach to include inferences of single-cell signaling pathway and transcription factor activity. We then identified protein-protein interaction networks that connect extra-cellular receptor-ligand interactions to intra-cellular signal transduction pathways. Using this approach, we compared inflammatory versus genetic models of colorectal cancer and identified cancer-associated-fibroblasts as drivers of a partial epithelial-to-mesenchymal transition in tumor cells via MAPK1 and MAPK14 signaling. Overall, the methods developed in this thesis provide a foundational computational framework for constructing "multi-scale" models of communication networks in multi-cellular tissues. / by Manu Prajapati Kumar. / 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 171-181). / Vaccination against infectious diseases has long been heralded as one of the greatest advancements in public health, yet its application to other clinical indications has fallen short of expectations. In this thesis, we apply engineering principles to develop more potent vaccines in the treatment of cancer and autoimmunity. Both major components of molecular vaccines, antigen and adjuvant, are independently explored as a part of this work. Our antigen studies sought to improve the delivery of peptide epitopes to lymphoid organs by fusing epitopes to inert protein carriers with defined pharmacokinetic properties. To promote anti-tumor immunity, we found that antigen carriers should 1) protect peptide cargo from proteolytic degradation, 2) be appropriately bulky to drain into the lymphatics, and 3) be rapidly cleared once in the blood to prevent tolerization at distal poorly inflamed organs. / Applying these principles, we identified transthyretin as an optimal delivery protein, and demonstrated efficacy against a number of clinically relevant antigens. Because our protein-epitope fusion approach is fully recombinant in nature, we were able to convert our protein vaccines into nucleic acid modalities, including plasmid DNA and self-replicating RNA, which are significantly easier and cheaper to manufacture at scale. Finally, we applied our learnings to purposefully induce tolerization in the treatment of autoimmunity, and found that albumin is a particularly efficacious antigen carrier protein for this application due to its extended half-life. On the adjuvant front, we attempted to engineer novel Toll-like receptor 3 (TLR3) agonists via yeast surface display. Although we successfully developed high affinity TLR3 binders, all tested clones failed to agonize TLR3 despite the utilization of several multimerization strategies. / Separately, in an effort to better understand adjuvant biology, we conducted a detailed mechanistic study of lipo-CpG, a particularly potent amphiphilic CpG variant previously developed by the Irvine lab. We uncovered a cascade of inflammatory signals originating from monocytes that facilitates the induction of high magnitude T cell responses, largely by acting in trans rather than directly on the antigen-presenting cell. Overall, these studies have elucidated a number of design principles that should aid in the engineering of next generation vaccines to better treat cancer and autoimmunity. / by Naveen K. Mehta. / 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 93-108). / Kinase inhibitors targeting the MAPK pathway are often limited by lack of durable clinical responses or, in many cancer types, lack of even initial responses. While great headway has been made on characterizing mechanisms of resistance, understanding the full influence of complex intercellular interactions on drug resistance remains a challenge. Here, we combine computation with experiment to investigate the cellular and molecular contributions of the tumor microenvironment to MAPK inhibitor response. First, we employ a computational framework using published bulk and single-cell patient gene expression data to investigate immune cell correlates of MAPK inhibitor resistance, and subsequently quantify potential intercellular ligand-receptor interactions between cell populations of interest. Next, we use multiplex proteomic immunoassays and co-culture experiments to characterize the impact of these interactions on tumor-intrinsic bypass signaling and phenotype. To assess the in vivo relevance of these multicellular and multidirectional signaling networks, we develop an intravital imaging strategy to monitor the influence of tumor-associated macrophages on cancer cell kinase activity dynamics. Finally, we rationally design a nanotherapy to exploit inhibitor-induced immunomodulation and crosstalk. Overall, we present a paradigm to systematically dissect signaling pathways between tumors and their microenvironments, validate these interactions in various models of disease, and design therapeutic strategies to target them. / by Stephanie Joy Wang. / 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. / Chimeric antigen receptor (CAR) T cells are a promising cancer therapeutic, as they can specifically redirect the cytotoxic function of a T cell to a chosen target of interest. CAR T cells have been successful in clinical trials against hematological cancers, but have experienced low efficacy against solid tumors for a number of reasons, including a paucity of tumor-specific antigens to target and a highly immunosuppressive solid tumor microenvironment. In chapter 2 of this thesis, we develop a strategy to target multiple solid tumor types through markers in their microenvironment. The use of single domain antibody (VHH)-based CAR T cells that recognize these markers circumvents the need for tumor-specific targets. Chapter 3 will describe methods to overcome the immunosuppressive microenvironment of solid tumors. Here, we have developed VHH-secreting CAR T cells that can modulate additional aspects of the tumor microenvironment, including the engagement of the innate immune system through secretion of a VHH against an inhibitor of phagocytosis. We show that this strategy of VHH-secretion by CAR T cells can lead to significant benefits in outcome. We also demonstrate that delivery of therapeutics by CAR T cells can improve the safety profile of the therapeutic. Chapter 4 of this thesis explores strategies to increase the targeting capacity of CAR T cells by building logic-gated CARs. Finally, chapter 5 will describe work in imaging CAR T cells specifically, longitudinally, and non-invasively through PET imaging. Our results demonstrate the flexibility of VHHs in CAR T cell engineering and the potential of VHH-based CAR T cells to target the tumor microenvironment, modulate the tumor microenvironment, and treat solid tumors. / by Yushu Joy Xie. / Ph. D. / Ph.D. Massachusetts Institute of Technology, Department of Biological Engineering

Thesium L. (Santalales: Santalaceae) is a large (360 species) genus of hemiparasitic perennial or annual species with a mainly Old-World distribution and a greatest concentration in southern Africa (ca. 186 species). Although Thesium is a major component of southern African flora, it often goes unnoticed and is poorly studied. The last revision of the entire genus was done by De Candolle in 1857. South African Thesium was last revised by Hill almost a century ago. Since Hill's revision, the number of collections have grown, and 49 new species have been described. Currently, no comprehensive Thesium taxonomic key exists, and species delimitation remains difficult due to a high variation in character states, rendering the genus in need of major revision. Within southern Africa, ca. 103 species occur in the Greater Cape Floristic Region (GCFR), of which about 72 are regional endemics. The GCFR Thesium, including ecologicalspecialists and generalists, offers an appropriate system for evaluating both the correlates of range extent, specialisation and the relative extinction risks associated with both ecological strategies. Here, it is predicted that a combination of edaphic, elevation and climate variables influence the geographic range of Thesium in the GCFR. Recent phylogenetic hypotheses revealed that Thesium is paraphyletic with respect to Austroamericium, Chrysothesium, Kunkeliella and Thesidium, suggesting the need for generic realignment. In addition, existing subgeneric and sectional classifications of this large genus lack a phylogenetic basis, thus compromising their predictive value. Using an expanded taxon sampling and a combination of nuclear (ITS) and chloroplast (matK, rpl32- trnL and trnL-F) DNA sequence data, chapter two re-assesses the phylogenetic relationships of Thesium and uses these as the basis of a new subgeneric classification of the genus. The phylogeny obtained confirms the need to place the four segregate genera into synonymy, resulting in a monophyletic Thesium. In addition, it resolves five, well-supported major clades within Thesiumwhich I recognize as subgenera. The South African endemic subgenus Hagnothesium is sister to the Eurasian subgenus Thesium (including Thesium, Kunkeliella and Mauritanica). The subgenus Psilothesium, occurring in tropical South America (formerly genus Austroamericium) and tropical Africa, is sister to the rest of the subgenera, which are all confined to South Africa. Within the latter, the subgenus Discothesium consists of subtropical and temperate species, whereas subgenus Frisea, comprising previously recognized sections Annulata, Barbata Frisea, Imberbia and Penicillata, is restricted to the GCFR. To facilitate identification of subgenera, I present identification keys, assigned species, provide brief diagnoses, identified ancestral morphological characters and, supply distribution and ecological data. Thesium subgenus Hagnothesium is endemic to the GCFR. In the past, there has been a propensity in revisionarywork ofthe subgenus Hagnothesium to split taxa into distinctspecies or vice-versa. Consequently, 15 different names exist although only six are accepted formally. Following recent molecular phylogenetic studies, the monophyly of the subgenus Hagnothesium is now well-established, but the circumscription of species within the section remains problematic given the complicated nomenclatural history which has added further confusion. Chapter three presents a revision of subgenus Hagnothesium using a total evidence approach to propose a modern taxonomy. I studied both herbarium collections and plants in their natural populations to circumscribe species boundaries, geographical ranges and estimates of their conservation status. Species of the subgenus Hagnothesium are dioecious, generally having four- merous, campanulate flowers, spikes borne in bract axils and arranged along the length of branchlets, with valvate perianth lobes and a short to absent hypanthial tube. The following eight species were recognized, of which one is here described as new: T. fragile L.f., T. fruticulosum (A.W.Hill) J.C.Manning & F.Forest, T. hirtum (Sond.) Zhigila, Verboom & Muasya comb. nov., T. leptostachyum A.DC., T. longicaule Zhigila, Verboom & Muasya nom. nov., T. microcarpum A.DC., T. minus (A.W.Hill)J.C.Manning & F.Forest and T. quartzicolum Zhigila, Verboom & Muasya sp. nov. I provide updated taxonomic keys, species descriptions, illustrations, distribution maps, new combinations, synonyms, and notes on the red list status for each species. In addition, six new species of Thesium endemic to the GCFR (but not included in subgenus Hagnothesium) are described and illustrated in chapter four. These are: Thesium aspermontanum Zhigila, Verboom & Muasya sp. nov., T. dmmagiae Zhigila, Verboom & Muasya sp. nov., T. neoprostratum Zhigila, Verboom & Muasya sp. nov., T. nigroperianthum Zhigila, Verboom & Muasya sp. nov., T. rhizomatum Zhigila, Verboom & Muasya sp. nov., and T. stirtonii sp. nov. Also, Thesium assimile var. pallidum is elevated to species rank as T. sawae Zhigila, Verboom & Muasya stat. nov. Morphological and ecological differences between species, along with their putative affinities, preliminary conservation status, phenology, etymology and distributional maps are presented. Narrow-ranged species are expected to be more at greater risk of extinction than generalists due to climate change. Such risk is greatest in biodiversity hotspots such as the GCFR, which house both ecological specialists and generalists. It was hypothesized that range size, ecological specialization and consequent climatically-modulated extinction-risk are all phylogenetic structured, such that climate change will precipitate a disproportionate loss of phylogenetic diversity. Past and future species distribution ranges were developed using MaxEnt models based on present-day occurrences and environmental conditions. There was a strong positive correlation between the ecological niche breadth of species, as determined by large-scale environmental variables, and their range extents. One hundred and one Thesium species were modelled, of which 71 species (83%) were predicted to have had broad range sizes during the Last Glacial Maxima, and 27 species (17%) recorded range contractions historically to the present. Similarly, 45 species (44%) will potentially expand their ranges, while 51 species (50%) are predicted to reduce their ranges in the future. Of the 65 species currently ranked as Least Concern or Data Deficient in the South African Red list, 24 species will likely shift into higher extinction risk categories. Interestingly, five ecological specialists (5%), although having experienced a range reduction from the LGM to the present, are predicted to persist in the face of future climate change. However, the range extent, ecological specialisation and extinction risk are phylogenetically random and therefore should have a negligible impact on the phylogenetic diversity of the GCFR Thesium. Overall, this study confirms the monophyly of the genus Thesium and sets its infrageneric classification scheme in place. The context of this classification framework allows the systematic revision of the genus, one clade at a time. Towards this goal, I revised the Hagnothesium clade and additionally described six new species from other clades. The climate, elevation and soil variables influence the distribution range and specialism of GCFR Thesium clades. However, ecological specialism of species and extinction risks were predicted to be phylogenetically random.

Pinniped focused tourism has grown rapidly both globally and in South Africa. In a 2002 survey South Africa was identified as having earned the most revenue from seal ecotourism of all the countries that engage in this activity. Seal tourism includes approaching colonies on foot, by kayak, on large and small motorised boats and more recently in the water through snorkelling and scuba diving activities. Like most wildlife tourism seal snorkelling operators can use their tours to educate clients about seal biology and threats, while providing them with a memorable physical experience with minimal impact on the seals. The goal of this research was to explore aspects of the demography, attitudes and values of tourists participating in two different seal viewing activities at the same island. Additionally, I investigated levels of satisfaction with each tour type, differences in the style of education provided by operators and which tour provided tourists and what facts tourists found most interesting. In the second part of the study I quantified the behavioural response of seals to tourists who entered the water to snorkel with seals in an attempt to assess potential impacts of immersive trips on seal behaviour. The study was conducted at Duiker Island, near Hout Bay in the city of Cape Town between November 2019 and January 2020. This period coincides with the breeding season of Cape fur seals (Arctocephalus pusillus pusillus) and the peak tourist season. Questionnaires were distributed to both seal viewing tourists (n = 53) who had viewed seals from a large boat and seal snorkelling participants who had swum with seals at the island (n = 90). More women than men undertook both types of tour, though the proportion of men increased on snorkelling compared to viewing tours. Probit models revealed key behavioural and attitudinal differences between seal viewing and seal snorkelling participants with the latter being more pro-environmental. Seal snorkelling guides used a more interpretive style when educating tourists including the use of visual aids while seal viewing tours announced facts through a loudspeaker on the vessel. Seal snorkelling guides would also include information to awareness about plastic pollution and seal entanglement, with a donation box for a seal disentanglement program run by the Two Oceans Aquarium. Seal snorkelling participants listed an average of two facts they had learnt on the tour compared to a mean of 1.24 facts for seal viewing tours. Both tours were rated very highly for overall levels of satisfaction (seal snorkelling mean score = 9.17/10; seal viewing = 8.58/100). Surface observations of seal behaviour in response to seal snorkelers in the water close to the boat suggested a minimal impact with most seals (88%) behaving neutrally and only 0.2 % engaged in avoidance behaviour. Below water observations revealed that seal numbers declined with increasing number of people in the water and seals adjusted both their position in the water column (more diving) and their activity (more active) in response to snorkeler presence. These findings suggest that both the number of snorkelers and the area over which they spread should be controlled so that seals can choose to avoid snorkelers and behavioural changes are localised to select demarcated areas. The presence of the guides in the water together with the tourists ensured there were no inappropriate interactions between seals and snorkelers (e.g. touching and biting by seals in response). This is the first study on the thriving seal ecotourism industry at Duiker Island and reveals high levels of satisfaction by both seal viewing and seal snorkelling tourists. While there were measurable impacts of seal snorkelers on seals these were highly localised and unlikely to present an adverse impact on seals at the Island more generally. Controlling the number of operators and ensuring snorkelers are always accompanied by guides should ensure that this valuable business continues, potential impacts are minimised and negative interactions (e,g. bites from seals or people touching or feeding seals) are prevented.

This thesis investigates the evolution of Pleistocene insular proboscideans from the centralwestern Mediterranean (Palaeoloxodon species from Sicily, Malta, Favignana) and a mammoth (Mammuthus lamarmorai) from Sardinia, with a particular emphasis on the anatomy of the limbs. Differences in the morphology of the limbs are examined across a tenfold reduction in mass (from 3,5m-tall P. antiquus from Germany to 1,2 m-tall P. ex gr. P. falconeri from Spinagallo Cave, Sicily), revealing insights into significant morphological changes in the long and foot-bones, particularly appendicular changes evident in SiculoMaltese P. ex gr. P. falconeri. Notable morphological differences between P. antiquus and its insular descendent P. ex gr. P. falconeri include the functional morphology of the ankle-joint (especially the calcaneus' articular facet for the tibia). Furthermore, morphological similarities found between the femur of young continental elephants (P. antiquus and L. africana) and adult insular dwarfs (P. ex gr. P. falconeri and its probable ancestor Palaeoloxodon sp. from Lparello Fissure, Sicily) suggest evidence of paedomorphism in the limbs. Similarly, comparisons of the ontogenetic allometry of the tibia in L. africana and P. ex gr. P. falconceri include changes which are also consistent with paedomorphism, although other factors could not be ruled out. In the humerus large differences are evident in the morphology of the deltoid tubercule between co-generic insular Palaeoloxodon species, suggesting interspecific differences in the musculo-skeletal system. Furthermore, on the basis of dimensions, morphology and stratigraphy, the large Palaeoloxodon sp. remains from Luparello Fissure, north-western Sicily are suggested to belong to the ancestral chronospecies of P. ex gr. P. falconeri from Sicily, which may have subsequently colonized Malta during the reduced sea-levels of a Middle Pleistocene glacial lowstand (following a corridor with reduced distances between the two islands). Additionally, morphological differences in the calcanei of elephants from Luparello Fissure, Sicily, and Benghisa Gap, Malta may be the result of allopatric speciation between similar-sized elephants during the Middle Pleistocene, or alternatively relate to ecomorphology. These findings suggest that the morphology of the calcaneus may be more informative than hitherto recognised for resolving systematics and taxonomy among the Elephantini. Furthermore, although the absolute chronology of SiculoMaltese elephants remains poorly constrained, preliminary U-Th dating at Alcamo Quarry, western Sicily suggests a tentative early Middle Pleistocene age for Palaeoloxodon sp.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, May, 2020 / Cataloged from student-submitted PDF version of thesis. / Includes bibliographical references (pages 111-130). / Much of the diversity we observe in cellular and organismal phenotypes can be attributed to epigenetic and genetic variation. DNA provides the instructions for life, while epigenetic modifications regulate which parts of the genetic information contained in DNA can be read out in a given cell and how this information is interpreted. In recent years, epigenetic and genetic variation has been profiled on a large scale with sequencing-based assays, generating many datasets to be explored. In this thesis, I present three projects which apply computational techniques to identify and characterize epigenetic mechanisms that may contribute to the regulation of phenotypic variance. First, we mine a dataset charactering the epigenomes of diverse cell types in order to discover signatures of adult stem cell differentiation. / We identify a novel marker of the multipotent state, a chromatin state characterized by the histone marks H3K36me3 and H3K9me3, and describe biological processes that may be linked to the loss of this chromatin state in fully differentiated cell types. Next, I present what we learned from profiling the epigenetic state of cells before and after transplantation into Xenopus oocytes, a process that transcriptionally reprograms the cells. This analysis elucidates how the initial epigenetic state of a cell influences the success of cellular reprogramming and identifies transcription factors that help regulate this process. Finally, we integrate studies measuring the effects of genetic variants on disease with studies measuring the effects of genetic variants on transcriptional and epigenetic activity. This identifies specific mechanisms underlying disease processes, and demonstrates that transcriptional and epigenetic mechanisms may independently contribute to disease pathogenesis. / Together, these projects demonstrate the biological insights that can be gained from epigenetic profiling, and expand our understanding of the potential effects of epigenetic modifications. / by Khoi Thien Nguyen. / Ph. D. / Ph.D. Massachusetts Institute of Technology, Department of Biological Engineering

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, May, 2020 / Cataloged from student-submitted PDF version of thesis. / Includes bibliographical references. / The human gastrointestinal tract is home to a dense and dynamic microbial community. The composition and metabolic output of the human gut microbiota have been implicated in many diseases: from inflammatory bowel disease, colorectal cancer, and diarrheal diseases to metabolic syndromes like diabetes. Treatment of these diseases will likely require targeted therapeutic interventions aimed at modulating the abundance and metabolism of specific commensal microbial species or probiotics. A promising avenue for such interventions is through diet, where the dietary components act as substrates for the species producing beneficial metabolites one wishes to enrich. In this thesis, I focus on a dietary intervention study in healthy individuals. Since the human gut microbiota is known for its highly heterogeneous composition across different individuals, it comes as no surprise that a more personalized approach is preeminent. / We first test effects of multiple micronutrients spiked into a fixed diet. Using a highly controlled diet within the cohort, we identify strong and predictable responses of specific microbes across participants consuming prebiotic spike-ins. However, select macronutrient spike-ins like unsaturated or saturated fat and protein, produce no predictable response. We next investigate prebiotic supplement in diet further as well as its downstream products, short chain fatty acids, in the digestive tract. We look to alleviate the stress of a highly controlled, low complexity diet on participants by testing the effect of different prebiotics simultaneously ex vivo. We show that individuals vary in their microbial metabolic phenotypes (as in they produce different quantities and proportions of short chain fatty acids from the same prebiotic inputs) mirroring differences in their microbiota composition. / Finally, we run a pilot study to elucidate how closely our ex vivo experiment results may reflect the in vivo changes following a short-term dietary fiber supplementation. In addition to obtaining preliminary data on this direct comparison, we also explore different parameters for generating high-throughput data on personalized dietary interventions. Together, these projects provide the framework for building a predicative model for the effect that prebiotic dietary supplementation will have on gut microbiota's composition. Such a prediction model would be equally helpful in both enhancing individuals' gut health and improving gut dysbiosis in cases of disease. / by Le Thanh Tu Nguyen. / Ph. D. / Ph.D. Massachusetts Institute of Technology, Department of Biological Engineering

Clam populations globally have declined, or been depleted, with one of the major causes being uncontrolled human exploitation. This thesis investigates the population structure, growth, reproduction and exploitation of the beaked clam Eumarcia paupercula in Maputo Bay. The substantial commercial harvesting of this clam may lead to overexploitation of the resource. In this regard, some fundamental knowledge is necessary for the management of future exploitation. The data collection was based on an 18-months (November 2012 - April 2014) sampling for population structure across a tidal flat. The growth analysis was performed on FiSAT II, using mark-recapture experiments and length-frequency data. Monthly reproduction analysis was based on the fluctuation of body weight and gonad smear analysis. Furthermore, interviews were used to ascertain the importance of the resources for collectors and estimate landings of E. paupercula. The von Bertalanffy growth function and length-frequency analysis revealed that E. paupercula has a fast growth rate and a short life span. Eumarcia paupercula is a year-round spawner with higher peaks in the summer; recruitment follows a similar pattern, occurring over the year and after the spawning peaks. The clam collectors, the majority of whom are women, have experienced an increase in the effort required to collect clams, resulting in a decline of catches over the season. This study highlights that temporal population dynamics are influenced by collection and reproductive patterns, and that single environmental parameters do not explain the patterns of growth, reproductive cycles and spatial distribution. Findings of this study have relevance and application for the livelihood of the collectors, as well as the sustainability of the Eumarcia paupercula stock, by providing a basis for fishery governance. While recommendations are presented for the management of E. paupercula collection, the Ministry of Fishery in Mozambique also has to consider collecting data on bivalves, as they are an important source of income for artisanal fishers. Future research should include monitoring of a less exploited population, so as to understand better how collection impacts on the population dynamics of E. paupercula. Furthermore, laboratory studies of the larval cycle are necessary to gain thorough understanding of the species life cycle.