VictorJuiz/thesis huggingface dataset:
Structure-guided engineering of CD47 and stem cell factor for immunotherapeutic applications [electronic resource]

Protein biologics have revolutionized the clinical landscape in recent decades. In particular, abundant attention and momentum has been generated in the immunotherapy space, which directs one's immune system to combat diseases. Protein immunotherapeutics are currently evolving at a rapid pace as a result of growing efforts into applying protein-engineering strategies to improve their therapeutic index. In this dissertation, two independent immunoregulatory ligands, CD47 and stem cell factor (SCF), were engineered using structure-guided approaches for immunotherapeutic applications. CD47/SIRPα (Signal regulatory protein α) immunoregulatory pathway is a key inhibitor of macrophage effector function (i.e. phagocytosis) in homeostatic cell clearance and in immune evasion by cancer cells. Due to ubiquitous CD47 expression, current CD47-targeted cancer therapies are handicapped by large antigen sinks in vivo that could reduce bioavailability and increase the risk of toxicity. We engineered a high-affinity variant of human CD47 (Velcro-CD47) as a competitive antagonist of SIRPα, which has restricted tissue expression compared to CD47. To do so, we developed a novel approach that we termed 'Velcro' engineering, which enhances protein-protein interaction by a 'molecular velcro'. Specifically targeting a subset of myeloid cells in human blood ex vivo, Velcro-CD47 enhances tumor-specific antibody mediated antibody-dependent cellular phagocytosis (ADCP) of tumor cells in vitro, demonstrating its potential therapeutic use as an adjuvant to antibody therapy for cancer. On the other hand, SCF is a homodimeric growth factor that dimerizes c-Kit receptors to elicit therapeutic expansion of hematopoietic progenitors, but is toxic in vivo because it concurrently activates mast cells. We modified the SCF ligand in order to 'cripple' c-Kit dimerization efficiency and truncate downstream signaling as a strategy to decouple SCF efficacy and toxicity. We engineered a high-affinity monomeric SCF variant that behaves as a partial agonist of c-Kit and shows selectivity for hematopoietic progenitors over mast cells in vitro. Tests in pre-clinical mouse models of SCF-mediated anaphylaxis, radioprotection, and cancer therapy revealed that our SCF variant exhibits therapeutic efficacy in vivo with virtually no anaphylactic effects, thus unlocking SCF's clinical potential. This approach has broad applications in dimeric receptor-ligand systems. Collectively, the work presented in this dissertation has generated two potential immunotherapeutic leads that encourage further preclinical and clinical investigations. In addition, it has expanded the repertoire of protein-engineering strategies to create novel protein biologics with potentially improved therapeutic value.

Keywords: Bioengineering.
Advisor: Garcia K. Christopher
Weissman Irving
Cochran Jennifer R. advisor. Thesis advisor
Shizuru Judith Anne advisor. Thesis advisor
Structuring peer interactions for massive scale learning [electronic resource]

Massive open online classes (MOOCs) offer an opportunity to dramatically broaden access to education. However, dramatically broadened access also creates challenges. Classes enroll tens of thousands of students, all of whom participate remotely and asynchronously based on their own schedule. This large, asynchronous and remote access in turn makes it challenging to scale effective teaching techniques that rely on personal interactions between teacher and student, such as open-ended assessment and discussion, and rapid formative feedback. This dissertation brings the benefits of effective teaching techniques to massive online classes, by introducing computational systems that replace hard-to-scale teacher-student interactions with peer interactions. Because peer interactions rely on interactions between students, they can potentially scale to any classroom size. In this dissertation first, I first study the causal mechanisms that lead to the learning benefits of classroom techniques like feedback and discussion. Then, I introduce interfaces that combine these operative mechanisms with the properties of online classes, such as mediated communication and the large number of students. This dissertation develops these ideas through two large-scale systems, PeerStudio and Talkabout, which target fast, revision-oriented feedback, and global-scale student discussions, respectively. This dissertation also includes the first large-scale evaluation of a global peer-assessment system. PeerStudio uses the temporal overlap in student schedules at large scale so that students receive fast, revision oriented feedback from classmates at any time of day. Talkabout leverages the globally distributed student participation to create discussions where students speak with peers with diverse experience and viewpoints. Controlled experiments show both systems improve both students' learning experience and their grades. These systems, and the large-scale evaluations that led to their design, point to a future in which classrooms rely on the collective experiences of their students, and students around the world have access to education that is as effective as it is accessible.

Advisor: Bernstein Michael S.
Klemmer Scott primary advisor. Thesis advisor
McFarland Daniel (Daniel A.) advisor. Thesis advisor
Studies of skeletal muscle satellite cell death and renewal [electronic resource]

Skeletal muscle is an abundant tissue, accounting for 42% of the body mass of the average adult male human, and is required to generate the force necessary for movement and posture. The regeneration of skeletal muscle tissue in mammals is accomplished by a rare population of muscle-resident adult stem cells known as satellite cells. These satellite cells primarily reside in a dormant state and are prompted to carry-out their regenerative function by an injury to the neighboring muscle tissue. Atrophy and impaired regeneration of skeletal muscle are significant comorbidities of aging and chronic illness. This dissertation describes and interprets studies of skeletal muscle stem cell biology with particular attention paid to molecular mechanisms that influence stem cell-fate during regeneration in healthy and diseased contexts. The hypothesis that changes in stem cell regenerative potential underlie the poor tissue repair observed in aged animals is explored through comparisons of young and old cells. These studies identify a unique mitotic cell death phenomenon that occurs specifically within old cells as a consequence of age-associated changes in the satellite cell environment. In additional analyses, broad characterizations of gene expression in human satellite cells are leveraged to develop pharmacologic methods for controlling cell-fate to enhance the regenerative potential of purified cells. These analyses also provide insight into human-specific mechanisms of satellite cell-mediated myogenesis. The final series of studies uses satellite cells as a model system to examine cell-fate determination during stem cell divisions that give rise to daughter cells with divergent fates. These so-called asymmetric cell divisions are characterized specifically in terms of the unique patterns of chromosome segregation observed to coincide with regulation of muscle stem cell fate.

Keywords: Developmental Biology.
Advisor: Rando Thomas A.
Kim Seung K. advisor. Thesis advisor
Nusse Roel
Studies toward improved RNA detection and isolation [electronic resource]

As our understanding of molecular biology continues to develop, both general improvements and specialized tools are needed for the progression of RNA detection and analysis. This thesis describes three distinct projects within the broad goal of expanding the techniques available for better interrogation of RNA. Chapter II illustrates the development of a new technique for microRNA detection. MicroRNAs are a class of small regulatory RNAs present in eukaryotes. MicroRNAs pose a challenge for detection because of their small size and sequence homology, but have drawn increasing interest due the predicted prevalence of their gene targets and links between their expression levels and cancer. The development of an isothermal detection strategy is described which incorporates two nucleic acid templated steps, two amplification steps, and fluorescent detection. Up to 3000-fold amplification can be obtained, and 200 pM quantities of let-7a microRNA and above can be detected reliably. In Chapter III, two distinct approaches are taken for the detection of N(6)-methyladenosine, the most common modification in RNA: harnessing base-pairing selectivity, and developing selective reactions. A successful method was developed that harnessed the inherently higher efficiency of a commercially-available polymerase, Tth DNA polymerase, when pairing T with adenosine as opposed to N(6)-methyladenosine. Kinetic analysis showed that the selectivity of the enzyme ranged from 4-18-fold, depending on sequence context. Detection of m6A in ribosomal RNA and in a highly-expressed messenger RNA are demonstrated. The final chapter focuses on a mechanistic approach for enhancing RNA recovery after formaldehyde treatment using an organocatalyst. Mass spectrometry and gel electrophoresis showed an enhanced rate of adduct reversal in the presence of the catalyst, and effects of temperature, buffer, and pH were also analyzed. The results from monomeric and oligomeric test systems have subsequently been applied to recovery of RNA from fixed samples, showing ~2-fold enhancement in recovered RNA due to the catalyst and up to 25-fold enhancement when the catalyst was used in conjunction with optimized time and temperature.

Keywords: Chemistry.
Advisor: Kool Eric T. primary advisor. Thesis advisor
Cimprich Karlene advisor. Thesis advisor
Khosla Chaitan
A study of input and output field quantification in heavy civil construction [electronic resource]

When I began writing this thesis, it was two years after I had completed my time as a Laborer. Many of my ideas formed during my time as a field engineer at the Granite Heavy Construction Division when I was only a year out of the Laborers. The early drafts of this thesis read like it was written by someone that had never written more than three pages at any one time -- and nobody had ever read those three pages and provided feedback. After numerous revisions, while I may have improved the writing style I began to realize that the improvement is at the cost of losing the core knowledge the thesis contains. So, for the most part, this thesis contains an understanding of construction from the mind of a laborer. In this thesis, I convey the methods of measuring the status of a construction project. In the case of this thesis, I focus on the vertically integrated project style typical of heavy construction projects though the methods I present are general to other construction types such as buildings. The basic block of construction is the activity. The difference between two activity status points is equal to the project progress. The progress is a fundamental metric of success that is as old as the construction profession. Without knowing the progress of activities, it is not possible to predict the expected sequence and duration of future activities. This means it is not possible to assemble the activity aspects of workspace, labor, material, and equipment to converge at the right time and place to achieve a safe activity that has a low risk of neither damaging the environment nor impacting the surrounding community. The methodology I used as to study the methods of measuring status is a mixed approach. At the core, I used an ethnography to form my understanding from a first person perspective. I then compared my firsthand knowledge with publications to fill the gaps in my knowledge. The limitation is the small observation sample characteristic of the ethnography. I had remaining observations that I had not found in the published knowledge. To resolve these I used a questionnaire survey and interviews of experts to understand the relation of these outliers. I found that activity progress status is formed by two components -- these are the status and the context of the activity. The two are inseparable. Within those two topics, I found numerous approaches of estimating and measuring status and context. Further, I found sources of errors in the status and context measurements. The significance of my findings is rooted in first the lack of an existing unified source of this knowledge and second I provide a complete knowledge within the domain of heavy construction, particularly for the vertically integrated project organization.

Keywords: Civil and Environmental Engineering.
Advisor: Fischer Martin
Studying Myotonic Dystrophy Pathogenesis using Engineered Patient Derived Fibroblasts

Myotonic dystrophy type 1 (DM1), a neuromuscular degenerative disorder, is
characterized by a CTG repeat expansion (>100 CTGs) in the 3’ untranslated region of the
Dystrophia myotonica protein kinase (DMPK) gene. The leading hypothesis regarding DM1
pathology is that this expansion leads to the sequestration of splicing factors, which is
responsible for aberrant splicing events in insulin receptor gene (INSR), Cardiac troponin T
(Tnnt2), and DMPK itself, resulting in severe muscular degeneration at the clinical level.
However, studying genetic diseases such as DM1 has historically been complicated by the
inherent genetic bias between cells from different sources, a complication that can now be
removed by using gene targeting technologies. The purpose of this research is to generate a
genetically unbiased model to study myotonic dystrophy’s pathology by targeting the expanded
CTG repeat in the DMPK locus in patient-derived fibroblasts. A set of engineered nucleases,
transcription activator-like effector nucleases (TALENS) and RNA-guided nucleases
(CRISPRs), were constructed to target the CTG expansion. To repair the CTG expansion, donor
DNA with arms of homology to the DMPK gene and a normal number of CTG repeats were
introduced to serve as a template for correction via homologous recombination. By correcting
this expanded CTG repeat through gene targeting, aberrant alternative splicing events caused by
the sequestering of splicing factors at the expanded repeat can be further studied by analyzing the
mRNA isoforms of genes known to be affected by the expanded repeat, such as INSR, Tnnt2,
and DMPK. Creating this genetically unbiased model of DM can provide insight into what other
genes are involved in the pathology of the disease, providing an invaluable tool in developing
potential therapeutics for this disease.

Keywords:  Department of Biology
Myotonic dystrophy
Genetic Engineering
Gene targeting
CRISPRS
TALENs
CTG Repeat Expansion
DMPK
Thesis
Advisor: Porteus Matthew
Hanawalt Philip
Supply chain management through cascading [electronic resource]

Supply chain disruptions affect companies across all industries. For highly specific products, such as semiconductors, contingency planning is necessary in advance. This is aggravated by supply chain invisibility as a result of a multi-tier supply chain structure. In this research, we identify how supply chain performance can be improved in the presence of disruption risk and limited visibility. By analyzing stylized supply chain models, we identify possible reasons for the particular inefficiencies observed in semiconductor supply chains. We investigate the optimal capacity decisions of different players in a supply chain and the effects of multiple emergency sources. We then explore what type of contracts can be used to coordinate multi-tier supply chains. Further, we introduce the concept of incentive cascading and analyze whether cascading can be used to coordinate multi-tier supply chains.

Keywords: Management Science and Engineering.
Advisor: Erhun Feryal primary advisor. Thesis advisor
Lee Hau Leung primary advisor. Thesis advisor
Hausman Warren H. advisor. Thesis advisor
Surrogate modeling and active subspaces for efficient optimization of supersonic aircraft [electronic resource]

This dissertation presents approaches for surrogate based optimization of supersonic vehicles analyzed with high fidelity flow simulations. It integrates several developments in surrogate modeling to enable a robust regression procedure in the presence of sparse data and inaccurate gradients. A series of hyperparameter constraints are developed which encourage the learning process to generate a physically representative fit of the data. It identifies the existence of subspaces based on linear combinations of inputs called "active subspaces" that reasonably model the behavior of objectives within aerospace design problems such as lift coefficient, drag coefficient and an equivalent area functional. Coherent physical features were found across several design problems for both two and three dimensional geometries. This dissertation further proposes an approach for adaptive refinement by conditioning the traditional expected improvement sampling criterion to avoid exploration of the design bounds. To begin work on applying active subspaces to optimization, inverse maps were developed to enable the linking of separate active subspaces for objectives and constraints, enabling surrogate based optimization in high dimension. Several design problems are explored, and it is shown that surrogate based optimization in active subspaces could enable the optimization of problems otherwise intractable via gradient based optimization alone.

Keywords: Aeronautics and Astronautics.
Advisor: Alonso Juan José
Constantine Paul G.
Kochenderfer Mykel J.
Kroo Ilan advisor. Thesis advisor
Synthetic biochemical production of medicinal natural products in yeast [electronic resource]

Natural products, organic molecules made by Nature, are the principal source of and inspiration for pharmaceuticals. Because of their complex regio- and stereo-chemistry, many natural products are most economically made by cultivating the producer. Plant natural products from opium poppy, which include morphine and codeine, are produced in this way. Agricultural supply of drugs and drug precursors from opium poppy is an annual process with a large demand on land, subject to high variability due to weather, pests, soil conditions, and natural variation, and requiring intensive mechanical and chemical processing for product isolation. Therefore, there is a need for alternative processes to provide opiates with greater supply stability and less resource use. Heterologous microbial biosynthesis has been used successfully to make plant natural products, most notably the sesquiterpene artemisinic acid, and could provide a more stable supply of opiates due to greater process control and shorter batch times. The objective of this research was to design and build yeast strains to biosynthesize two classes of plant natural products found in opium poppy, morphinan and protoberberine alkaloids, using a synthetic biology framework. One challenge to implementing these pathways is the decrease in titers observed with increasing pathway length, which must be tackled with strain engineering efforts. Another challenge was that a key enzyme in morphine biosynthesis was not yet discovered. Both target classes of alkaloids were successfully accessed. Using metabolic engineering approaches, we optimized a yeast strain to convert a commercially available substrate to protoberberine alkaloids. This strain provided a 70-fold increase in titer relative to its predecessor. We developed yeast strains to convert either sugar or a commercially available substrate to morphinan alkaloids, which required the discovery of a novel epimerase from opium poppy. We co-discovered this missing enzyme and demonstrated its activity in the context of our engineered yeast strain. Rational protein engineering of a plant cytochrome P450 enzyme increased product titers. The engineered yeast strains are able to biosynthesize the morphinan alkaloid thebaine and the opiate-derived semi-synthetic drug hydrocodone de novo at proof-of-principle levels. Further protein and strain engineering will lead to improved strains for microbial biosynthesis of opium poppy alkaloids and their congeners.

Keywords: Chemistry.
Advisor: Smolke Christina D. primary advisor. Thesis advisor
Kool Eric T. advisor. Thesis advisor
Sattely Elizabeth advisor. Thesis advisor
Systematic discovery of human gene function and principles of modular organization through phylogenetic profiling [electronic resource]

A significant fraction of human genes remain functionally uncharacterized despite major advances in perturbation and mapping techniques. The correlated gain and loss of orthologous genes across species, reflecting a shared evolutionary history, can be used to infer novel functional interactions in a technique known as phylogenetic profiling. Advances in sequencing technology and plummeting costs have enabled the sequencing of hundreds of eukaryotic genomes, hugely increasing the potential utility of phylogenetic profiling in providing systematic functional predictions for the human genome. However, genome-wide profiling has been hindered by challenges in orthology assignment caused by the large number of human genes related to each other through duplication events and the lack of robust scalable methods to assess the degree of coevolution. I overcame these challenges in automatically profiling over 30,000 groups of homologous human genes (orthogroups) representing the entire protein-coding genome across 177 eukaryotic species. By generating a full pairwise orthogroup phylogenetic co-occurrence matrix, I derived unbiased genome-wide predictions of functional modules and identified topological constraints on evolutionary modularity, with metabolic genes and physical protein complexes more likely to coevolve than signaling and transcriptional networks. Importantly, the algorithm predicts functions for hundreds of poorly characterized genes. To demonstrate its utility in guiding experimental gene function discovery, I validated two subsets of candidates experimentally for their predicted link to the actin-nucleating WASH complex and cilia/basal body function.

Keywords: Chemical and Systems Biology.
Advisor: Meyer Tobias primary advisor. Thesis advisor
Ferrell James Ellsworth advisor. Thesis advisor
Skotheim Jan
Wandless Thomas advisor. Thesis advisor