VictorJuiz/thesis huggingface dataset:
Investigating Dendritic Cell Subset Changes in Response to Irradiation of Induced and Transplanted Mouse Melanomas

The treatment of melanoma has seen significant advances recently with the introduction of immunotherapies such as immune checkpoint inhibitors or dendritic cell vaccines. Dendritic cells (DCs) are crucial for successful immunotherapy due to their ability to initiate and modulate T-cell responses. Current research suggests that combining immunotherapies with targeted radiotherapy could be highly effective treatment strategies for melanoma. However, recent research has also indicated that ionizing radiation causes immunosuppression by disproportionately inducing cell death in immunogenic dendritic cell subsets that generate an immune response. This effect was not observed in tolerogenic dendritic cell subsets, which produce immunological tolerance. This project extended the existing knowledge by examining the effect of targeted radiotherapy on DC subsets localized in the tumor microenvironment. Our ultimate goal is to determine whether radiotherapy can impair or amplify DC function. To achieve that goal, DC subset numbers were evaluated using flow cytometry at different time points after targeted radiotherapy in two tumor mouse models, i.e., a transplantation tumor model and a genetically engineered mouse model. The genetically engineered mouse model can be chemically induced to develop melanomas, allowing for the exploration of melanomas developed from the mouse’s own cells. Our results show a transient decrease in the overall number of leukocytes. Interestingly, the number of immunogenic DCs, but not tolerogenic DCs, was rapidly decreased after radiotherapy but return to normal levels after 3 days. These results were consistent both in the transplantable model and in the genetically engineered mouse model, suggesting that both models could be used for subsequent studies combining radiotherapy and immunotherapy strategies. Importantly, our results suggest that combinatory immunotherapeutic strategies should be administered at least 72 hrs after radiotherapy when immunogenic DC subsets numbers are back to normal levels. Altogether, our comprehensive characterization of immune responses and DC subsets during radiotherapy allows for the rational design of combinatory therapeutic strategies.

Keywords:  Department of Biology
biology
dendritic cells
radiotherapy
murine models
melanoma
immunotherapy
Thesis
Advisor: Idoyaga Juliana
Jones Patricia
Investigating Novel Super Enhancer Sequences for Cardiac Development

Cardiac development, physiology and function depend on the dynamic control of tissue specific gene expression by distant acting transcriptional enhancers. Super enhancers (SEs) are recently discovered enhancer clusters in the mammalian genome with unusually strong enrichment of various transcriptional co-activators (Young et al., 2013). Although recent studies suggested that the SEs are critical for development and disease mechanism, the role of cardiac-specific SEs is still unclear.
Here, I identified a potential SE that regulates the transcription of cardiac transcription factor, NKX2.5, and the tropomyosin-binding subunit of the troponin complex, TNNT2, during cardiomyocyte (CM) differentiation. I utilized the chemically defined monolayer method to generate beating CMs from human embryonic stem cells (hESCs). Chromatin immune-precipitation sequencing (ChIP-seq) and RNA sequencing (RNA-seq) of each stage of CM differentiation revealed the target super enhancer to be located upstream of the NKX2.5 gene. I implemented the CRISPR/Cas9 system to generate mutant hESC lines by deletion of target super enhancer (NKX2.5SE-/-). The expression of NKX2.5 and TNNT2 genes was severely decreased in NKX2.5SE-/- lines during CM differentiation, demonstrating the effect of the candidate SE in transcriptional regulation of key cardiac marker genes.
Collectively, our current results and further studies may help identify the potential mechanisms of crucial cardiac-specific SEs that regulate cardiac development through transcriptional regulation of key regulatory genes.

Keywords:  Department of Biology
Biology
Stem Cells
Cardiomyocytes
Super Enhancers
Epigenetics
Molecular Genetics
Thesis
Advisor: Wu Joseph
Luo Liqun
Investigating the biophysical mechanisms of substrate rigidity-induced hippo pathway regulation in human embryonic stem cells

Mechanobiology, the study of how cells sense and transduce mechanical information into chemical signals, has become an integral aspect of both basic and applied biological research over the past decade. Of particular interest to researchers is the Hippo signaling pathway, a mechanosensitive pathway that participates in diverse cellular processes ranging from embryonic development to cancer progression. The transcriptional coactivator YAP (yes-associated protein) is a central effector in the Hippo pathway. In embryonic stem cells, YAP activation promotes cell survival and pluripotency. YAP also directs cell fate in mouse embryos and mesenchymal stem cells in a manner that depends on both cell position within tissues and substrate rigidity. Despite YAP's central biological importance, the mechanisms underlying its regulation by mechanical inputs remain incompletely understood. We used human embryonic stem cells (hESCs) as a model system to determine how mechanical cues such as those that are present in mammalian embryos regulate YAP activity. To do so, we performed an unbiased proteomics screen to identify a Hippo pathway interactome, and found that the set of proteins interacting with YAP and its upstream kinase large tumor suppressor (Lats) was highly enriched for proteins associated with the actin cytoskeleton. Using engineered hydrogels with tunable stiffness, we observed that hESCs adopted drastically different colony morphologies, actin cytoskeleton organization, and YAP nuclear versus cytoplasmic localization on substrates of differing stiffnesses. Further investigation revealed that an intact actin cytoskeleton was required for the regulation of Hippo pathway activity as a function of cell position within hESC colonies, but that signaling downstream of RhoA and Rho-associated coiled-coil kinase (ROCK) was specifically required for Hippo pathway modulation in response to substrate stiffness. Contrary to results reported for other cell types, we found that both bundled F-actin and nonmuscle myosin II activity were dispensable for YAP mechanoregulation in hESCs. In addition, we found that YAP phosphorylation by Lats was required for the modulation of YAP activity in response to substrate stiffness. Lastly, we evaluated the biological relevance of YAP activation and found that YAP cytoplasmic sequestration in cells adhering to soft substrates did not induce changes in cell fate or cell proliferation. Instead, nuclear localization of YAP and its interaction with the transcription factor TEAD were crucial for hESC survival. A quantitative model relating substrate stiffness and ROCK activity to YAP phosphorylation and nuclear localization can account for our results and highlights the unique mechanisms that modulate Hippo pathway signaling in pluripotent cells.

Keywords: Chemical Engineering.
Advisor: Dunn Alexander Robert 
Fuller Margaret T.
Khosla Chaitan
Sebastiano Vittorio
Investigating the Effects of Ubiquitin Linkage Topology on Protein Stability

The ubiquitin proteasome system is the primary method that cells use to degrade proteins. Breakdown of this important regulatory pathway is implicated in neurodegenerative disorders, cancer, and other debilitating human diseases. The small molecule ubiquitin can be appended to its target substrate via free amino groups on lysine. Because ubiquitin itself has seven lysines, a chain of ubiquitin molecules can be linked together in thousands of different ways. Ubiquitin chains linked together at the lysine 48 site promote degradation of a tagged substrate via the 26S proteasome, whereas other linkage types, such as lysine 63 linked chains, do not promote degradation in this way. This study seeks to determine why lysine 48 linked ubiquitin chains promote degradation. One motivating hypothesis is that the chains themselves physically destabilize the tagged substrate, which makes it easier for the 26S proteasome to degrade the protein. In order to test this hypothesis, we created mutant ubiquitin monomers with only one lysine residue, rather than the seven lysine residues typically found in wild type ubiquitin. Using a protecting group strategy and in vitro biochemistry, we developed a system of stepwise ubiquitination that allowed us to create ubiquitin chains of variable linkage type and length. Lysine 48 linked chains are only pro-degradation when at least four molecules are linked together, and we were only able to create linkages of one, two, and three molecules so our hypothesis could not be fully tested in this study. Optimization of our protocol will likely increase the yield of tri-ubiquitin chains so that specific chains of greater length can be generated in the future.

Keywords:  Department of Biology
Ubiquitin
protein degradation
lysine 48
Stanford Department of Biology
Stanford Department of Chemical and Systems Biology
ChEM-H
Thesis
Advisor: Wandless Thomas
Dixon Scott
Investigating the mechanism for MYC and Twist1-driven hepatocellular carcinoma metastasis

Hepatocellular Carcinoma (HCC) is the most prevalent form of liver cancer, the fifth most common cancer, and the third most common cause of cancer-related deaths worldwide. The vast majority of HCC patient mortality is due to the inability to surgically resect the tumor. c-Myc (MYC) is a proto-oncogene that is frequently overexpressed in HCC. Previously, the Felsher lab found that in transgenic mouse models, MYC-driven HCC (MYC HCC) is non-metastatic. Now we have found that overexpressing MYC and Twist1 leads to metastatic HCC (MYC/Twist1 HCC) in vivo and in vitro. However, the pathway by which these genetic events facilitate metastasis is not well characterized. Many studies show that a high density of macrophages in the tumor microenvironment correlates strongly with tumor aggressiveness and poor prognoses. Macrophages can be polarized to adopt the alternative growth promoting M2 phenotype, which may play a key role in altering the tumor niche to promote metastasis. To determine the molecular pathway for HCC metastasis, we investigated candidate genes that are differentially upregulated in MYC/Twist1 HCC compared to MYC HCC that also have regulatory functions in the immune system, specifically with macrophages. In our in vivo mouse model, we found that both MYC and Twist1 are necessary and sufficient for HCC metastasis. In our transgenic mouse models, we show that MYC/Twist1 HCC recruits macrophages to the primary tumor site compared to MYC HCC. In vivo, we demonstrate that macrophages are necessary for HCC metastasis to occur. Furthermore, MYC/Twist1 HCC cells polarize naïve macrophages to adopt the M2 pro-tumoral phenotype compared to MYC HCC. Our data provides insight into the mechanism by which HCC relies on molecular crosstalk with macrophages to induce metastasis, which can later be exploited for clinical advancements and create treatments to improve patient prognoses.

Keywords:  Department of Biology
Stanford University Department of Biology
Biology
Hepatocellular Carcinoma
Metastasis
MYC
Twist1
Macrophages
Thesis
Advisor: Felsher Dean
Dixon Scott
Investigating the role of mTOR in ferroptosis

Bioactive compounds are useful probes for cellular activity. From a library of 1,833 bioactive compounds we identified dozens of small molecule inhibitors of ferroptosis, including investigational and FDA-approved drugs, with unexpected off-target antioxidant activities. ATP-competitive mechanistic target of rapamycin (mTOR) inhibitors, by contrast, were on-target ferroptosis inhibitors. These results highlight widespread bioactive compound pleiotropy and link mTOR signaling to ferroptosis regulation.

Keywords: Biology.
Advisor: Dixon Scott
Mudgett Mary Beth
Stearns Tim
"Investigating the Role of ssDNA AAV6 in the DNA Damage Response"

The Cas9/AAV6 gene editing technique has been utilized to correct mutations in genetic diseases and is now being transferred to the clinic, undergoing clinical trials. In this technique, electroporation is used to introduce the components of the Cas9/AAV6 machinery into the cells being treated: complexed Cas9 protein and guide RNA (RNP), and adeno-associated virus (AAV). This treatment may be contributing to the observed cytotoxicity, eventually leading to cell death, in the treatment of hematopoietic stem cells. It is unknown exactly which component or combination of components is the largest contributor to the cells’ DNA damage response that leads to the cell deciding whether to temporarily enter cell cycle arrest and repair itself or to undergo apoptosis. Given the single-stranded nature of AAV6 (ssAAV6), we hypothesize that the cell mistakenly interprets this gene-editing component as a damaged piece of DNA, triggering the DNA damage response in a treated cell. Following the treatment of cells that separates the individual components of the Cas9/AAV6 machinery, a series of assays, measuring the amount of DNA damage in the cells and the percentage of the treated cell population that decides to opt between repair or death, demonstrate which component is the greatest contributor to cytotoxicity. As genome engineering techniques are used more frequently to treat patients, it is imperative that the efficiency and effectiveness of the techniques are maximized and that cytotoxicity of the treatment is brought to a minimum.

Keywords:  Department of Biology
Biology
Cas9
gRNA
AAV
hematopoietic stem cells
DNA damage
cytotoxicity
Thesis
Advisor: Porteus Matthew H
Gozani Or
Investigating the Role of TMEM106B, a Genetic Modifier of Frontotemporal Dementia, in the African Turquoise Killifish

Frontotemporal Dementia (FTD) is an aging-related neurodegenerative disease characterized by the preferential atrophy of the frontal and temporal lobes of the brain and the accumulation of protein aggregates. Recently, variants of the lysosomal transmembrane gene TMEM106B have been identified by a genome-wide association study as protective for FTD in humans. Though TMEM106B holds promise for future therapeutic purposes, it remains uncharacterized in the context of neurodegeneration with age, mostly due to the lack of appropriate and feasible vertebrate animal models for aging. A new model organism, the African turquoise killifish, is especially advantageous for addressing this question in aging due to the killifish’s short life-span, ease of genetic manipulation, and large number of offspring. Using CRISPR technology, we have created a knockout of tmem106b, the killifish ortholog of TMEM106B. Though we are unable to definitively conclude from our experiments whether the removal of tmem106b increases or decreases lysosomes in the killifish brain, preliminary data suggests that knocking out tmem106b in the killifish alters lysosomal levels of the brain differently depending on age. Preliminary data also suggests that the rate of lysosomal decline with age is slower in tmem106b knockout killifish than in wildtype killifish. In exploring the mechanisms of how TMEM106B protects against diseases of the aging brain, we may be able to target relevant pathways in the future to fight old-age dementia.

Keywords:  Department of Biology
Biology
Frontotemporal Dementia
protein homeostasis
protein aggregates
African turquoise killifish
TMEM106B
Thesis
Advisor: McKay Andrew
Brunet Anne
Kopito Ron
Investigating the role of translation initiation machinery in mutant NPM1 driven leukemogenesis

Nucleophosmin 1 (NPM1) mutations are implicated as the driver mutation in approximately one-third of adult acute myeloid leukemia (AML) patients. Despite the prevalence of this mutation, however, targeted therapies remain elusive for patients with this subtype of leukemia because the molecular mechanism of NPM1 mutations is unknown. The principle model is that mutant NPM1 (mutNPM1) leads to p53 degradation, allowing cells to escape from p53-mediated cell cycle arrest. However, the distinct clinical characteristics of AML with NPM1 mutations and AML with p53 mutations do not support a simple link between mutNPM1 and p53. This discrepancy stimulated our investigation of mutNPM1's involvement in alternative mechanisms for leukemogenesis. We first conducted a screen for novel binding partners of mutNPM1 through immunopurification of the protein complexes formed by wild-type (WT) NPM1 and mutNPM1 in human leukemia cells followed by mass spectrometry (MS) analysis. MutNPM1 exhibited novel or enriched binding to eukaryotic translation initiation factors (eIFs) compared to WT NPM1. These findings implicated a putative role of mutNPM1 in the upregulation of translation through direct interactions with the initiation machinery, consistent with the theory that cancer cells require aberrantly high levels of protein synthesis to sustain rapid proliferation. However, inability to validate these interactions through co-immunoprecipitation warrants further investigation of the molecular mechanism of mutNPM1 and how it promotes leukemogenesis, in order to potentially reveal unexplored therapeutic targets.

Keywords:  Department of Biology
Biology
NPM1
leukemia
AML
translation
Thesis
Advisor: Artandi Steven
Feldman Jessica
Investigating the Urban-Rural Divide in California Transportation Sales Taxes

Since the 1970s, California counties have used transportation sales taxes measures to fill the gap between available funds and local need. This thesis identifies and explores a limitation of this trend – rural counties face particular difficulties in passing such measures. A combination of historical analysis, case studies, and quantitative methods are used to demonstrate the significance of this phenomenon. It is found that there have been tensions in urban-rural equity since the beginning of the California transportation system and that rural counties have long objected to the state’s promotion of transportation sales taxes as a viable funding mechanism. Quantitative analysis of voting records confirms that rural counties face higher obstacles in passing new transportation sales tax measures compared to other counties renewing their measures. Collectively, these results point to the need for increased state transportation funding and amplify the significance of Proposition 6 on the fall 2018 California ballot.

Keywords:  Earth Systems
School of Earth Energy &amp; Environmental Sciences
transportation
taxes
rural
Thesis
Advisor: Anderson Michelle
Nall Clayton
Investigation of advanced clinical PET system designs

Positron emission tomography (PET) is a functional radionuclide imaging modality commonly used for managing diseases such as cancer. High-resolution, high-sensitivity, quantitative PET imaging could open up potential new uses in early stage cancer management such as guiding biopsy or surgical resection. Locoregional PET systems with reconfigurable field-of-views (FOV) can further increase the sensitivity and resolution for a specific organ of interest. We will start this thesis by introducing the physics and basic detector designs of PET systems. It will then discuss design and performance of whole-body and locoregional PET system designs that are being investigated in the field. Then, the thesis will describe in detail a 1 mm resolution clinical locoregional PET scanner that is being constructed at Stanford. The design choices and electronics of this system will be discussed, and then system calibrations and performance test results will be shown. Finally, we will discuss additional data correction algorithms specific to this system, such as randoms correction and limited angle tomography image artifact correction algorithms. The results presented in this thesis show that the prototype subsystem of the 1 mm resolution clinical locoregional PET system can achieve spatial resolution of 0.73 mm at the center of the FOV, energy resolution of 11.34%, and coincident timing resolution (CTR) of 13.92 ns. The unique 3D position sensitive scintillation (3D-PSS) detectors used in the system allows for intercrystal scatter positioning algorithms that can be used to improve contrast-to-noise ratio (CNR) by 8.4%, or reduce the patient dose by 12% while maintaining the same image quality. This thesis may be useful for readers that are interested in the physics, electronics, and image quality of high-resolution PET scanners with small scintillation crystal elements. Interesting topics include investigations of detector signal multiplexing across a system, limited angle tomography effects in the reconstructed image, and ICS positioning in a system with multiplexed detectors.

Keywords: Electrical Engineering.
Advisor: Levin Craig 
Horowitz Mark (Mark Alan) 
Nishimura Dwight George
Investigation of n-heptane ignition in shock tubes by endwall imaging, speciation, and temperature measurements

With the aim of increased energy efficiency and reduced pollutant emissions from internal combustion engines which burn petroleum and other liquid fuels, several modern alternative engine concepts, termed low-temperature combustion (LTC) engines, have recently been proposed. These engines have shown promising results with regard to increasing energy efficiency and reducing pollutant emissions, but have encountered challenges in controlling the timing of ignition and the rate of heat release. These difficulties have stemmed from an inherent reliance on the global chemical reaction rate of the fuel to determine the timing of these parameters in LTC engines. Current work to better understand the reaction rates of petroleum fuels at low temperatures is critical to the development of LTC engines. Some previous experimental work measuring the global reaction rates of petroleum fuels at low temperatures relevant to LTC engines exists in the literature. However, inconsistent or scattered results are often observed in the measured ignition delay times due to complications from impurities and non-ideal effects in the experimental reactors used. In the current work, a shock tube reactor was used to measure the rate of reaction of the primary reference fuel, \norm-heptane, at low temperatures. These measurements were largely free of the influence of particle impurities that have been found to influence the low-temperature combustion behavior of \norm-heptane. High-speed imaging and laser diagnostic measurements were performed to characterize under which conditions particle impurities were problematic, and to show when experiments were free of particle effects. Following the removal of particle impurity effects, species time-histories, ignition delay times, and temperature time-histories were measured during \norm-heptane combustion. Chemical reaction models which attempt to predict the global reaction rates for LTC fuels, often disagree with experimental measurements made at low temperatures and low pressures. These chemical models are under continuous development and require additional experimental measurements as validation targets for further improvement. The data provided here may serve as validation targets for future mechanism optimizations, and may lead to an improved ability to model LTC combustion, thereby aiding in the creation of LTC engines that are better able to control their ignition timing and heat release rate.

Keywords: Mechanical Engineering.
Advisor: Hanson Ronald 
Bowman Craig T. (Craig Thomas)
Davidson David F. (David Francis)