VictorJuiz/thesis huggingface dataset:
Investigating the biophysical mechanisms of substrate rigidity-induced hippo pathway regulation in human embryonic stem cells
Keywords: Chemical Engineering.

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.
Investigating the Effects of Ubiquitin Linkage Topology on Protein Stability
Keywords:  Department of Biology
Ubiquitin
protein degradation
lysine 48
Stanford Department of Biology
Stanford Department of Chemical and Systems Biology
ChEM-H
Thesis

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.
Investigating the mechanism for MYC and Twist1-driven hepatocellular carcinoma metastasis
Keywords:  Department of Biology
Stanford University Department of Biology
Biology
Hepatocellular Carcinoma
Metastasis
MYC
Twist1
Macrophages
Thesis

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.
Investigating the role of mTOR in ferroptosis
Keywords: Biology.

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.
"Investigating the Role of ssDNA AAV6 in the DNA Damage Response"
Keywords:  Department of Biology
Biology
Cas9
gRNA
AAV
hematopoietic stem cells
DNA damage
cytotoxicity
Thesis

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.
Investigating the Role of TMEM106B, a Genetic Modifier of Frontotemporal Dementia, in the African Turquoise Killifish
Keywords:  Department of Biology
Biology
Frontotemporal Dementia
protein homeostasis
protein aggregates
African turquoise killifish
TMEM106B
Thesis

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.
Investigating the role of translation initiation machinery in mutant NPM1 driven leukemogenesis
Keywords:  Department of Biology
Biology
NPM1
leukemia
AML
translation
Thesis

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.
Investigating the Urban-Rural Divide in California Transportation Sales Taxes
Keywords:  Earth Systems
School of Earth Energy &amp; Environmental Sciences
transportation
taxes
rural
Thesis

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.
Investigation of advanced clinical PET system designs
Keywords: Electrical Engineering.

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.
Investigation of n-heptane ignition in shock tubes by endwall imaging, speciation, and temperature measurements
Keywords: Mechanical Engineering.

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.
The invisible Romans : an archaeological investigation of the poorest Roman burials and their contexts in imperial period Italy
Keywords: Classics.

ABSTRACT Contemporary perspectives on the ancient Mediterranean tend to be top down and because of this, many segments of Roman society are effectively invisible in modern scholarship. The problem is due at least in part to the challenges of attributing past activity and material culture to the members of Roman society who had the least. The lack of traditional sources has resulted in a treatment of the non-elite in simplistic terms or as an essentially homogeneous entity. This research uses human remains analyses and multiscalar contexts to address the invisibility problem of the poor in three settings: the very urban context of the burials at Casal Bertone just outside Rome, the burials outside the city of Aesernia high in the Apennines which arguably straddles the urban and rural divide, and the rural context of the Vagnari vicus cemetery. This interdisciplinary approach juxtaposes bioarchaeological assessments, digital mapping and the work of economists with ancient texts, epigraphic evidence and more traditional archaeological reports to learn more about the poorest members of Roman society. The results of the comparison show a clear diversity in the lifestyles and life experiences of the non-elite, help explore ritual activity even among the simplest graves in the Roman archaeological record and raise important questions about how Roman cemeteries can be interpreted.
Iron and copper active sites in zeolites and their correlation to metalloenzymes
Keywords: Chemistry.

Metal-exchanged zeolites are a class of heterogeneous catalysts that perform important functions ranging from selective hydrocarbon oxidation to remediation of NOx pollutants. Among these, copper and iron zeolites are remarkably reactive, hydroxylating methane and benzene selectively at low temperature to form methanol and phenol, respectively. In these systems, reactivity occurs at well-defined molecular active sites. In this thesis, I discuss significant advances in the spectroscopic characterization of these active sites and their reactive intermediates. Site selective spectroscopy plays a critical role, making it possible to focus on active sites that exist within a distribution of inactive 'spectator' metal centers. Work presented here advances our understanding of the geometric and electronic structures of metallozeolites to the level attained in bioinorganic chemistry, enabling direct comparison of metallozeolites to functionally analogous Fe and Cu active sites in biology. Parallels and differences between metallozeolites and metalloenzymes are identified, leading to a number of new mechanisms to tune the performance of synthetic catalysts.
Is the United States Ready for a Female President? An Examination of American Media Culture and Current Political Evaluations
Keywords:  Department of Communication
Stanford University
Stanford University Communication Department
media studies
media culture
media representation
presidential election
gender politics
political evaluations
gender stereotypes
female presidential candidates
Thesis

The 2016 presidential election was a pivotal moment for the United States of America. For the first time in history, a woman ran as an elected major-party candidate in the race for President. Despite her deep political background, Hillary Clinton lost the election to Donald Trump, a man who had only recently entered the world of politics, and whose brash character is analyzed more than his actual policies. To fully understand the outcome of the election requires an examination of American culture. In particular, the victory of Donald Trump, and more importantly, the loss of Hillary Clinton cannot be explained without discussing the role in which gender played in the election, and more broadly, the role in which gender continues to play in American culture. By assessing America’s long history with traditional gender roles as well as how media has continued to reinforce those roles over time, this paper demonstrates how crucial the genders of these two candidates were in 2016. More specifically, gender provided a framework by which Americans evaluated the presidential candidates throughout the campaign. Despite critiques and other potential explanations, it is necessary to include gender when discussing the campaign and subsequent outcome of the 2016 presidential election. Before a woman can successfully hold the office of the President, American society and all of the institutions within it must commit to challenging and expelling traditional gender stereotypes.