VictorJuiz/thesis huggingface dataset:
Calcineurin regulates a Cdc42 GAP, Rga2, to modulate Cdc42 signaling during the yeast pheromone response [electronic resource]
Keywords: Biology.

Rho GTPases are central regulators of cell polarity whose activity must be tightly regulated. In S. cerevisiae, the essential small type Rho GTPase Cdc42 regulates cell polarity during vegetative growth and coordinates cell signaling with morphogenesis in the mating response. Three Rho GTPase activating proteins (GAPs), Bem3, Rga1, Rga2, contribute to the specific spatial and temporal regulation of Cdc42, serving both overlapping and unique regulatory functions. Phosphorylation of these GAPs are known to regulate their function as during the G1 phase of the cell cycle, the cyclin-dependent kinases phosphorylate and inhibit Rga2 at S/TP sites to promote Cdc42 activation during bud emergence. Regulation of these phosphosites by phosphatases and other kinases remains to be examined. A recent systematic screen identified Rga2 as a substrate for the Ca2+-activated phosphatase, calcineurin. Our studies reveal that calcineurin dephosphorylates Rga2 during pheromone signaling to activate its function in limiting Cdc42 signaling. Calcineurin regulation of Rga2 is important to dampen pheromone-induced gene expression and promote cell survival during prolonged exposure to mating factor. Together with the known functions of calcineurin in promoting endocytosis of the pheromone receptor and inhibiting transcription factor activity, calcineurin signaling provides critical negative feedback during the pheromone response that is essential for cell survival.
Catalytic strategies for the synthesis of environmentally-responsive poly(aminoesters) [electronic resource] : applications for the delivery of messenger RNA
Keywords: Chemistry.

Polyesters are a remarkable class of polymeric materials that have been featured as biodegradable textiles, implantable biomedical devices, and drug delivery vehicles for therapeutic applications. However, the synthesis of polyesters with rich functional group diversity, narrow molecular weight distributions and targeted molecular weighs remains a significant challenge. The selective aerobic oxidation of 1,5-diols with [(neocuproine)Pd(OAc)]2(OTf)2 (neocuproine = 1,9-dimethylphenanthroline), A, yields cyclic lactones. A selection of functionalized 1,5-diols was investigated to explore the substrate scope and to determine compatible functional groups for the oxidative lactonization reaction using A. These studies led a general strategy for the synthesis of functionalized lactones, specifically N-substituted morpholin-2-ones. The organocatalytic ring-opening polymerization of N-acyl morpholin-2-ones occurs readily to generate functionalized poly(aminoesters) with N-acylated amines in the polyester backbone. The thermodynamics of the ring-opening polymerization depends sensitively on the hybridization of the nitrogen of the heterocyclic lactone. N-Acyl morpholin-2-ones polymerize readily to generate polymorpholinones, but the N-aryl or N-alkyl substituted morpholin-2-ones do not polymerize. Experimental and theoretical studies reveal that the thermodynamics of ring opening correlates to the degree of pyramidalization of the endocyclic N-atom. A wide range of novel polyesters was synthesized. Deprotection of the N-Boc protected polyaminoesters yields water-soluble, cationic materials. Three novel cationic water-soluble polyaminoesters (PAEs) were synthesized. These materials were shown to be stable in neutral D2O for over two days, however, upon exposure to alkaline or buffered conditions, two of the PAEs degraded rapidly and quantitatively to single small molecule products. An immolative mechanism is proposed to account for the exquisite selectivity of the observed in the controlled degradation. The rate of degradation is sensitive to pH, providing a new class of water-soluble materials which can be induced to degrade under specific environmental conditions.
Causal inference with random forests [electronic resource]
Keywords: Statistics.

Random forests, introduced by Breiman [2001], have become one of the most popular machine learning algorithms among practitioners, and reliably achieve good predictive performance across several application areas. This has led to considerable interest in using random forests for doing science, or drawing statistical inferences in problems that do not reduce immediately to prediction. As a step in this direction, this thesis studies how random forests can be used for understanding treatment effect heterogeneity as it may arise in, e.g., personalized medicine. Our main contributions are as follows: - We develop a causal forest algorithm for heterogeneous treatment effect estimation, and find our method to be substantially more powerful at identifying treatment heterogeneity than traditional methods based on nearest-neighbor matching, especially when the number of considered covariates is large. - We provide an asymptotic statistical analysis of causal forests, and prove a Gaussian limit result. We then propose a practical method for estimating the noise scale of causal forests, thus allowing for valid statistical inference with causal forests. - In a high-dimensional regime where the problem complexity and the number of observations jointly approach infinity, we identify the signal strength at which tree-based methods become able to accurately detect treatment heterogeneity. Perhaps strikingly, we find that the required signal strength only scales logarithmically in the dimension of the problem. Taken together, these results show that random forests -- despite often being understood as a mere black box predictive algorithm -- provide a powerful toolbox for heterogeneous treatment effect estimation in modern large-scale problems.
Cell envelope functions of Sinorhizobium meliloti
Keywords:  Department of Biology
Biology
Microbes
Thesis

Background: S. meliloti is a nitrogen-fixing bacteria that infects the roots of its legume host plants to form nodules, where it converts atmospheric nitrogen into ammonia that plants can use in a symbiotic relationship. Much of the mechanism of this symbiotic relationship is still unknown, but genes required for proper cell envelope function have been implicated, and more of such genes likely exist. At the same time, recent research has uncovered possible genes that may be involved in cell growth and cell envelope synthesis in the closely related A. tumefaciens, which may also play a role in S. meliloti cell envelope function and symbiosis.
Methods: Tn5 mutagenesis followed by screening on chlorophenol red-β-D-galactopyranoside (CPRG) was conducted on S. meliloti strain MB782. Mutants with possible cell envelope defects were selected and the location of Tn5 insertions identified by arbitrary PCR and sequencing. Mutant sensitivities to detergents were tested by spot assays with SDS or deoxycholate. SMc00038 and SMc00039 were identified to be possible candidates required for cell growth and cell envelope synthesis based on recent research of A. tumefaciens, and were deleted from the chromosome of S. meliloti CL150 to create the strain D217. Effects of deletion on symbiosis, growth rates, cell shape, and deoxycholate and NaCl sensitivity were studied.
Results: 3 genes previously reported to affect both cell envelope function and symbiosis were amongst the list of genes identified using CPRG screening, and 48% of mutants identified showed increased sensitivity to detergents. D217 demonstrated slower growth and abnormal cell branching, but no effects on symbiosis, deoxycholate, or NaCl sensitivity were observed.
Conclusion: Tn5 mutagenesis followed by screening on CPRG is a promising method of identifying genes required for cell envelope integrity and symbiosis. SMc00038 and SMc0039 may play a role in proper cell growth.
Cell Intrinsic and Microenvironmental Etiologies of Chemotherapy-Induced White Matter Damage
Keywords:  Department of Biology
Biology
Chemobrain
Oligodendrocytes
OPCs
Microglia
Stanford Biology Department
Thesis

The majority of cancer survivors with a history of chemotherapy treatment endure long-term deficits in neurological performance. This chemotherapy induced cognitive and motor decline is known colloquially as chemobrain. The symptoms associated with chemobrain may be attributed to damage to myelin, the insulating sheath surrounding neurons allowing for rapid saltatory conductions, or myelin-forming cells, such as oligodendrocyte precursor cells (OPCs) thus altering brain function. To confirm the extent of OPC population depletion in children receiving chemotherapy, we examined post-mortem brain samples from the frontal lobes of children treated with multi-agent traditional chemotherapy. We found that OPCs are depleted specifically in subcortical white matter. In contrast, grey matter OPCs are preserved in comparison to age-matched control subjects. Methotrexate (MTX), an antimetabolite chemotherapeutic, is a commonly used agent in pediatric cancer therapy and is particularly associated with white matter injury and cognitive dysfunction. We have developed a mouse model of juvenile methotrexate chemotherapy exposure in which mice are treated with MTX or PBS (one dose each week from P21-35 for a total of three doses). One month following the last dose of MTX, mice exhibit behavioral deficits in motor speed and attention, as well as depletion of deep cortical grey matter and subcortical white matter OPCs. As in human subjects, superficial grey matter OPC density was preserved. Concomitant with deep cortical and subcortical OPC depletion, we observed an increase in immature oligodendrocytes, suggesting increased but incomplete differentiation of OPCs. Experiments allografting healthy, GFP-labeled OPCs into the environment of the previously chemotherapy-treated brain suggest a persistent change to the gliogeneic microenvironment underlies the accelerated OPC differentiation. OPCs isolated from whole brain exhibit an IC50 less than the measured MTX concentration achieved in the brain with this paradigm, suggesting that a direct cytotoxic effect on OPCs may also plays a role in depletion of the subcortical OPC population. Also, MTX treated mice exhibit increases in the number of activated microglia within the corpus callosum in MTX treated mice. Additionally, we found an increase in TGFβ signaling in MTX-exposed OPCs as indicated by an increase in phosphorylated Smad2/3 (pSmad2/3) and a subsequent increase in TGFβ gene expression in the activated microglia. Collectively, these findings suggest MTX chronically activates microglia and elevates TGFβ signaling to inhibit the ability of OPCs to proliferate and accelerate OPC differentiation respectively.
Character and causes of changing North Pacific climate extremes [electronic resource]
Keywords: Earth System Science.

The geographically and socioeconomically diverse state of California is a region characterized by frequent hydrological cycle extremes. Located near the southern margin of the prevailing Pacific storm track, California receives most of its annual precipitation over the course of a small number of heavy precipitation events during its well-defined winter rainy season. This dependence upon a handful of powerful storms for the bulk of its water supply makes California uniquely sensitive to the high year-to-year precipitation variability caused by relatively subtle shifts in the Pacific storm track—ultimately leading to wide swings between drought and flood. Severe drought in California during 2012-2016 has highlighted the state's substantial vulnerability to hydroclimatic extremes, and the extraordinary character of this extreme climate event has prompted a range of physical science questions regarding the drought's underlying physical causes—including the potential role of human-caused global warming in increasing drought risk in this already water-stressed region. Of particular interest has been the remarkable multi-year persistence of atmospheric high pressure over the northeastern Pacific, which deflected the Pacific storm track and generated very dry conditions over several consecutive winters. Questions regarding the behavior and dynamics of anomalously recurring high pressure events, in addition to their role in preventing moisture-bearing winter storms from reaching California, have therefore featured prominently in the recent scientific discourse. The work presented in this dissertation uses the 2012-2016 California drought as a springboard to explore the atmospheric mechanisms linked to Pacific Southwest precipitation extremes, and in doing so to develop a broadly-applicable methodological framework for quantifying complex changes in atmospheric circulation in a warming world.
Characteristics of bolus formation and propagation from breaking internal waves on shelf slopes [electronic resource]
Keywords: Civil and Environmental Engineering.

A series of laboratory experiments was conducted to study the formation of internal boluses through the run-up of periodic internal wave-trains on a uniform slope/shelf topography in a two-layer stratified fluid system. In the experiments, the forcing parameters of the incident waves (wave amplitude and frequency) are varied for a constant slope angle and layer depths. Simultaneous particle image velocimetry (PIV) and planar laser-induced fluorescence (PLIF) measurements are used to calculate high resolution, two-dimensional velocity and density fields. Over the range of wave forcing conditions, four bolus formation types were observed: backward overturning into a coherent bolus, top breaking into a turbulent bolus, top breaking into a turbulent surge, and forward breaking into a turbulent surge. Wave forcing parameters, including a wave Froude number Fr, a wave Reynolds number Re, and a wave steepness parameter ka0, are used to relate initial wave forcing to a dominant bolus formation mechanism. Bolus characteristics, including the bolus propagation speed and turbulent components, are also related to wave forcing. Results indicate that for Fr > 0.20 and ka0 > 0.40, the generated boluses become more turbulent in nature. As wave forcing continues to increase further, boluses are no longer able to form.
Characterization and therapeutic modulation of mesenchymal and myeloid lineages [electronic resource]
Keywords: Regenerative Medicine and Stem Cell Biology.

Fibrosis and regeneration are intimately connected. While fibrotic processes create nonfunctional tissue through the deposition of connective tissue, regenerative processes have the potential to generate new functional tissue. In this manner, efforts to better understand and potentially inhibit fibrosis should be complemented by regenerative approaches. In the first half of this dissertation, mechanisms and targeted inhibition of fibrosis are investigated primarily in the context of mesenchymal lineages. Regenerative approaches focused around cell-based therapies are then explored in the second half of this dissertation.
Characterization of a multimode CQED system [electronic resource]
Keywords: Applied Physics.

Cavity quantum electrodynamics (CQED) is a proven testbed for studies of the fundamental light- matter interaction and in recent years has become increasingly relevant in quantum simulation. By engineering the electromagnetic properties of the cavity it is possible to tailor unique interatomic interactions resulting in the possibility of observing novel phase-transitions and self-organization phenomena. These transitions distinguish themselves from existing ultra-cold atom optical lattice experiments in that the order exhibited by the atoms is not externally-imposed but emergent in the atom-cavity interaction. Increasing the variety of cavity modes available for crystallization towards a complete basis leads to the possibility of observing quantum soft-condensed matter phenomena like phonons, dislocations, and frustration in this system. This thesis introduces a novel cold-atoms apparatus designed and constructed with the aim of observing and characterizing emergent quantum phenomena. Details of the experimental setup and procedure are presented along with recent experimental and theoretical results pertaining to a novel self-organization phenomenon. Additionally, the technical aspects of an approach to couple light selectively to arbitrary cavity modes using digital holography will be presented. This tool will be useful for seeding the cavity towards, for example, the promotion of self-organization in a particular mode.
Characterization of nemitin as a novel microtubule-related protein involved in axonal and dendritic developmental growth
Keywords:  Department of Biology
Biology
Neurology and Neurological Sciences
Neurodegenerative Diseases
Microtubule
Nemitin
Thesis

Neurons are an astonishing component of our nervous system that intricately control our intended, and unintended, actions. A highly organized microtubule network is essential for multiple cellular functions in neurons including axonal transport, neuronal polarity, and synaptic activity. Disrupted microtubule networks are a hallmark of several neurodegenerative diseases, like Alzheimer’s disease. In order to understand these diseases more comprehensively, the mechanisms behind microtubule organization and regulation must be elucidated.
This research aims to provide more insight into microtubule processes. The Yang lab has recently identified nemitin as a novel protein involved in microtubule regulation. Nemitin is enriched in neurons, colocalizes with microtubules and is essential for viability, since knockout of nemitin in mice is lethal embryonically. Using molecular and cellular neuroscience techniques, we further characterize nemitin’s cellular functions and suggest a model for its mechanism of action. Nemitin localizes to the centrosome in Cos-7 cells but no neurons. Using a conditional knockout mouse line, we show that Cre-mediated conditional knockout of nemitin in embryonic neurons suppresses the growth of axons and primary dendrites. To address a potential mechanism, we show using 2D gel electrophoresis that nemitin is phosphorylated in neurons. Finally, we characterize nemitin’s interaction with a putative kinase, STK25, and suggest that phosphorylation may be involved in nemitin’s mechanism of action. Altogether these results suggest that nemitin is necessary for microtubule organization, while also being important for neurite development. This research will strengthen our current knowledge of microtubule regulation in neurons, and could provide novel therapeutic approaches in treating neurodegenerative diseases that exhibit abnormal microtubule networks.
Characterizing bacterial physiology and diversity using microfluidic systems [electronic resource]
Keywords: Electrical Engineering.

Bacteria are ubiquitous and play essential roles across the biosphere from environmental nutrient cycling to human health. Despite their importance, most bacterial diversity remains unknown. Among known species, traditional techniques measure bulk properties of a bacterial culture and fail to account for single-cell variations. Microfluidic technology enables single-cell manipulation and alleviates technical limitations associated with traditional methodologies. In addition to enabling single-cell resolution, integrating microfluidic platforms in experimental approaches increases throughput, which benefits statistical analyses. This thesis discusses two applications for which high-throughput microfluidic systems enable data acquisition with single-cell resolution. The first application addresses growth dynamics of Synechocystis, a model photosynthetic bacterium. In this investigation, a custom microfluidic setup facilitates long term, single-cell tracking under various illumination conditions. Single-cell growth curves demonstrate rapid response to light-dark transitions and reveal an adder like mechanism for cell-size regulation. The second microfluidic-based experimental method investigates the diversity of uncultivable bacteria from environmental samples. The throughput of the microfluidic platform allows the generation of many sub-samples, enabling genome binning based on presence of genomic contigs across sub-samples. Multiple known and novel bacterial phylogenies are identified in hot spring samples from Yellowstone National Park. Finally, this thesis discusses prospects of continued scaling of microfluidic systems and associated technical challenges. Both on-chip and off-chip schemes are explored, including an on-chip microfluidic component for digital to analog pressure conversion and various off-chip pressure control interfaces. The increased functional integration of microfluidic platforms and their application in microbial investigations offer an exciting future for the scientific community.
Characterizing protein interactions of putative and novel photosynthetic components in Chlamydomonas reinhardtii
Keywords:  Department of Biology
Biology
Chlamydomonas
photosynthesis
carbon concentration
affinity purification
mass spectrometry
Thesis

Population growth places great strain on our natural resources, bringing to the forefront the pressing problems such as limited food supplies. One way to address this problem is by improving plant photosynthetic efficiency, resulting in increased plant productivity and higher crop yields. To study photosynthesis and carbon concentration, we use the model organism Chlamydomonas reinhardtii, a single-celled green alga with an inducible carbon concentrating mechanism (CCM) that allows it to perform efficient photosynthesis: our long-term goal is to introduce the algal CCM into higher land plants, which would reduce photorespiration and enhance photosynthesis. It is necessary, therefore, to identify the key components of the CCM and to understand their molecular interactions in order to transfer this mechanism into crop plants. To this end, we have developed a pipeline for large-scale characterization of the algal CCM components and map how these components function together. With fluorescent and affinity tagging coupled to immunoprecipitation (IP) and mass spectrometry to explore these mechanisms, we took the localization data for 40 putative photosynthesis and CCM components to present 381 confident interactions between the proteins involved. The developed pipeline and the foundational knowledge reported here provide valuable tools for continuing work towards bioengineering of the CCM.