VictorJuiz/thesis huggingface dataset:
Study of the Yeast Vacuolar-type ATPase by Electron Cryomicroscopy

Vacuolar-type ATPases (V-ATPases) are rotary enzymes that use energy from hydrolysis of adenosine triphosphate (ATP) to pump protons across membranes and control the pH of many intracellular compartments. ATP hydrolysis in the soluble catalytic region of the enzyme is coupled to proton translocation through the membrane-bound region by rotation of a rotor subcomplex. Studies of ATP synthases, V-ATPases, and vacuolar/archaeal ATPases (V/A-ATPases) have suggested that flexibility is necessary for the catalytic mechanism of rotary ATPases but the structures of different rotational states have never been observed experimentally. V-ATPases also serve an important role in the immune system and are targeted by intracellular pathogens. Legionella pneumophila, the causative agent of Legionnaires' Disease, secretes a protein effector called SidK that binds and inhibits V-ATPases, preventing acidification of lysosomes and promoting survival of the bacterium inside macrophages. The mechanism of V-ATPase inhibition by SidK is not known. The structure of V-ATPases can be analyzed by single particle electron cryomicroscopy (cryo-EM), an imaging technique involving the identification of tens to hundreds of thousands of individual proteins in micrographs. A partially-automated image selection program was developed to increase particle image identification throughput. A method was also developed to correct for magnification anisotropy that was identified in our images. Cryo-EM maps were obtained for structures of three rotational states of the V-ATPase from the yeast Saccharomyces cerevisiae. The three different maps reveal the conformational changes that occur to couple rotation in the soluble catalytic region to the symmetry-mismatched membrane-bound proton-translocating region. The structures of these states provide direct evidence that deformation during rotation enables the smooth transmission of power through rotary ATPases. To investigate the molecular mechanism of V-ATPase inhibition by SidK, we determined structures of the V-ATPase:SidK complex by cryo-EM. The cryo-EM maps reveal SidK to be an elongated protein composed of a structured region that interacts with the soluble catalytic region of V-ATPase and a flexible region of unknown function. The structures suggest a novel inhibition site for V-ATPases and provides evidence that the activity of these proton pumps is linked to the flexibility of the A-subunit.

Keywords: cryo-EM
magnification anisotropy
SidK
TMaCS
V-ATPase
Advisor: Rubinstein, John L
Picosecond Infrared Lasers (PIRL): Applications in Biodiagnostics and Towards Quantitative Mass Spectrometry

A new concept based on the Picosecond Infrared Laser (PIRL) has been introduced to selectively excite water to directly drive ablation of biological tissue faster than any competing energy dissipation process that could damage surrounding tissue. This thesis work showed that this mechanism can be exploited for a new form of spatially resolved mass spectrometry with the prospect of near instant biodiagnostics with mass spectroscopic sensitivity. The ion source for imaging mass spectrometer was based on PIRL ablation with an electrospray pick up source to entrain the ablated species. This hybrid Picosecond Infrared Laser Assisted Electrospray Ionization (PIR-LAESI) showed a limit of detection of 100 nM and a spatial resolution of ~ 100 Îźm, limited only by the current focusing optics. The longitudinal spatial resolution is approximately determined by the absorption depth. At the wavelengths of PIRL pulse, the light is absorbed within a few microns at the ablation threshold, resulting in removal of ~ 4 Îźm thick of material per pulse, which is the size of a single cell. Most importantly no additional treatment was needed as in Matrix Assisted Laser Desorption/Ionization (MALDI). Molecular images using PIR-LAESI for both plant and animal tissues could be collected directly and matched to optical images of the same region. The image quality was compared to nanosecond LAESI versions. Smaller crater size with PIR-LAESI was noted with similar molecular image quality, indicating PIR-LAESI a more sensitive approach with higher spatial resolution. The question is how far the sensitivity can be improved. In principle, single protein detections is possible if there is no thermal fragmentation as shown in this work. New direct in vacuum sample presentation to mass spectrometry should enable this ultimate limit to single molecular detection, using an electric field for ion separation. Molecular dynamics simulation on the ablation process driven by PIRL on ionic polymer water solutions revealed the effect of electric field cannot be observed in the early stage of plume expansion but that the polymers stay intact with shedding of the water solvation layers. These results illustrate that selective excitation of the water provides the desired ablation physics.

Keywords: Biodiagnosis
Desorption via Impulsive Vibrational Excitation ablation
Imaging Mass Spectrometry
Laser Ablation Electrospray Ionization
Picosecond Infrared Lasers
Quantitative Mass Spectrometry
Advisor: Miller, Dwayne
Rural Youth within Changing Education, Formal Labour Market and Informal Economic Conditions during Post-Soviet Transition Period in the Kyrgyz Republic

This qualitative case study examines the experiences, expectations and aspirations of education, employment, and future prospects of a group of rural youth growing up in the postsocialist era in southern Kyrgyzstan. Despite popular conceptions that youth are the authors of their life projects with an abundance of choice (Du Bois Reymond, 1995), the socially determining categories of social class, gender, ethnicity, and place still structure these choices and shape the life chances of contemporary youth (Roberts, 2010). This study shows how the postsocialist transformation has increased the burdens and uncertainties of young people and undermined the stability and security of their futures (DeYoung, 2010; Korzh, 2014; Roche, 2014; Walker, 2011). It identifies educational and social inequalities persisting from traditional Kyrgyz and Soviet society, as well as those emerging in the post-Soviet period, and analyzes the effects of these on the experiences and aspirations of rural Kyrgyz youth. Three types of rural Kyrgyz youth emerged from the data set of the study and three illustrative cases were: the trajectory of youth from the rural intelligentsia class with aspirations for a professional future; that of youth from the emerging successful stratum of kommersants (merchants) with aspirations for an entrepreneurial future, and that of youth from the former kolkhoz (Soviet collective farm) working class, who face the precarious future of the migrant worker. These three social trajectories are explained, following Bourdieu, as manifestations of the class and gendered habitus of the rural Kyrgyz youth and their families. The findings of the study reveal the ways that the social resources of kinship-oriented families, specifically their economic, cultural, social, and symbolic forms of capital, determine rural youth experiences and future chances in postsocialist transformation in Kyrgyzstan.

Keywords: Bourdieu
Educational inequalities
International education
Post socialist education
Rural Kyrgyz youth
Youth aspirations and lives
Advisor: Sawchuk, Peter
A Microfluidic Approach to Tissue Vascularization

From the inception of tissue engineering, tissue vascularization has been one of the greatest challenges in this field. Our inability to capture the daunting complexity of this natural biological system has slowed down the advancement of this field. But the emergence of microfluidic technology/micro-fabrication has expanded engineers' toolbox, allowing us to create structures that are as small as a capillary and as complex as a vascular bed. My thesis hypothesis is that functional, high-density tissues can be vascularized by microfluidic bio-scaffolds based on synthetic biodegradable polymer with built-in, endothelialized, permeable 3-D vascular networks. The synthetic vessel network is also mechanically stable enough to establish immediate blood perfusion upon implantation by direct surgical anastomosis. Successful development of a robust vascularization strategy would serve as the foundation to both further advance tissue engineering in clinical application as well as establish a more complete in-vitro model for drug discovery. In the first specific aim, we established a protocol for culturing endothelial cells in small conduit such as a PDMS networks with circular channel cross-sections. In these micro-networks, endothelial cells naturally coat the inner luminal surface and form tubular structure with tight-intercellular junction. However, PDMS is not an implantable material and also does not allow us to incorporate a parenchymal space. Then in the following specific aims, we established a 3-D stamping technique to create a bio-scaffold with build-in micro-channel network that is mechanical stable, yet permeable and permits intercellular crosstalk and extravasation of monocytes and endothelial cells on biomolecular stimulation through the incorporation of nano-pore and micro-holes on the channel walls. With this technology (hereafter referred to as AngioChip), we successfully engineered vascularized functional rat and human hepatic and cardiac tissues that can process clinically relevant drugs delivered through the vasculature. Furthermore, millimetre-thick cardiac tissues can be engineered in a scalable manner. Finally, we demonstrated direct surgical anastomosis of the engineered tissues in both artery-to-artery and artery-to-vein configuration.

Advisor: Radisic, Milica
Biophysical Analysis of the C9orf72 Hexanucleotide Repeat Associated with ALS/FTD

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are two extremes of a spectrum of neurodegenerative disorders. Recently, it was shown that expansion of (GGGGCC)n. (GGCCCC)n in the C9orf72 gene is the main cause of ALS/FTD. Being G-rich on one strand, and C-rich on the other strand, the C9orf72 repeat is a great candidate for secondary structure formation. To date, it has been shown that different repeats of the G-rich DNA and RNA, can form G-quadruplexes in-vitro. Our studies have been mainly focused on biophysically characterizing G-rich RNA and DNA strands, and C-rich DNA strands by using CD and UV spectroscopy and gel electrophoresis. We have shown that G-rich RNA forms parallel G-quadruplexes and C-rich DNA can form an array of secondary structures including i-motifs at acidic pH, monomolecular hairpins at basic pH and G-quadruplexes at neutral pH. This range of structure is due to the ability of cytosines to get hemiprotonated. Non-canonical structures have long been known to act as mutagenic intermediates leading to genetic instability. Therefore, biophysically assessing these structures not only may provide a basis for mechanistic pathways leading to the disease, but may help find ways to prevent the disease. One of the proposed disease mechanisms is through toxic gain-of-function where neurodegeneration is caused by formation of nuclear RNA aggregates which sequester proteins. Based on this, we also identified two proteins which exhibit structure-specific binding to the RNA G-quadruplexes. Subsequently, we offered proof-of-principle of a ligand-based therapeutic approach where TMPyP4, a G-quadruplex binding ligand, was shown to disrupt the interaction between the RNA G-quadruplexes and ASF/SF2 and hnRNPA1. Lastly, we studied the effect of methylation on the secondary structures formed by the repeat. Recently, it was shown that the repeat itself is methylated in ALS patients, which also rationalized the observed 50% transcription reduction seen in expansion carriers. We have shown that while methylation at CpG sites stabilizes the structures on both G-rich and C-rich DNA strands, hydroxymethylation destabilizes them.

Keywords: ALS
C9orf72
CD spectroscopy
G-quadruplex
Methylation
Nucleic acid
Advisor: Macgregor Jr., Robert B
DNA-mediated Gold Nanoparticle Assembly: a Universal Platform for Therapeutic and Diagnostic Applications

Unique physicochemical properties of nanomaterials offer novel methodologies for disease treatment and diagnosis. Dynamic nanoparticle assemblies are adding an additional level of functionality and flexibility by combining multiple nanoparticle types into structures with stimuli-responsive size and surface chemistry. DNA-mediated gold nanoparticles assemblies are offering the most functional and versatile platform, which combines DNA addressability with large surface area, monodispersity, and easy functionalization of gold nanoparticles. Assembly size determines their application, with smaller nanoassemblies particularly suitable for drug delivery, and larger microassemblies used for biosensing. Despite a number of published studies, no in vivo examples or clinically ready biosensors have been reported. Therefore, the goal of this thesis was to identify the barriers for translation in both fields and to develop methods to overcome these limitations. To this end core-satellite nanoassemblies were designed for in vivo delivery, which efficiently protected loaded cargo and minimized macrophage recognition. Their controllable serum stability could be used as the mechanism of cargo release. In vivo nanoassemblies showed longer circulation half-life, improved tumour targeting, and renal bioelimination. In parallel, larger microassembly structures were combined with DNA enzyme signal amplification to generate a novel infectious pathogen biosensor. The assay was particularly suited for point-of-care setting with cheap and stable components and simple, fast, and specific colourimetric readout that could assay multiple targets in parallel. To demonstrate its clinical readiness, the assay was adapted for detection of a panel of five urinary tract infection bacteria. All targets were successfully identified in parallel at clinically relevant concentrations. Preliminary tests with E. coli patient samples confirmed clinical detection with good sensitivity and excellent specificity. This work presents a novel DNA-mediated gold nanoparticle assembly platform that is ready for in vivo translation and clinical in vitro diagnostics.

Keywords: Cancer therapy
Diagnostics
Gold nanoparticle
Nanoparticle
Nanoparticle assembly
Nanotechnology
Advisor: Chan, Warren CW
Genetics of Obsessive-compulsive Disorder: From Phenotypes to Pharmacogenetics

Background: Obsessive-compulsive disorder (OCD) is a debilitating neuropsychiatric disorder that is characterized by a diverse clinical presentation. Evidence suggests genetic involvement in the etiology of OCD; however, genetic association studies have yielded mixed results partly due to clinical heterogeneity. Aims: We therefore investigated the genetics of OCD subphenotypes: age at onset (AAO), Yale-Brown Obsessive-Compulsive Scale (Y-BOCS) severity score and symptom dimensions, family history of obsessive-compulsive and related disorders (OCRDs), psychiatric comorbidities, and drug response. We first examined these subphenotypes to ascertain clinically homogeneous dimensions of OCD. We then analyzed these subphenotypes and antidepressant response for genetic association to identify marker(s) for each subphenotype. Methods: The sample consists of 560 OCD individuals. For the subphenotypic analyses, admixture analysis (STATA) was performed to analyze AAO and factor analysis (SPSS) was applied to reduce the Y-BOCS symptom checklist. Family history and psychiatric comorbidity were obtained using the modified Family History Index and SCID-IV interviews respectively. The candidate gene study investigated markers, mostly in the remote regulatory regions, of 17 candidates for association with subphenotypes in 497 OCD participants. Genome-wide association study was conducted in a subset of this sample to examine AAO and Y-BOCS severity. Retrospective antidepressant response data was available in 222 OCD individuals. Statistical analyses were performed using SPSS, PLINK, and R programs, comparing genotype frequencies between different subphenotype groups. Results: We identified early (

Keywords: Candidate genes
Genome-wide association study
Molecular genetics
Obsessive-compulsive disorder
Pharmacogenetics
Advisor: Kennedy, James L
Richter, Margaret (Peggy) A
Stabilization, Extension and Unification of the Lattice Boltzmann Method Using Information Theory

A novel Lattice Boltzmann method is derived using the Principle of Minimum Discrimination Information (MinxEnt) via the minimization of Kullback-Leibler Divergence (KLD). Approximations of this method yield the single relaxation time (SRT-LBM), two relaxation time (TRT-LBM), multiple relaxation time (MRT-LBM) Lattice Boltzmann methods as well as Entropic Lattice Boltzmann Method (ELBM) and Ehrenfest Step LBM (EF-LBM). Specically it is shown that these methods can be understood as approximations of a method for constrained KLD minimization. By carrying out the actual single step Newton-Raphson minimization (MinxEnt-LBM) a more accurate and stable Lattice Boltzmann Method can be implemented. To demonstrate this, 2D Poiseulle flow, 1D shock tube and lid-driven cavity ow simulations are carried out and compared to SRT-LBM, TRT-LBM MRT-LBM and EF-LBM.

Keywords: Cavity Flow
Entropy
Information Theory
Kullback-Leibler Divergence
Lattice Boltzmann Method
Stability
Advisor: Pugh, Mary
Dawson, Francis P
Non-intrusive Virtual Systems Monitoring

In this thesis, I discuss why existing intrusive systems monitoring approaches are not a good fit for the modern virtualized cloud, and describe two alternative out-of-band solutions that leverage virtualization for better systems monitoring. My first solution employs Virtual Machine Introspection (VMI) to gain access to a VM's runtime state from the virtualization layer. I develop new VMI techniques to efficiently expose VM memory state from outside the VM boundary, which can be readily employed in existing cloud platforms as they are designed to operate with no new modifications or dependencies. While there exist a variety of other competing alternatives, their latency, overhead, complexity and consistency trade-offs are not clear. Thus, I begin my thesis with addressing this gap by organizing the various existing VMI techniques into a taxonomy based upon their operational principles, and performing a thorough exploration of their trade-offs both qualitatively and quantitatively. I further present a deep dive on VMI consistency aspects to understand the sources of inconsistency in observed VM state, and show marginal benefits for consistency with commonly employed VMI solutions despite their prohibitive overheads. Then, I present NFM (Near Field Monitoring)- a new approach that decouples system execution from monitoring by pushing monitoring components out of the target systems' scope. By extending and combining VMI with a backend cloud analytics platform, NFM provides simple, standard interfaces to monitor running systems in the cloud that require no guest cooperation or modification, and have minimal effect on guest execution. By decoupling monitoring and analytics from target system context, NFM provides always-on monitoring, even when the target system is unresponsive. My second solution- CIVIC (Cloning and Injection based VM Inspection and Customization)- avoids NFM's functionality duplication effort and overcomes its VMI-related limitations arising out of its raw memory byte level visibility into the guest. CIVIC operates at a logical OS level and reuses the vast stock monitoring software codebase, but in a separate isolated environment thus avoiding guest intrusion and interference hassles. CIVIC enables a broader usage scope in addition to NFM's passive (read-only) monitoring, by supporting actuation or on-the-fly introduction of new functionality. It restricts all impact and side-effects of such customization operations inside a live clone of the guest VM. New functionality over the replicated VM state is introduced using code injection. I present four applications built on top of NFM using its `systems as data' monitoring approach, to showcase its capabilities for across-systems and across-time analytics. I also highlight CIVIC's versatility in terms of enabling hotplugged and impact-free live customization, by employing it to monitor, inspect, troubleshoot and tune unmodified VMs.

Advisor: de Lara, Eyal
School Function In Children After Traumatic Brain Injury: Developing A New Outcome Measure

Trauma remains the leading cause of morbidity and mortality for children. Traumatic brain injury (TBI) is responsible for large population-level costs, and protracted burden on family and caregivers. TBI can significantly impact a child's ability to learn and attain important educational and social milestones. Coordinated reintegration into the school environment is paramount in order to achieve optimal outcome. To date, no validated instrument exists for teachers to be able assess the function of children in school after suffering TBI. By using Food and Drug Administration guidelines on Patient Reported Outcomes (PRO), and National Institutes of Health guidelines on mixed methods research, this thesis aims to develop a validated measure of school function in children after TBI, as assessed by the educational professional most closely involved with that child's education. There are multiple intended uses of this instrument. It will serve as a means for teachers to assess the function of their injured students in the classroom. It will also serve as a vehicle to provide tailored rehabilitation services to injured students. Clinicians will be able to assess the recovery of their patients who have returned to school using this outcome measure. Finally, it will serve as a validated outcome measure for clinical trials in pediatric TBI. This work was carried out in three phases. First, qualitative research methodology was used to develop a measurement concept of school function after TBI. School function was defined as the observable traits and behavioural manifestations of multiple cognitive, psychosocial, and neurologic processes, as well as performance on in-classroom academic tasks that represent a child's ability to achieve expected academic and social milestones. The qualitative data informed the second phase of instrument development, in which items were generated and reduced to form a 95-item prototype questionnaire. In the third phase, field testing was performed in order to validate the concept of school function and further reduce items. Only 58 questionnaires were completed; much further work is necessary to achieve the goal of generating a valid and reliable instrument. When complete, it will fill a large gap in the outcome assessment of this vulnerable population.

Keywords: Measurement
Pediatric
Qualitative research
Questionnaire
School function
Traumatic brain injury
Advisor: Kulkarni, Abhaya V
Beaton, Dorcas E