VictorJuiz/thesis huggingface dataset:
Analysis of Microtubule Network Organization and Dynamics in the Yolk Cytoplasm during Zebrafish Epiboly

Morphogenesis describes the coordinated spatial distribution and rearrangement of cells in an organism. To study morphogenesis, I focused on zebrafish epiboly – the coordinated spreading of the blastoderm, which originally sits on top of a large yolk cell, over the yolk surface. Previous work implicated microtubule networks in the yolk cytoplasm (YCL) in this process, but their exact role is unclear. Here I report constant nucleation of new yolk microtubules during epiboly. I found extensive vegetal microtubule growth in the YCL. Furthermore, I demonstrated that the networks can move both animally and vegetally with high variability. In addition, I found evidence to suggest that yolk syncytial nuclei pulling may be facilitated by newly formed microtubules, rather than by existing YCL networks. Together, these results provide insights to the organization and dynamics of the YCL microtubule networks, and in turn their potential function during epiboly.

Keywords: epiboly
zebrafish
microtubule
yolk
Advisor: Bruce, Ashley
Spontaneous DNA Extracellular Trap Formation and Th2-biased Immune Responses in Cystic Fibrosis-like Airways of Mice with Lung-specific Nedd4-2 Ubiquitin Ligase Deletion

Extracellular DNA exerts major pathological effects in cystic fibrosis (CF) airways. However, the cause for increased DNA and the contribution of neutrophil extracellular traps (NETs) in cystic fibrosis (CF) airways is uncertain. Additionally, suitable mouse models to study CF lung disease are lacking. We investigated the inflammatory profile and the presence of NETs in a novel mouse model with cystic fibrosis-like lung disease (Nedd4L lung-specific knockout; NL-KO). Our studies show that NL-KO mice have a Th2-biased background inflammation, spontaneous leukocyte infiltration and NET formation in their airways. Overall, inflammatory changes in NL-KO mouse airways occur age-dependently and spontaneously in the absence of detectable infection. Furthermore, NL-KO mice challenged with LPS and P. aeruginosa have an exacerbated inflammatory profile and impaired bacterial clearance, respectively. In conclusion, we show the relevance of NL-KO mouse model to study inflammation, infection, and the role of NETs in CF-like lung disease.

Keywords: cystic fibrosis
neutrophil
neutrophil extracellular traps
DNA extracellular traps
mouse model
Nedd4-2
Th2
cytokines
LPS inflammation
P. aeruginosa infection
Advisor: Palaniyar, Nades
Three-degrees-of-freedom MEMS Electrostatic Out-of-plane Comb-drive Actuator for Auto-focus and Image Stabilization in Phone Cameras

Two versions of a novel three-degrees-of-freedom MEMS electrostatic vertical comb-drive actuator were developed, fabricated, and demonstrated. The novel actuator utilizes a flexure mechanism in which the in-plane (x-y) stiffness is high compared to the out-of-plane (z-axis) stiffness, allowing the actuator to achieve large stroke along the z-axis. The first actuator was able to achieve a large pure translation of 28 µm at 80 V without a stroke magnification mechanism, and also achieve an angle of rotation of 0.78º at 70 V. The second actuator was able to vertically displace a 3 mg mass for 13.5 µm at 80 V. Two enhanced designs of the 3-DOF actuator were also proposed and modelled and analyzed using CoventorWare. These actuators are developed specifically for Auto-Focus (AF) and Optical Image Stabilization (OIS) in phone cameras. Both designs meet the requirements of AF and OIS in phone cameras based on the numerical simulations results.

Keywords: MEMS, Comb-drive, Image stabilization, Auto-Focus
Advisor: Mrad, Ridha Ben
Structural and Functional Characterization of the Multi-domain Protein PelA: Implications for Biofilm Formation by Pseudomonas aeruginosa

The Pel exopolysaccharide (PEL) provides structural integrity and antibiotic resistance to Pseudomonas aeruginosa PA14 biofilms. The periplasmic protein PelA is essential for PEL biosynthesis. I have established that PelA has at least five domains, three of which have structural similarities to proteins with known functions. I resolved the structure of the glycosyl hydrolase PelA48-300 at 1.53 Å revealing a β8/α7 TIM-barrel fold with a highly electronegative cleft lined with conserved acidic residues ideal for polysaccharide binding and hydrolysis. When added to growing bacteria overexpressing PEL, PelA48-300 exhibits hydrolase activity that prevents pellicle formation: an activity that appears to be dependent on residues D160 and E218. In vitro characterization of full-length PelA using p-nitrophenyl acetate suggests that PelA also exhibits deacetylase activity. Site-specific mutagenesis of putative active site residues, D528, D530, H600 and H604 reduced the in vitro activity by over 10-fold and were found to abolish biofilm formation in vivo.

Keywords: Pseudomonas aeruginosa
Glycosyl hydrolase
Deacetylase
PelA
Bacterial biofilm
Pellicle
Structure
TIM-Barrel
Pel exopolysaccharide
Cystic fibrosis
Advisor: Howell, Dr. P. Lynne
Disruption of RAC1 in Murine Intestinal Enterocytes Results in Cytoplasmic Microvillus Containing Inclusions

RAC1 is a versatile effector that plays critical roles in cellular adhesion, differentiation, migration and death. As we recently demonstrated a role for RAC1 in ulcerative colitis, we examined the function of RAC1 in the intestinal epithelium by generating conditional knockout (cKO) Villin Cre – ERT2 Rac1mice. This model allows for the selective deletion of Rac1 in intestinal epithelial cells following intraperitoneal tamoxifen injection. In this study, we show that ablation of intestinal Rac1 results in weight loss, severe dehydration and intestinal inflammation, eventually resulting in death within 7 days of Tamoxifen induction. Most strikingly, transmission electron microscopy images depict the loss of apical microvilli, large cytoplasmic vacuoles and cytoplasmic microvilli containing inclusions in the small intestinal crypt-villus transition zone, which represent characteristic hallmarks of a rare enteropathy known as the known as of the Microvillus Inclusion Disease. Here we present a novel association for RAC1 in the pathogenesis of MVID.

Keywords: Rac1
Microvillus Inclusion Disease
Crohn's Disease
Ulcerative Colitis
Intestinal Disease
Myo5b
Advisor: Aleixo, Muise
ERp44 is Critical for Cardiac Development and Function

Dysregulation of Ca2+ cycling in the heart leads to defective cardiogenesis, ER stress, and eventually heart failure. The molecular mechanisms involved in all of these processes are not well characterized. Previously, our laboratory found that ERp44 was one of the proteins highly up regulated in a dilated cardiomyopathy model, R9Ctg mouse. ERp44 is a resident endoplasmic reticulum (ER) protein that is involved in the ER stress response and is a member of the protein disulfide isomerase (PDI) family. In order to characterize the function of ERp44 in cardiomyocytes, we generated and characterized ERp44 knockout mice. A lack of ERp44 in the mice led to significant embryonic lethality, abnormal heart development, dysfunction in Ca2+ dynamics, activation of ER stress gene profiles, and hypertrophic cardiomyopathy. Therefore, these results suggest that ERp44 plays a critical role in embryo and heart development and is crucial in regulating cardiac cell Ca2+ signaling.

Keywords: Heart failure
Calcium
Advisor: Gramolini, Anthony
Vsx2 Inhibits Dmbx1a to Regulate Retinal Progenitor Cell Proliferation in Zebrafish

Vsx2 is a gene expressed in RPCs during development and in a subset of bipolar cells and Müller glia of the differentiated neural retina. Dmbx1a is involved in the development and maintenance of numerous cell types and is important for the development of the midbrain, hindbrain, and retina. Since the mRNA expression patterns of vsx2 and dmbx1a are non-overlapping, the present study investigates the molecular networks regulating the transition between RPCs to post-mitotic neurons with these genes. The primary objective is to test the hypothesis of mutual antagonism between vsx2 and dmbx1a. Results suggests that vsx2 is required on and off in a carefully timed balance for the proper expansion of progenitor cells to occur during retina development. It also implicates that this gene has an effect on proper retina cell differentiation and laminar organization. Finally there is evidence that dmbx1a is needed but not sufficient in regulating vsx2 expression.

Keywords: Retina Progenitor Cells
vsx2
Advisor: Tropepe, Vincent
The Molecular Mechanism of Alginate O-Acetylation in Pseudomonas aeruginosa

Pseudomonas aeruginosa is an opportunistic, gram-negative pathogen that is involved in a variety of infections. The bacterium secretes and chemically modifies alginate to form a protective biofilm matrix. One protein implicated in the O-acetylation of alginate is AlgJ. In this study, we have successfully crystallized AlgJ and determined its structure to 1.83Å. The structure reveals that AlgJ is a SGNH hydrolase-like protein. AlgJ possesses a Ser-Asp-His catalytic triad which suggests that AlgJ is catalytically active. The structure of AlgJ demonstrated that the previously identified conserved residues of the AlgJ signature motifs form a local network with the proposed catalytic triad and are important for alginate O-acetylation in vivo. We have assayed the proposed Ser-Asp-His catalytic triad in vitro and observed impairment in acetyl esterase activity. In vitro binding studies demonstrated that AlgJ was unable to bind polymannuronate and that was it not capable of O-acetylating the polymer.

Keywords: Biochemistry
X-ray Crystallography
Advisor: Howell, Lynne
Replaceable Cast Steel Links for Eccentrically Braced Frames

The application of eccentrically braced frames (EBFs) as the lateral load resisting system of a structure has become increasingly popular for their stiffness and ductility. The EBF link acts as the energy dissipating element of the frame. However, the occurrence of fracture, local buckling, or lateral-torsional buckling limits the ductility of the link. This thesis presents the development of a replaceable cast steel link which incorporates the advantages associated with decoupling the link from the EBF beams with the geometric freedom inherent of steel castings. Its unique geometry distributes flexural yielding over the length of the link and prevents the failure modes observed of links composed of rolled or built-up sections to achieve a larger rotation capacity and longer low-cycle fatigue life for increased energy dissipation. Results from an extensive numerical analysis study demonstrate that the cast link significantly improves the performance and safety of EBFs.

Keywords: steel
earthquake
structural
seismic
braced frames
design
Advisor: Christopoulos, Constantin
Transmyocardial Revascularization Enhances Mesenchymal Stem Cell Engraftment in Infarcted Hearts Through SCF--c-kit and SDF-1--CXCR4 Signaling Axes

Objective: We investigated the roles of stem cell factor (SCF)–c-kit and stromal derived factor-1 (SDF-1)–CXCR4 signaling in transmyocardial revascularization (TMR)-enhanced engraftment of transplanted mesenchymal stem cells (MSCs) in infarcted hearts. Methods: 3 weeks after LAD ligation, female Lewis rats underwent 10-channel needle TMR, followed by daily IV injections of 1-million male donor MSCs for 5 days, either wild type (WT) or with knockdown of c-kit or CXCR4. Three days and 1 week after transplantation, we evaluated MSC engraftment, SCF—c-kit and SDF-1—CXCR4 expression, and left ventricular (LV) function. Results: MSC engraftment was affected by both TMR and donor cell type at 3 days and 1 week. LV ejection fraction improved when WT MSCs were infused, but knockdown of either receptor abrogated TMR-mediated augmentation of MSC reparative effect. Conclusions: Downregulation of either c-kit or CXCR4 on MSC decreased engraftment of circulating MSC and inhibited reparative effects of TMR.

Keywords: Cardiology
Stem Cells
Advisor: Yau, Terrence M
Structural and Functional Characterization of the Yeast R2TP Complex

Rvb1 and Rvb2, two closely related essential AAA+ helicases, play important roles in various cellular pathways. Our group found that they form a complex with novel Hsp90 chaperone interacting proteins, Tah1 and Pih1. We named the complex R2TP. It was found to be required for box C/D snoRNP biogenesis in yeast and stabilizing phosphatidylinositol 3-kinase-related protein kinase (PIKK) and RNA polymerase II assembly in mammalian cells. In order to obtain detailed functional and structural characterization of the R2TP complex, Rvb1/Rvb2 and Tah1/Pih1 complexes were successfully co-expressed and purified. Oligomeric states and ATPase activity of the co-expressed Rvb1/Rvb2 complex were determined by mass spectrometry, EM and ATPase assay. Through tagging and negative stain EM, the alternating layout of the yeast Rvb1/Rvb2 ring complex has been determined. Preliminary 3D reconstruction (~18Å) of the untagged Rvb1/Rvb2 was also obtained. Finally, six Tah1/Pih1 heterodimers were found to bind to one Rvb1/Rvb2 hexamer.

Keywords: protein
yeast
rvb
EM
purification
SEC
I domain
Liang Zhao
3D
cryo-EM
AAA+
R2TP
Advisor: Houry, Walid
Cross-platform Screening for Novel Fluvastatin and Kinase Inhibitor Co-therapies in Cancer Cell Lines

Statins are widely used to manage hypercholesterolemia, and clinical trials have demonstrated that statins as monotherapies are effective anticancer agents in some patients. We aim to identify combination treatments that will expand the anticancer benefit of statins. We hypothesize that screening cancer cell lines with a genomic shRNA library and a small-molecule library will identify novel compounds to use in co-therapies with fluvastatin. A pooled shRNA dropout screen was performed to find genomic targets to inhibit alongside fluvastatin, which revealed several kinase targets as dropouts. This suggested that knocking down specific kinases can potentiate fluvastatin-induced cell death. To identify known kinase inhibitors that interact with fluvastatin to elicit cancer cell kill, we screened with a library of 320 kinase inhibitors, which revealed several putative hits. We anticipate that continued validation of candidates from both screens will lead to the identification of novel anticancer statin-based drug cocktails.

Keywords: Cancer
Statins
Kinase Inhibitors
High-Content Screening
Genomic Screening
Advisor: Penn, Linda
Improved Peripheral Nerve Recording with a Small Form-factor Nerve Electrode; A Novel Bipolar Nerve Cuff Design

Electrical nerve stimulation has been successfully used to treat a wide range of neurological disorders (e.g., epilepsy, obstructive sleep apnea) by surgical implantation of nerve cuff electrodes (NCEs). However, limited progress has been made in using this single interface as a closed-loop controlled neuroprosthetic system. Poor signal-to-noise ratio (SNR) of the recorded neural signal is one of the primary limitations of using NCEs to measure peripheral nerve activity. To this end, we investigated the feasibility of improving SNR by optimizing the design of a small form-factor, bipolar NCE. The computer simulations and animal studies were used to test specific design parameters of the NCE: electrode length, electrode edge length and an external conductive shielding layer. Both computational and experimental results suggest that each electrode parameter can significantly improve the SNR of recorded neural signals. Based on these findings, a novel electrode design for achieving bi-directionally-sensitive recording activity was also investigated.

Keywords: Action Potential
Nerve Cuff Electrode
Peripheral Nerve Recording
Signal to Noise Ratio
Advisor: Yoo, Paul B
Design and Validation of a Magnetic Resonance-conditional Robot for the Delivery of Transcranial Focused Ultrasound in Infants

Current treatment for post-hemorrhagic hydrocephalus (PHH) involves cerebral shunt placement or an invasive brain surgery. Magnetic resonance-guided focused ultrasound (MRgFUS) applied to the brains of pediatric patients presents an opportunity to treat PHH in a non-invasive manner, termed "incision-less surgery". A clinical Phillips Sonalleve MRgFUS system is expanded to perform transcranial treatment. Design criteria are defined for a robot to perform the surgery. A five degree-of-freedom MR-conditional robot is designed and manufactured. The robot is validated to the design criteria. Experimental results show the robot has achieved 0.5~mm accuracy, does not produce severe MR-imaging artifacts, has workspace providing sufficient coverage of a neonatal brain, can manipulate a 5 kg payload, and moves with enough speed to perform the surgery. A full system demonstration shows these characteristics apply in an application environment. The thesis concludes that the robot has demonstrated a capability to perform a transcranial MRgFUS thrombolysis treatment.

Keywords: Focused Ultrasound (FUS/HIFU)
Magnetic Resonance Imaging (MRI)
Mechanical Design
Medical Robotics
MR-Compatible Robotics
Surgical Robotics
Advisor: Drake, James
Development of Rayleigh Index Measurement Techniques for Swirl-stabilized Lean Premixed Combustion

The research presented in this work was conducted with the aim of using OH* chemiluminescence measurement techniques to aid in the understanding of heat release dynamics in premixed flames exhibiting thermoacoustic instabilities. Research was conducted on two experimental flames, one operating at atmospheric pressure and the second at engine-relevant high pressure. 3D reconstructions of the atmospheric flame were created for cases when a non-axisymmetric helical vortex core was observed, and thermoacoustic energy transfer fields were produced from these results. Positive and negative energy transfer regions were found to precess with the vortex core. In the high-pressure combustor, characteristics of the heat release fluctuations were determined in 2D, finding that the oscillation in heat release rate occurred as an axial wave propagating from the fuel nozzle. An analysis of the fluctuations in coupling between acoustics and heat release over time was also performed, indicating significant changes in the axial wave behaviour.

Keywords: Chemiluminescence
Combustion
Premixed
Advisor: Steinberg, Adam M
Targeted MicroRNA-126 and VEGF Gene Therapy for Chronic Ischemia in Diabetes

MicroRNAs are involved in many critical biological functions, including angiogenesis. miR-126 is an important endothelial specific angiomiR which targets negative inhibitors of the VEGF-A angiogenesis pathway. We applied ultrasound mediated gene delivery (UMGD) for miR-126 and VEGF-A delivery in a clinically relevant chronic ischemia model with Fischer and Zucker diabetic fatty (ZDF) rats. In vitro, miR-126 transfected HUVECs showed greater angiogenic potential with increased tube formation and migration response. UMGD of miR-126 produced targeted vascular transfection in rat hindlimb skeletal muscle lasting for >3 days, and resulting in inhibition of SPRED-1 and PIK3R2 protein. ZDF rats showed a significant decrease of miR-126. The dual delivery of miR-126 and VEGF-A to the ischemic leg with UMGD resulted in greatest microvascular blood improvement, vessel density, enhanced arteriolar formation than either VEGF-A or miR-126 therapy alone. UMGD is therefore a promising platform for multiple gene and miRNA delivery, with applications for therapeutic angiogenesis.

Keywords: Angiogenesis
Diabetes
Ischemia
micro RNA
Peripheral artery disease
Ultrasound Mediated Gene Delivery
Advisor: Leong-Poi, Howard
Characterization of the Ultra-short Echo Time Magnetic Resonance (UTE MR) Collagen Signal Associated with Myocardial Fibrosis

Although late-stage heart failure is often identified in the clinic, prevention of the disease before its full onset would be beneficial for improving patient outcomes. Specifically, the imaging of diffuse myocardial fibrosis, a precursor of heart failure, would enable more efficient identification of patients susceptible to this disease. Herein, I address the clinical problem by using an ultra-short echo time (UTE) technique to characterize the magnetic resonance (MR) signal properties of collagen associated with myocardial fibrosis. Via a model of bi-exponential T

Keywords: collagen
magnetic resonance imaging
myocardial fibrosis
ultra-short echo time
Advisor: Wright, Graham A
Development of a Novel Device for High-throughput Screening of Cell Contraction

Cell contraction plays a major role in promoting crucial body functions such as wound healing by contractile fibroblasts and heart beating by cardiac muscle cells. The regulation of cell contraction is vital for the tensional homeostasis of healthy tissue; dysregulation is associated with disease states such as fibrosis and chronic hypertension. Our lab has identified an increasing demand of pharmaceutical companies for a functional in vitro test to evaluate compounds that target cell contraction. None of the currently available techniques are efficient and quantitative on large cell populations. Specialized methods such as traction force microscopy and are limited for use in specifically equipped laboratories and incompatible with high-throughput methods. We have engineered a novel quantitative assay system which tests cell contractility in a high- throughput screening (HTS) format that is suitable for use on a variety of different cell types. This assay measures the amount that cells are able to deform, or wrinkle, the soft silicone substrates below them. The wrinkling assay has been transferred to a multi-well plate format compatible with industry HTS imaging systems with an automated image analysis protocol. The assay has been adapted for isometrically contracting fibroblasts and cyclically contracting cardiomyocytes. The wrinkling assay will have applications in wound healing, fibrosis, diabetes and many other contraction related conditions. Its high throughput format will allow this assay to be used by pharmaceutical companies and researchers in non-specialized laboratories alike.

Keywords: cardiomyocytes
contraction
myofibroblasts
wrinkling
Advisor: Hinz, Boris
Nurses' Experiences with Providing Newborn Screening Education to Mothers in the Hospital: An Exploratory, Qualitative Research Study

Newborn screening (NBS) parent education is an important component of a well-functioning NBS program. Postpartum nurses are in an important position to educate parents about NBS, but their experiences have not yet been captured in the literature. This qualitative study sought to explore nurses' experiences with providing NBS education to mothers on the postpartum unit by conducting one-on-one interviews with postpartum nurses and nursing leaders. An explanatory framework was developed which showed that although postpartum nurses were actively involved in providing both written and verbal information about NBS to mothers and viewed it as their responsibility, organizational factors and nurses' desire to be responsive to mothers' perceived preferences and needs limited the amount of time postpartum nurses spent on this education and the depth to which they provided it. This research illuminated the context in which NBS education occurred and suggests several research and policy implications.

Keywords: hospital
newborn screening
nurse leader
patient education
postpartum nurse
qualitative
Advisor: Miller, Fiona A