VictorJuiz/thesis huggingface dataset:
Effects of Sam68 on HIV-1 RNA Processing and Gene Expression
Keywords: Sam68
HIV-1 gene expression

The unspliced 9kb HIV-1 RNA (encoding Gag and GagPol) can undergo multiple splicing events to produce members of the 4kb (encoding Env, Vif, Vpr, and Vpu) or 2kb (encoding Tat, Rev and Nef), respectively. The incompletely spliced 9 and 4kb viral RNAs are exported by HIV-1 Rev which interacts with the RRE (Rev responsive element) in these RNAs as well as the nuclear export receptor Crm1. Several proteins can modulate Rev function and/or HIV-1 gene expression, including the nuclear phosphorprotein Sam68. We have found that overexpression of Sam68 stimulates HIV-1 structural gene expression and increases the proportion of unspliced, 3’ end processed viral RNA. This activity requires the RNA binding activity of Sam68. Surprisingly, Sam68 overexpression does not increase the proportion of unspliced, cleaved RNA found in the cytoplasm, suggesting that Sam68 alters the viral RNP to increase its translation. The Sam68 related proteins Slm1 and Slm2 also stimulate 3’ end cleavage and expression of unspliced HIV-1 RNAs. Sam68 and Slm2 were expressed in Hela cells, whereas Slm1 was not. Therefore, we reduced Sam68 expression alone or in combination with Slm2 to determine if these proteins were required for HIV-1 RNA processing or expression. Knockdown of Sam68 and/or Slm2 had little to no effect on viral RNA cleavage or structural gene expression from transiently transfected reporters. Furthermore, depletion of Sam68 only slightly reduced Gag expression from a stably expressed proviral reporter. These results suggest that additional redundant proteins may be present that functionally replace Sam68 and Slm2. We defined a region encompassing the N-terminal GSG (GRP33, Sam68, Gld1) and KH RNA binding motif as the minimal region of Sam68 required to stimulate HIV-1 gene expression in 293 and 293T cells. The minimal mutant enhanced unspliced RNA cleavage in 293T, but not in 293 cells suggesting that Sam68 may act at other stage of the viral lifecycle to increase gene expression.
Examination of Unspliced HIV-1 mRNA Translation
Keywords: HIV
RNA
Sam68
Translation

Replication of HIV-1 requires nuclear export and translation of the incompletely spliced 4 and 9 kb classes of HIV-1 mRNA, which encode the structural and enzymatic proteins of the virus. HIV-1 Rev binds to the Rev-responsive element (RRE) contained in the introns of incompletely spliced HIV-1 mRNAs and mediates their nuclear export via the Crm1 pathway. Sam68C, is a C-terminal deletion mutant of the endogenous human protein Sam68, and has been shown to be a potent inhibitor of Rev-dependent reporters. In this study we have performed deletion analysis of Sam68C, and determined the minimal mutant required for inhibition of Rev-dependent expression is Sam6814(45-54)-300. Sam68C inhibition is specific to RRE/Rev/Crm1 transported mRNAs: the Rev/Crm1 exported reporter construct GagRRE is inhibited while the Tap/p15 transported GagCTE reporter construct is not. Previous work from our lab showed that Sam68C co-localized with the Rev-exported mRNAs in perinuclear bundles. Further investigation has shown that Sam68C inhibition of incompletely spliced HIV-1 mRNAs is independent of the perinuclear bundling of the viral mRNA. We go on to show that Sam68C specifically inhibits the translation of the incompletely spliced HIV-1 mRNAs. Translational inhibition by Sam68C is correlated with a loss of PABP1 binding with no attendant change in abundance, polyadenylation or polyadenosine tail length of the affected mRNAs. The selective inhibition of Crm1 exported HIV-1 mRNAs by Sam68C suggests that it is able to recognize unique characteristics of these viral mRNPs. We show that Rev and the RRE are required, but individually neither is sufficient for complete Sam68C inhibition. Study of the incompletely spliced HIV-1 mRNP revealed that the nuclear cap binding complex, CBP20/80, is not exchanged for eIF4E. Additionally, in cells expressing the HIV-1 provirus, CBP80 relocalizes to the cytoplasm and co-sediments with polysomes. This supports the hypothesis that incompletely spliced HIV-1 mRNAs are translated in an eIF4E-independent, CBP20/80-dependent fashion. This property of the 9kb and 4kb HIV-1 mRNAs could be utilized to develop new therapeutic approaches to controlling HIV-1 infection.
The Putative Transcription Factor Prdm8 is Required for the Differentiation and Maintenance of Rod and Cone Bipolar Cells in the Mammalian Retina
Keywords: Genetics
Molecular
Neuroscience

Highly conserved genes that are abundantly and specifically expressed in the retina are likely to be important for retinal physiology or development. Prdm8 is expressed abundantly in retina and belongs to the PRDM (PR domain containing) protein family, conserved transcription factors important in the regulation of development and cancer. To begin to understand the role of Prdm8 in the retina, I cloned the cDNA and characterized Prdm8 expression in the mouse. Prdm8 encodes a PR domain and three zinc finger domains and is expressed predominantly in the developing and mature nervous system, including the retina, hippocampus and cerebellum. In the developing retina, Prdm8 mRNA is enriched in differentiating neurons. Together, these findings suggested that Prdm8 might be important for both neural development and maintenance. To determine whether Prdm8 is necessary for neural development, I generated Prdm8 null mice. Prdm8eGFP/eGFP mice are born at expected Mendelian ratios and viable, but have a hypocellular retina, develop prominent ventral skin lesions and display defects in hindlimb coordination, impaired spatial learning and memory, and changes in the volume of brain substructures. To better understand the role of Prdm8 in development, I focused on the Prdm8eGFP/eGFP retinal phenotype. I determined that Prdm8 is expressed in subsets of bipolar, amacrine and ganglion cells, and at a low level in photoreceptors. In the Prdm8eGFP/eGFP retina, cell loss was restricted to the inner nuclear layer, due to a lack of rod bipolar and cone bipolar subtypes. I found that bipolar cells were specified, but lost due to a failure in maturation, including a decrease in Vsx1 and Bhlhb4 expression, two transcription factors necessary for bipolar cell development. Consistent with this defect, Prdm8eGFP/eGFP mice exhibited impaired electroretinogram b-wave responses. Given the abundant expression of Prdm8 in the developing and mature nervous system and the array of neurobehavioural phenotypes identified, Prdm8 is likely to be necessary for development in other regions of the brain. My work provides novel insight into the genetic control of visual disorders and a useful framework for understanding the role of Prdm8 and this class of transcription factors in the development of other parts of the nervous system.
Embryonic Stem Cell Technologies for Understanding the Complexity of VEGF Function
Keywords: VEGF
embryonic development
embryonic stem cells
gastrulation
hematopoiesis
vasculogenesis
transgenes

Newly established F1 hybrid Embryonic Stem cells allow the production of ES cell-derived animals at a high enough efficiency to directly make ES cell based genetics feasible. An F1 hybrid ES cell line, G4 was used to generate transgenic over-expressing cell lines. The consequence of the expression of a panel of transgenes was assessed directly from ES cell-derived embryos produced by the tetraploid complementation assay. The generation of ES cell-derived embryos/animals was very efficient. A sufficient number of mutants for initial phenotypic analyses was derived only a few weeks after the establishment of the cell lines. The genes used in the study had either angiogenic/vasculogenic, anti-angiogenic or unknown properties. Of these transgenic mouse lines VEGF-A and Flt-Fc were used to further elucidate the effects of altered VEGF signaling on cell fate decisions in embryonic development and ES differentiation in two experimental systems. A. Early but transient Flk-1 activation led to enhanced generation of blood progenitors, whereas continuous activation of Flk-1 abolished this effect and enhanced endothelial cell generation. Ex vivo analysis of cells derived from E7.5 embryos demonstrated that sFlt-1-mediated control of Flk-1 activity also impacted the fate of hematopoietic and endothelial cells. The Flt-1-Fc transgenic mouse model was used to alter Flk-1 activation in vivo and show the relevance of the in vitro observations. These results demonstrate that sFlt-1 regulates Flk-1 activation in an oxygen responsive manner. Inhibition of Flk-1 activation by sFlt-1 increases the specification of hemangioblasts to blood cells consistent with a VEGF-independent default mechanism. B. Ubiquitous over-expression of VEGF164 isoform led to E8.75 embryonic lethality. The primary cause of lethality was the failure to form an organized cardiovascular system, which was manifested in three ways: the absence of yolk sac blood vessels, the lack of embryonic-maternal circulation due to the failure of allantochorionic fusion and improper cardiac function. The described phenotypes suggest that VEGF does not inhibit embryonic or extra-embryonic mesoderm formation at gastrulation but perturbs the balance amongst the mesodermal components.
Phosphorylated Motif Recognition and Mechanisms of Cell Signaling in Actin-cytoskeletal Regulation
Keywords: Nck
srGAP
actin cytoskeleton
nephrin
cellular signaling
podocyte cells

The actin cytoskeleton is critical to the proper function of cells and its misregulation can lead to human disease states. As a consequence, actin dynamics is tightly controlled. To gain further insight into the mechanisms controlling actin dynamics, my studies have focused on two families of proteins implicated in actin regulation. The Nck proteins act as molecular adaptors in signal propagation by linking upstream mediators, which they recognize through the Nck SH2 domain, to downstream effectors, which bind the Nck SH3 domains. I have found that Nck is required in podocyte cells for proper foot process formation, a process involving actin cytoskeletal reorganization, and therefore for proper kidney function. Furthermore, I show that Nck links the podocyte adhesion protein nephrin to actin polymerization. In cell-based assays, nephrin-induced actin polymerization is dependent on an interaction with functional Nck, which occurs through binding of three phosphorylated tyrosine sites within the cytoplasmic tail of nephrin to the Nck SH2 domain. Finally, I demonstrate that the enteropathogenic E.coli protein Tir reorganizes the cytoskeleton by molecular-mimicry of nephrin-like signaling. The srGAP proteins are GTPase activating proteins that attenuate the activity Rho GTPases, proteins directly involved in actin cytoskeletal control. The regulatory mechanisms that control srGAP activity are unclear. I have found that the srGAP family members srGAP1, srGAP2, and srGAP3 interact, through their carboxy-terminal region with 14-3-3 proteins, and that this interaction is dependent on protein kinase C-induced phosphorylation of srGAP. 14-3-3 binding does not affect the activity of srGAP2, as determined using cell-based GAP assays. Further studies are required to clarify the biological significance of this interaction to srGAP regulation. The data presented in this thesis furthers our understanding of signaling networks that control the actin cytoskeleton, and brings us closer to the goal of fully understanding actin dynamics and cellular signaling.
Development of Immunotherapy Against Prostate Cancer Using Lentivirally-transduced Dendritic Cells Expressing Murine erbB2 as a Model Tumor-associated Antigen
Keywords: Immunotherapy
Cancer

Prostate cancer is a leading cause of cancer deaths in North American men. Current treatments are often not curative, particularly in cases of advanced metastatic disease. Immunotherapy is a promising approach to treating cancer as it harnesses the immune system’s ability to mount potent responses against tumor-associated antigens (TAAs). Dendritic cells (DCs) play a central role in mediating antigen-specific immunity and have been recently used with some success in clinical trials. The difficulties associated with obtaining sufficient quantities of DCs from cancer patients provided the rationale for developing low-dose DC-based immunotherapy approaches in my thesis project. DCs were genetically engineered using a lentiviral vector (LV) to express erbB2tr, a kinase-deficient version of erbB2. The human form of erbB2, HER2/neu, is overexpressed in 20% of primary prostate tumors and 80% of their metastases, making this TAA an attractive target. Using this LV system, efficient transgene delivery into DCs was achieved without compromising DC function or phenotype. Administering low prime and boost doses (2x10^5 or 2x10^3) of LV-transduced DCs to mice yielded potent and long-term anti-tumor responses against murine prostate tumors engineered to overexpress erbB2tr. The 2x10^5 DC dose yielded complete tumor protection and was associated with humoral and cellular responses. The 2x10^3 dose also offered complete protection in some mice, suggesting that we had reached a lower threshold DC dose. This novel finding prompted us to determine if co-transducing DCs with an additional LV carrying the cDNA for an immunomodulatory factor could augment the efficacy of our low-dose strategy. We chose to test both the DC survival-enhancing RANKL protein and DC function-enhancing IL-12 in combination with erbB2tr. Although DCs co-transduced with the LV/RANKL and LV/erbB2tr did not appear to offer enhanced anti-tumor benefits in a prophylactic setting, co-transduction with LV/IL-12 and LV/erbB2tr did. The incorporation of IL-12 into the low-dose immunization strategy led to robust long-term tumor protection and relatively high levels of Th1 immunity. This is the first demonstration of the efficacy of low-dose DC-mediated immunotherapy using lentiviral vectors as gene transfer tools. These studies establish a platform for DC-mediated therapies that can be realistically translated to the clinic.
Cardiac Tissue Characterization Following Myocardial Infarction Using Magnetic Resonance Imaging
Keywords: myocardial infarction
magnetic resonance imaging

This thesis describes the development of new magnetic resonance imaging (MRI) methods to characterize cardiac tissue with myocardial infarction (MI). Wall motion imaging (for visualizing myocardial contraction) and viability imaging (to identify MI) are two components of cardiac tissue characterization used for prognosis and treatment planning. MRI-based wall motion and viability methods are considered the gold standard in imaging, and characterization of MRI viability images has been correlated with inducibility for ventricular tachycardia (VT). However, viability imaging with MRI has limitations such as difficulty visualizing the blood-infarct border. Wall motion and viability images are acquired separately, each requiring cardiac gating and breath holds, leading to long scan times. A novel multi-contrast delayed enhancement (MCDE) sequence was developed that simultaneously acquires wall motion and viability images. In a patient study, the MCDE sequence was demonstrated to provide improved visualization of MI compared to the conventional inversion-recovery gradient echo (IR-GRE) sequence, particularly for small infarcts adjacent to the blood pool. MCDE images also provided accurate wall motion images that could be used to calculate the left ventricular ejection fraction. An image processing algorithm was developed to analyze MCDE images to segment and classify the infarct gray zone, which is hypothesized to represent heterogeneous infarct responsible for causing VT. In a study of 15 patients with MI, the MCDE-derived gray zone was shown to be less sensitive to image noise than the IR-GRE-derived gray zone, and did not require manual contours of the blood pool which contributes to additional variability in the IR-GRE gray zone analysis. Finally, a real-time delayed enhancement (RT-DE) method was developed to provide black-blood viability images without requiring cardiac gating or breath holds. RT-DE imaging was shown to have a high sensitivity for detecting MI in a study of 23 patients. The methods described in this thesis help expand the patient population that can undergo a cardiac viability exam and help improve the visualization of myocardial infarct. Further modifications in the pulse sequences to improve the temporal and spatial resolutions are proposed with the goal of predicting and guiding treatment of ventricular tachycardia resulting from myocardial infarct.
Structural and Kinetic Characterization of Cell Surface and Internalized Alzheimer Amyloid Peptides in Neuronal Cells
Keywords: Alzheimer's disease
cell culture

Alzheimer’s disease is linked to the formation of amyloid fibrils, which are primarily composed of two Alzheimer amyloid peptides, Abeta40 and Abeta42. The peptides start to deposit in brains as plasma membrane-bound diffuse plaques. Current detection methods utilize dyes or antibodies that bind specific conformations of the peptides. However, these observation tools are limited, as they fail to detect the whole ensemble of pre-amyloid structural conformations. We adopted the approach of covalently attaching a fluorescent molecule to the N-terminus of Abeta, which allows for following the aggregation profile and examination of the association of Abeta with neuronal cells lines in real time. With the aid of confocal microscopy and flow cytometry, the rate of Abeta association to neuronal cell lines was found to correlate directly with their aggregation propensities. A non-aggregating mutant of Abeta42 did not bind to live cells and none of these peptides were found to associate with a non-neuronal human lymphoma cell line, U937, which is resistant to Abeta toxicity. Aggregation of Abeta42 on the surface of cells was characterized over time using photobleaching Forster resonance energy transfer, fluorescence quenching, and photobleaching recovery. Furthermore, exposed regions of Abeta aggregates on the cell surface were identified with sequence-specific antibodies. Two populations of aggregates were revealed; the first population displayed reduced energy transfer, showed fluorescence quenching, and bound antibodies specific for the C-terminal of Abeta, whereas the second population of aggregates was capable of energy transfer, was resistant to quenching, and increased in number over time. Interestingly, neither population of aggregates displayed photobleaching recovery. Addition of Abeta monomers to neuronal cells lead to the formation of cell surface aggregates that were eventually internalized into endosomes and lysosomes. The rate of internalization was greatly enhanced when the peptides were partially aggregated by exposure to conditions similar to the lumen of endosomes. Highly aggregated Abeta did not bind to neuronal cells. These studies have mapped out the aggregation pathway of Abeta in the physiologically relevant milieu of neuronal cells in culture, and have potentially revealed information about the formation of the pathological hallmark of Alzheimer’s disease, the senile plaque.
On the Relation Between Quantum Computation and Classical Statistical Mechanics
Keywords: Quantum Computation
Statistical Mechanics
Ising model
Potts model
Linear Codes
Graph Theory

We provide a quantum algorithm for the exact evaluation of the Potts partition function for a certain class of restricted instances of graphs that correspond to irreducible cyclic codes. We use the same approach to demonstrate that quantum computers can provide an exponential speed up over the best classical algorithms for the exact evaluation of the weight enumerator polynomial for a family of classical cyclic codes. In addition to this we also provide an efficient quantum approximation algorithm for a function (signed-Euler generating function) closely related to the Ising partition function and demonstrate that this problem is BQP-complete. We accomplish the above for the Potts partition function by using a series of links between Gauss sums, classical coding theory, graph theory and the partition function. We exploit the fact that there exists an efficient approximation algorithm for Gauss sums and the fact that this problem is equivalent in complexity to evaluating discrete log. A theorem of McEliece allows one to turn the Gauss sum approximation into an exact evaluation of the Potts partition function. Stripping the physics from this result leaves one with the result for the weight enumerator polynomial. The result for the approximation of the signed-Euler generating function was accomplished by fashioning a new mapping between quantum circuits and graphs. The mapping provided us with a way of relating the cycle structure of graphs with quantum circuits. Using a slight variant of this mapping, we present the final result of this thesis which presents a way of testing families of quantum circuits for their classical simulatability. We thus provide an efficient way of deciding whether a quantum circuit provides any additional computational power over classical computation and this is achieved by exploiting the fact that planar instances of the Ising partition function (with no external magnetic field) can be efficiently classically computed.
Lie Algebras of Differential Operators and D-modules
Keywords: Representation theory
Algebraic geometry

In our thesis we study the algebras of differential operators in algebraic and geometric terms. We consider two problems in which the algebras of differential operators naturally arise. The first one deals with the algebraic structure of differential and pseudodifferential operators. We define the Krichever-Novikov type Lie algebras of differential operators and pseudodifferential symbols on Riemann surfaces, along with their outer derivations and central extensions. We show that the corresponding algebras of meromorphic differential operators and pseudodifferential symbols have many invariant traces and central extensions, given by the logarithms of meromorphic vector fields. We describe which of these extensions survive after passing to the algebras of operators and symbols holomorphic away from several fixed points. We also describe the associated Manin triples, emphasizing the similarities and differences with the case of smooth symbols on the circle. The second problem is related to the geometry of differential operators and its connection with representations of semi-simple Lie algebras. We show that the semiregular module, naturally associated with a graded semi-simple complex Lie algebra, can be realized in geometric terms, using the Brion's construction of degeneration of the diagonal in the square of the flag variety. Namely, we consider the Beilinson-Bernstein localization of the semiregular module and show that it is isomorphic to the D-module obtained by applying the Emerton-Nadler-Vilonen geometric Jacquet functor to the D-module of distributions on the square of the flag variety with support on the diagonal.