CONTRIBUTION OF A SPERM PROTEIN, PAWP, TO THE SIGNAL TRANSDUCT PATHWAY DURING VERTEBRATE FERTILIZATION

PAWP, postacrosomal sheath WW domain binding protein, is a novel sperm protein identified as a candidate sperm borne, oocyte-activating factor (SOAF). PAWP induces both early and later egg activation events including meiotic resumption, pronuclear formation and egg cleavage. Based on the fact that calcium increase is universally accepted as the sole requirement for egg activation, we hypothesized that PAWP is an upstream regulator of the calcium signaling pathway during fertilization. Intracellular calcium increase was detected by two-photon laser scanning fluorescence microscopy following microinjection of recombinant PAWP into Xenopus oocytes, bolstering our hypothesis and suggesting the involvement of a novel PAWP-mediated signaling pathway during fertilization. The N-terminal of PAWP shares a high homology to WW domain binding protein while the C-terminal half contains a functional PPXY motif, which allows it to interact with group I WW domain proteins. These structural considerations together with published data indicating that PPXY synthetic peptide derived from PAWP inhibits ICSI-induced fertilization led to the hypothesis that PAWP triggers egg activation by binding to a group I WW domain protein in the oocyte. By far-Western analysis of oocyte cytoplasmic fraction, PAWP was found to bind to a 52 kDa protein. The competitive inhibition studies with PPXY synthetic peptide, WW domain constructs, and their point mutants demonstrated that the interaction between PAWP and its binding partner is specifically via the PPXY-WW domain module. The 52 kDa protein band crossreacted with antibodies against group I WW domain protein YAP in Western blot assay, indicating that this 52 kDa PAWP binding partner is either YAP or a YAP-related protein. In addition, the far-Western competitive inhibition studies with recombinant GST fusion protein YAP and another WW domain-containing protein, TAZ, demonstrated that the binding of PAWP to its binding partner was significantly reduced by TAZ, providing evidence that TAZ could be the 52 kDa protein candidate. Mass spectrometry was employed to identify this PAWP binding partner candidate. However, due to the low abundance of the candidate protein and the complexity of the sample, several strategies are still needed to enrich this protein. This study correlates PAWP induced meiotic resumption and calcium efflux at fertilization and uncovers a 52 kDa candidate WW domain protein in the oocyte cytoplasm that most likely interacts with PAWP to trigger egg activation.

Higher brain neurons succumb to acute stroke-like insult while lower brain neurons strongly resist

Pyramidal neurons (PyNs) in ‘higher’ brain are highly susceptible to acute stroke injury yet ‘lower’ brain regions better survive global ischemia, presumably because of better residual blood flow. Here we show that projection neurons in ‘lower’ brain regions of hypothalamus and brainstem intrinsically resist acute stroke-like injury independent of blood flow in the brain slice. In contrast `higher` projection neurons in neocortex, hippocampus, striatum and thalamus are highly susceptible. In live brain slices from rat deprived of oxygen and glucose (OGD), we imaged anoxic depolarization (AD) as it propagates through these regions. AD, the initial electrophysiological event of stroke, is a depolarizing front that drains residual energy in compromised gray matter. The extent of AD reliably determines ensuing damage in higher brain, but using whole-cell recordings we found that all CNS neurons do not generate a robust AD. Higher neurons generate strong AD and show no functional recovery in contrast to neurons in hypothalamus and brainstem that generate a weak and gradual AD. Most dramatically, lower neurons recover their membrane potential, input resistance and spike amplitude when oxygen and glucose is restored, while higher neurons do not. Following OGD, new recordings could be acquired in all lower (but not higher) brain regions, with some neurons even withstanding multiple OGD exposure. Two-photon laser scanning microscopy confirmed neuroprotection in lower, but not higher gray matter. Specifically pyramidal neurons swell and lose their dendritic spines post-OGD, whereas neurons in hypothalamus and brainstem display no such injury. Exposure to the Na+/K+ ATPase inhibitor ouabain (100 μM), induces depolarization similar to OGD in all cell types tested. Moreover, elevated [K+]o evokes spreading depression (SD), a milder version of AD, in higher brain but not hypothalamus or brainstem so weak AD correlates with the inability to generate SD. In summary, overriding the Na+/K+ pump using OGD, ouabain or elevated [K+]o evokes steep and robust depolarization of higher gray matter. We show that this important regional difference can be largely accounted for by the intrinsic properties of the resident neurons and that Na+/K+ ATPase pump efficiency is a major determining factor generating strong or weak spreading depolarizations.

Building a Rockfall Database Using Remote Sensing: Techniques for Hazard Management in Canadian Rail Corridors

The section of CN railway between Vancouver and Kamloops runs along the base of many hazardous slopes, including the White Canyon, which is located just outside the town of Lytton, BC. The slope has a history of frequent rockfall activity, which presents a hazard to the railway below. Rockfall inventories can be used to understand the frequency-magnitude relationship of events on hazardous slopes, however it can be difficult to consistently and accurately identify rockfall source zones and volumes on large slopes with frequent activity, leaving many inventories incomplete. We have studied this slope as a part of the Canadian Railway Ground Hazard Research Program and have collected remote sensing data, including terrestrial laser scanning (TLS), photographs, and photogrammetry data since 2012, and used change detection to identify rockfalls on the slope. The objective of this thesis is to use a subset of this data to understand how rockfalls identified from TLS data could be used to understand the frequency-magnitude relationship of rockfalls on the slope. This includes incorporating both new and existing methods to develop a semi-automated workflow to extract rockfall events from the TLS data. We show that these methods can be used to identify events as small as 0.01 m3 and that the duration between scans can have an effect on the frequency-magnitude relationship of the rockfalls. We also show that by incorporating photogrammetry data into our analysis, we can create a 3D geological model of the slope and use this to classify rockfalls by lithology, to further understand the rockfall failure patterns. When relating the rockfall activity to triggering factors, we found that the amount of precipitation occurring over the winter has an effect on the overall rockfall frequency for the remainder of the year. These results can provide the railways with a more complete inventory of events compared to records created through track inspection, or rockfall monitoring systems that are installed on the slope. In addition, we can use the database to understand the spatial and temporal distribution of events. The results can also be used as an input to rockfall modelling programs.

Multi-Scale Visualization and Interpretation of Geological Relationships

Visualization and interpretation of geological observations into a cohesive geological model are essential to Earth sciences and related fields. Various emerging technologies offer approaches to multi-scale visualization of heterogeneous data, providing new opportunities that facilitate model development and interpretation processes. These include increased accessibility to 3D scanning technology, global connectivity, and Web-based interactive platforms. The geological sciences and geological engineering disciplines are adopting these technologies as volumes of data and physical samples greatly increase. However, a standardized and universally agreed upon workflow and approach have yet to properly be developed. In this thesis, the 3D scanning workflow is presented as a foundation for a virtual geological database. This database provides augmented levels of tangibility to students and researchers who have little to no access to locations that are remote or inaccessible. A Web-GIS platform was utilized jointly with customized widgets developed throughout the course of this research to aid in visualizing hand-sized/meso-scale geological samples within a geologic and geospatial context. This context is provided as a macro-scale GIS interface, where geophysical and geodetic images and data are visualized. Specifically, an interactive interface is developed that allows for simultaneous visualization to improve the understanding of geological trends and relationships. These developed tools will allow for rapid data access and global sharing, and will facilitate comprehension of geological models using multi-scale heterogeneous observations.