In eukaryotes, the regulation of mRNA translation and decay provides a mechanism which can be finely tuned to control gene expression. This ability to control the life cycle of an mRNA begins with the control of mRNA export from the nucleus and extends to the processes which regulate the degradation of the message. In my work, summarized below, I describe how some of the proteins involved in cytoplasmic decay regulate many aspects of the control of mRNA and also describe a novel regulatory mechanism involving the relocation of cytoplasmic mRNA back into the nucleus of the cell.Firstly, I have identified that the protein Pat1, which has been shown to be critical for translational repression and activation of decapping, consists of essentially 3 major domains. By means of a deletional functional analysis, I show that two of these domains are the primary functional domains responsible for all of the currently ascribed function of Pat1. One domain promotes translational repression and P-body assembly, while the second domain promotes mRNA decapping after assembly of the mRNA into a P-body mRNP. Along with the first evidence that Pat1 binds to RNA, we also determine numerous domain-specific interactions with mRNA decapping factors.In eukaryotic cells mRNAs are produced in the nucleus followed by what is thought to be unidirectional export to the cytoplasm. In the cytosol, mRNAs either associate with ribosomes for translation or can be found in cytoplasmic RNP granules, termed P-bodies, when they are translationally repressed. I have now demonstrated that yeast mRNAs can be re-imported into the nucleus. Import of mRNAs into the nucleus is in competition with translation and increased in strains lacking specific components of cytoplasmic processing bodies, which also exhibit nuclear-cytoplasmic shuttling. This indicates that one function of cytoplasmic granules is to limit the import of cytoplasmic mRNAs back into the nucleus. These results demonstrate a novel pathway for mRNA import into the nucleus and suggest distinct pathways of mRNA export of nascent mRNAs and imported mRNAs.

Dorsal raphe nucleus (DRN) neurons projects to widespread areas throughout the brain and are involved in many physiological functions and neuropsychiatric disorders. In particular, DRN serotonin (5-HT) neurons are thought to be implicated in major depressive disorder (MDD) as are steroid hormones. Therefore, the aim of this thesis was to assess the effects of some neurosteroids on DRN neurons in non-anaesthetized rats. Initially, we examined electrophysiological properties of dorsal raphe cells across the sleep---wake cycle in non-anaethetized rats. In this first study we characterized six distinct neuronal populations in the DRN based on spike waveform and firing pattern. We then examined the effects of DHEA-S and testosterone (T) on the firing properties of DRN neuronal populations previously characterized. We observed that most populations exhibited an initial decrease in firing activity following one week of treatment. However, there was a great variability in responses across the populations.

The objective of this study was to investigate whether or not the treatment of nuclear donor cells with inhibitors of protein synthesis and cyclin-dependent kinases (CDKs) affect the development of swine embryos produced by somatic cell nuclear transfer. Host oocytes were derived from pre-pubertal females and matured in vitro for 42-46 h under standard conditions. Nuclear donor cells were fetal fibroblasts maintained in culture for 2 to 6 passages. Oocytes were reconstructed with cells treated for 22-24 hours with cycloheximide (CHX; 10mug/ml), roscovitine (ROS; 25 muM), the combination of CHX + ROS (CR), or untreated cells. Two hours after reconstruction, the oocytes were activated using ionomycin (15muM/5 min) and strontium chloride (10mM/4h), maintained for 6 h in the presence of cytochalasin B (7.5mug/ml) and CHX (10mug/ml), and then cultured in porcine zygote medium (PZM3) for 6 days. The cleavage rate, 63.7% (n=318), 55.2% (n=99), 56.7% (n=107) and 60.6% (n=347), at 48 h post-fusion were not significantly different between embryos derived from ROS, CHX, CR and control cells, respectively. Developmental rate to blastocyst stage was higher for embryos reconstructed with ROS (12.2%) and untreated cells (12.1%) when compared to CHX (5.7%) and CR (4.9%). Blastocysts produced with ROS treated cells had similar number of nuclei compared to embryos reconstructed with untreated donor cells (30.9+/-10.4 vs. 32.2+/-8.0). Phosphorylated H2A.X (gammaH2A.X) was highly expressed in donor cells treated with CR compared to non treated cells, but it was similarly expressed in most of 1-cell stage embryos reconstructed with control or treated cells. Flow cytometry analysis showed that the majority of the fibroblasts were at G 0/G1 phase of the cell cycle at the time of nuclear transfer. It was concluded that the treatment of nuclear donor cells with inhibitors of protein synthesis and CDKs did not improve the in vitro development of somatic cell nuclear transfer embryos in pigs.

No description available.

This thesis is concerned with very high energy γ-ray observations of blazars observable from the southern hemisphere. The data presented were obtained using the recently deployed University of Durham Mark 6 high sensitivity Čerenkov telescope. Chapters 1 and 2 are introductory; the subjects of γ-ray astronomy, extensive air showers, Čerenkov light production, the development of the atmospheric Čerenkov technique and the current status of TeV astronomy are discussed. Chapter 3 introduces the telescopes operated by the University of Durham. The Mark 6 telescope, designed to have a low threshold energy and a high resolution imaging system, is discussed in detail. Chapter 4 presents the calibration and analysis techniques routinely applied to data obtained with the Mark 6 telescope. The chapter concludes with a set of moment parameter selections designed to reject a significant fraction of the cosmic ray cascades, while retaining the majority of γ-rays cascades. These selections have resulted in a 5σ detection of PSR 1706-44. Chapter 5 discusses active galactic nuclei and in particular blazars. Topics included are recent high energy observations; the differences between the radio/X-ray selected BL Lacs and flat spectrum radio quasars; the infrared background; high energy flux variability and γ-ray production mechanisms within these objects. The chapter concludes with a list of possible very high energy γ-ray emitting blazars observable with the Mark 6 Čerenkov telescope. Chapter 6 presents the results from four of these very high-energy γ-ray blazar candidates; PKS 0548-322, PKS 1514-24, PKS 2005-489 and PKS 2155-304. There is no evidence for γ-ray emission from these sources, either in the form of a steady flux or variable activity. Three sigma integral flux upper limits above 300 GeV are produced for these objects and the implications of these observations are discussed. The conclusions are presented in Chapter 7 as well as ideas for future work.

The Advanced Camera for Surveys (ACS) Fornax Cluster Survey is a Hubble Space Telescope programme that imaged 43 early-type galaxies in the Fornax cluster, using the ACS F475W (~g) and F850LP (~z) bandpasses. We use this data set, which spans a range of ~600 in blue luminosity down to M_B ~ -16 mag, to study and characterize the properties of central galactic nuclei by fitting ellipses to the galaxy isophotes and examining their 1-dimensional surface brightness profiles. To test the robustness of this method, we perform a similar analysis with 2-dimensional surface brightness profile fitting using GALFIT, and find acceptable agreement between the derived structural parameters from the two techniques. We determine 72% of our sample (31 galaxies) to be nucleated, a significant increase from 28% found in the ground-based study of Ferguson et al. (1989). This high frequency of nucleation suggests that the creation of a compact nuclear component is a common outcome in early-type galaxy formation. Only three of the nuclei (10%) are observed to be significantly offset (by > 0.5") from their host galaxy photocentres, and a trend of increasing offset in fainter galaxies is observed, which indicates that nucleus formation timescales and/or pathways may vary with host luminosity. The nuclei are found to be larger and approximately 50 times brighter than typical globular clusters from our Fornax sample, and to follow different half-light surface brightness versus magnitude scaling relations. The colours of the nuclei are characteristic of intermediate to old stellar populations, and those residing in galaxies with B_T > 13 mag are observed to correlate with the nucleus luminosities. Both nucleus and galaxy colours become redder with increasing host luminosity, although the trend with the nuclei is steeper, and the nuclei in the brightest galaxies are found to be redder than their hosts. However, the majority of nuclei are bluer than their hosts, having an average colour difference of 0.27 +/- 0.25 mag. Comparison of our results to the complementary ACS Virgo Cluster Survey (ACSVCS) study of nuclei Côté et al. (2006), which examined 100 early-type galaxies in the Virgo cluster, yields strikingly similar results. Both samples show similar frequency of nucleation (68% in the ACSVCS), a constant nucleus-to-galaxy luminosity ratio (with a mean value of 0.41% +/- 0.04% derived from the combined samples), as well as excellent agreement in the nucleus luminosity functions and sizes (with median values of 6 pc in g and 7 pc in z in both studies). Since the Fornax cluster presents a much denser environment than Virgo, such consistency between the properties of the nuclei indicates that their formation and evolution may be influenced by local factors more than environmental ones. In particular, a constant nucleus-to-galaxy luminosity ratio suggests that a host galaxy's luminosity (or, more likely, mass) may be a key element in determining the properties of its nucleus. Since simulations have found the two main theorized nucleus formation pathways to be effective on different mass scales (with dissipationless infall of star clusters being more efficient in lower-mass galaxies, and in-situ gas accretion in higher-mass hosts), we propose that both processes may both in fact be responsible for nucleus formation, but varying in relative importance along the galaxy luminosity function. / Graduate

This thesis develops image segmentation methods for the application of automated cervical cancer screening. The traditional approach to automating this task has been to emulate the human method of screening, where every one of the hundreds of thousands of cells on each slide is analysed for abnormality. However, due to the complexity of cervical smear images and the low error tolerance imposed upon the segmentation stage, only limited success has previously been found. A different approach is to detect malignancy associated changes (MACs) in a relatively small sample of the total population of cells. Under this paradigm, the requirement to segment every cell is loosened, but delineation accuracy and error checking become essential. Following a review of generic and cervical smear segmentation, it is concluded that prior work on the traditional approach to automation is not suitable for a MACs solution. However, the previously proposed framework of a dual-magnification system is found to be relevant and is therefore adopted. Here, scene images are first captured at low resolution in order to rapidly locate the cells on a slide. Cells that are deemed to be suitable for further analysis are then imaged at high resolution for the more accurate segmentation of their nuclei. A water immersion algorithm is developed for low resolution scene segmentation. This method achieves a rapid and robust initial segmentation of the scene without the requirement of incorporating extensive a priori knowledge of the image objects. A global minimum searching contour is presented as a top-down method for segmenting the high resolution cell nucleus images where the image objects are well characterised by shape and appearance. This latter method is tested upon 20,000 images and found to achieve an accurate segmentation rate of 99.47%. An error checking method, that uses segmentation stability as an indicator of segmentation success, is developed that is capable of detecting 100% of the failures of the nucleus segmenter, at the expense of discarding only 9% of the data. Throughout this work, contemporary issues in the field of generic image segmentation are presented and some of these are addressed for the cervical smear application. Finally, an avenue of future work is proposed which may lead to the much wider proliferation of computer vision solutions to everyday problems.

Thesis (PhD) -- McMaster University, 1996. / Includes bibliographical references (p.260-271). Also available via World Wide Web.

Thesis (M. Phil.)--University of Hong Kong, 2007. / Also available in print.

Thesis (Ph. D.)--Virginia Polytechnic Institute and State University, 1994. / Vita. Abstract. Includes bibliographical references (leaves 130-161). Also available via the Internet.