My project has been involved in analysing a class of interneuron that expresses the calcium‐binding protein parvalbumin (PV). In my thesis, I will describe the application of a method that involves the local injection of Creactivated recombinant adeno-associated viruses (AAVs) into a transgenic mouse line that expresses Cre recombinase in PV positive cells. This will drive the expression of a transgene specifically in PV positive cells, at a specific brain region. In the first part of my project, I used this method to introduce the molecular trans-synaptic tracer proteins wheat germ agglutinin and tetanus toxin heavy chain specifically to PV positive neurons to visualize their postand pre‐synaptic connections, respectively. What I found is that while our technique of combining Cre-activated AAVs in transgenic mice has allowed specific labelling of neurons in a brain region and cell type specific manner, we could not definitively identify trans-synaptically traced neurons. In the second part of my project I have used these novel AAV‐based techniques in mice to introduce tetanus toxin light chain (TeLC) to PV neurons in the dentate gyrus. This has been previously used to functionally remove PV neurons from the CA1 of the hippocampus. This protein inhibits neurotransmitter release by cleaving the vesicle docking protein, VAMP2. The DG has been implicated in the separation of sensory inputs (pattern separation) which increases the resolution of the encoded memory and thereby assists in the accurate recall. The lateral inhibition of excitatory activity in the DG is believed to aid accurate encoding. Using our AAV method, I found that PV positive interneurons are required for spatial working and reference memory. Using a new behavioural assay that I developed, I could also show that these neurons are needed to enhance the resolution of spatial information. However, I also discovered that long term expression of TeLC could result in neuronal cell death. I have therefore demonstrated that local injection of Cre recombinase activated AAVs allows for a quick, versatile method of genetic manipulation, provided long term expression (greater than 2 months) is not required.

No description available.

No description available.

The issue of subject pronominal expression (SPE) in language contact situations, as illustrated in Nosotros somos muy buenos estudiantes [‘We are very good students’] vs. Ø somos muy buenos estudiantes [‘(we) are very good students’], has been the focus of decades of research in both variationist and generative studies (Bentivoglio, 1987; Cameron, 1992, 1993, 1995, 1996; Chomsky, 1981, 1986; Huang, 1984; Orozco & Guy, 2008; Travis, 2005a, 2005b; Otheguy & Zentella, 2007, 2012; Rizzi, 1982; Morales, 1980; Silva-Corvalán, 1982, 1994, 1997; inter alios). While generative studies have shown that null subject languages (NSLs) can either be licensed by rich verbal paradigms or by discursive mechanisms, variationist studies have shown that there are predictors that condition SPE in NSLs. Furthermore, they have argued that the rates of SPE reflect uniformity across different varieties of Spanish (Orozco, 2015). In spite of this significant body of evidence in monolingual varieties of Spanish, studies involving bilingual groups and indigenous languages are relatively sparse, as most studies have studied Spanish in contact with other Indo-European languages. This dissertation investigates the SPE in the Spanish of a group of highly proficient bilingual speakers of Uwa and Spanish in a language contact situation. The research reported in this dissertation also studies the nature of cross-linguistic influence between the two languages of the bilinguals. Specifically, the idiosyncratic morphosyntactic traces in SPE resulting from the contact between Uwa and Spanish and whether those traces evidence patterns of variation, and, if so, how they can be accounted for. Spanish and Uwa are both NSLs and thus both allow for referential null subject pronouns (SPs). However, each language has specific syntactic and discursive predictors responsible for null subjects. For instance, Uwa relies heavily on discursive clues whereas Spanish is a sentence-oriented language. This means that while in Uwa the subject reference is understood from context and discursive clues governed by chains of topics, the rich verbal paradigm of Spanish is responsible for licensing null subjects. The fact that Spanish and Uwa are both NLs but still have dissimilar typological status provides a rich testing ground for the mechanisms involved in SPE as well as for investigating the effects of bilingualism. This dissertation aims at enhancing our linguistic knowledge in relation to the principles and mechanisms involved in SPE in bilingual communities and at providing a better understanding of the nature of cross-linguistic influence in highly proficient bilinguals.

A dissertation submitted to the Faculty of Science, University of the Witwatersrand, Johannesburg, in fulfilment of requirements for the degree of Master of Science. Johannesburg, 2015. / Claudins are a family of proteins that are conserved amongst all vertebrates, they are integral in the formation and maintenance of the tight junctions between epithelial cells. Claudins are implicated in embryo morphogenesis, vertebrate evolution, solute movement, cell-cell adhesion, designation of cellular and tissue identity, and several diseases when mutated. Petromyzon marinus (the sea lamprey) is the most basal extant vertebrate and is a model organism in both developmental and evolutionary biology for this reason. In this study, the expression patterns and functions of novel claudin genes in P. marinus were examined with the aim of discovering more about the role of claudins in vertebrate evolution. Presumptive claudin genes in P. marinus were compared to all known claudins in the NCBI database. Primers were designed against all known P. marinus claudin genes and RT-PCR was performed in order to determine their expression levels at embryonic stages E8 to E18, as well as in adult eye, gill, heart, liver and skin tissues. Probes were designed against Claudin 1a, Claudin 9, Claudin 10 and Claudin 19b and RNA in situ hybridisation was performed on embryos at developmental stages E4 to E31 in order to determine their spatial expression patterns. Areas of common claudin gene expression appear to include the pharyngeal arches, otic placode, neural tube, notochord and ectoderm. Claudin 1a is uniquely expressed in the lamprey migrating neural crest. Morpholino-mediated gene knockouts were performed on P. marinus embryos and the loss of Claudin 19b appears to result in abnormal somite morphogenesis.

A dissertation submitted to the Faculty of Science, University of the Witwatersrand, Johannesburg, in fulfilment of requirements for the degree of Master of Science. October 2014. / Despite the fact that there has been quite a lot of research done in the eld of facial expression recognition, not much development has occurred in detecting the intensity of facial actions. In facial expression recognition, the intensity of facial actions is an important and crucial aspect, since it would provide more information about the facial expression of an individual, such as the level of emotion in a face. Furthermore, having an automated system that can detect the intensity of facial actions in an individual's face can lead up to a lot of potential applications from lie detection to smart classrooms. The provided approach includes robust methods for face and facial feature extraction, and multiple machine learning methods for facial action intensity detection.

Chow Hoi Yee. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (leaves 127-153). / Abstracts in English and Chinese. / Abstract --- p.iii / Acknowledgments --- p.vi / Abbreviations --- p.vii / Table of contents --- p.viii / List of Figures --- p.xi / List of Tables --- p.xiv / Chapter Chapter One --- Literature Review / Chapter 1.1 --- Introduction --- p.1 / Chapter 1.2 --- The life cycle of rice --- p.3 / Chapter 1.3 --- Physiological and molecular studies on rice leaf development --- p.6 / Chapter 1.3.1 --- Physiological study of rice leaf development --- p.6 / Chapter 1.3.1.1 --- Leaf primordium formation and SAM --- p.6 / Chapter 1.3.1.2 --- Leaf expansion and water status --- p.7 / Chapter 1.3.1.3 --- Leaf senescence and phytohormone --- p.8 / Chapter 1.3.1.4 --- Rice leaf and temperature --- p.9 / Chapter 1.3.2 --- Molecular study of rice leaf development / Chapter 1.3.2.1 --- Leaf primordium formation and SAM --- p.10 / Chapter 1.3.2.2 --- Leaf elongation and related genes --- p.13 / Chapter 1.3.2.3 --- Leaf senescence and related genes --- p.13 / Chapter 1.3.2.4 --- Photoreceptor genes --- p.14 / Chapter 1.3.2.5 --- Temperature-related genes --- p.17 / Chapter 1.4 --- Prospectus --- p.18 / Chapter Chapter Two --- Isolation of Genes Differentially Expressed During the Development of Rice by RAP-PCR / Chapter 2.1 --- Introduction --- p.20 / Chapter 2.2 --- Materials and Methods --- p.23 / Chapter 2.2.1 --- Strains and culture conditions --- p.23 / Chapter 2.2.2 --- Isolation of total RNAs --- p.23 / Chapter 2.2.3 --- cDNA Library construction --- p.24 / Chapter 2.2.3.1 --- First strand synthesis --- p.24 / Chapter 2.2.3.2 --- cDNA amplification by LD PCR --- p.25 / Chapter 2.2.3.3 --- Proteinase K digestion --- p.25 / Chapter 2.2.3.4 --- Sfi digestion --- p.26 / Chapter 2.2.3.5 --- cDNA size fractionation by CHROMA-SPIN´ёØ-400 --- p.26 / Chapter 2.2.3.6 --- Ligation of cDNA to λTripEx2vector --- p.26 / Chapter 2.2.3.7 --- Titering the unamplified library --- p.27 / Chapter 2.2.3.8 --- Determining the percentage of recombinant clones --- p.27 / Chapter 2.2.3.9 --- Library amplification --- p.28 / Chapter 2.2.3.10 --- Titering the amplified library --- p.28 / Chapter 2.2.3.11 --- Converting λ TripEx2 recombinant clones to pTripEx2 recombinant plasmids --- p.28 / Chapter 2.2.4 --- RNA fingerprinting by RAP-PCR --- p.29 / Chapter 2.2.5 --- Reverse dot-blot analysis --- p.30 / Chapter 2.2.5.1 --- Membrane preparation --- p.30 / Chapter 2.2.5.2 --- Probe preparation --- p.30 / Chapter 2.2.5.3 --- Hybridization --- p.31 / Chapter 2.2.5.4 --- Stringency washes and chemiluminescent detection --- p.31 / Chapter 2.2.6 --- Sequencing of differentially expressed genes --- p.32 / Chapter 2.2.6.1 --- Extraction of plasmid DNA --- p.32 / Chapter 2.2.6.2 --- DNA cycle sequencing --- p.33 / Chapter 2.3 --- Results --- p.34 / Chapter 2.3.1 --- Total RNA isolation --- p.34 / Chapter 2.3.2 --- cDNA library --- p.37 / Chapter 2.3.3 --- RNA fingerprinting --- p.40 / Chapter 2.3.4 --- Reverse dot-blot analysis --- p.45 / Chapter 2.3.5 --- Sequence analyses --- p.48 / Chapter 2.4 --- Discussion --- p.68 / Chapter Chapter Three --- cDNA Microarray Analysis and expression Pattern Analysis by Northern Blot Hybridization / Chapter 3.1 --- Introduction --- p.71 / Chapter 3.2 --- Materials and Methods --- p.76 / Chapter 3.2.1 --- RNA extraction by RNeasy® Mini Kit for cDNA microarray --- p.76 / Chapter 3.2.2 --- Microarray analysis --- p.76 / Chapter 3.2.2.1 --- Array preparation --- p.76 / Chapter 3.2.2.2 --- Probe preparation --- p.78 / Chapter 3.2.2.3 --- Hybridization --- p.79 / Chapter 3.2.2.4 --- Stringency washes and TSA Detection --- p.79 / Chapter 3.2.2.5 --- Microarray Analyses --- p.80 / Chapter 3.2.3 --- RNA extraction by Tri-reagent for Northern Blot hybridization --- p.81 / Chapter 3.2.4 --- RNA fragmentation by formaldehyde gel electrophoresis --- p.81 / Chapter 3.2.5 --- Northern blotting --- p.82 / Chapter 3.2.6 --- Preparation of probe --- p.83 / Chapter 3.2.7 --- Hybridization and stringency washes --- p.84 / Chapter 3.2.8 --- Stripping and reprobing of Northern Blot --- p.85 / Chapter 3.3 --- Results --- p.86 / Chapter 3.3.1 --- Total RNA isolation --- p.86 / Chapter 3.3.2 --- Microarray analysis --- p.86 / Chapter 3.3.3 --- Normalization of microarray data --- p.91 / Chapter 3.3.4 --- Detection of probe labeling efficiency --- p.92 / Chapter 3.3.5 --- Northern blot analysis --- p.100 / Chapter 3.3.5.1 --- Genes expressed highly in primordia stage --- p.100 / Chapter 3.3.5.2 --- Genes with low expression in primordia stage --- p.101 / Chapter 3.3.5.3 --- Genes with high expression in half expanded stage --- p.101 / Chapter 3.3.5.4 --- Genes steadily increased in expression throughout the development --- p.103 / Chapter 3.3.5.5 --- Genes with low expression in fully expanded stage --- p.104 / Chapter 3.4 --- Discussion --- p.114 / Chapter Chapter four --- General Discussion --- p.122 / References --- p.127

Wong, So-wan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (leaves 44-53). / Abstracts in English and Chinese. / Chapter I --- Abstract / English Version --- p.i / Chinese Version --- p.ii / Chapter II --- Acknowledgement --- p.iii / Chapter III --- Table of Contents --- p.v / Chapter IV --- Chapter 1: Introduction --- p.1 / Theories of Emotional Expressivity - Individual Level --- p.4 / Cross-Cultural Studies on Emotional Expressivity --- p.11 / Model of Emotional Expressivity of the Present Study --- p.17 / Chapter V --- Chapter 2: Method / Data Source --- p.22 / Data Analyses --- p.25 / Chapter VI --- Chapter 3: Results --- p.27 / Chapter VII --- Chapter 4: Discussion --- p.32 / Model of Emotional Expressivity --- p.32 / Future Direction --- p.40 / Concluding Notes --- p.42 / Chapter VIII --- References --- p.44 / Chapter IX --- Tables --- p.54 / Chapter X --- Figure --- p.60 / Model of Emotional Expressivity at the Individual Level --- p.61 / Dendogram of hierarchical cluster analysis of the three forms of emotional expressions for the emotion of anger --- p.62 / Dendogram of hierarchical cluster analysis of the three forms of emotional expressions for the emotion of joy --- p.63

Mak Wai Yan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves 95-115). / Abstracts in English and Chinese. / Declaration --- p.i / Abstract --- p.ii / Acknowledgements --- p.viii / Table of Contents --- p.ix / List of Tables --- p.xiii / List of Figures --- p.xiv / List of Abbreviations --- p.xvi / Chapter Chapter 1 --- Introduction and Literature Review / Chapter 1.1 --- Introduction --- p.1 / Chapter 1.2 --- Endocrine control of reproduction in crustaceans --- p.2 / Chapter 1.3 --- The X-organ sinus gland (XOSG) complex --- p.3 / Chapter 1.3.1 --- CHH/MIH/GIH family of neuropeptides --- p.4 / Chapter 1.3.2 --- Gonad inhibiting hormone (GIH) --- p.6 / Chapter 1.3.3 --- Molt inhibiting hormone (MIH) --- p.7 / Chapter 1.3.4 --- Crustaceans hyperglycemic hormone (CHH) --- p.9 / Chapter 1.3.5 --- The chormatophorotropins (RPCH and PDH neuropeptide family) --- p.10 / Chapter 1.4 --- Other mechanisms of reproduction control --- p.11 / Chapter 1.4.1 --- Methyl farnesoate (MF) and mandibular organ inhibiting hormone (MOIH) --- p.11 / Chapter 1.4.2 --- Gonad stimulating hormone (GSH) --- p.13 / Chapter 1.4.3 --- Serotonin (5HT) --- p.13 / Chapter 1.4.4 --- Dopamine --- p.14 / Chapter 1.4.5 --- Enkephalin (ENK) --- p.15 / Chapter 1.4.6 --- 17β-Estradiol --- p.16 / Chapter 1.4.7 --- Progesterone --- p.17 / Chapter 1.5 --- Androgenic hormone (AH) --- p.18 / Chapter 1.6 --- Objective and methodology of present research --- p.19 / Chapter 1.7 --- Reproductive biology of the shrimp Metapenaeus ensis --- p.20 / Chapter Chapter 2 --- Materials and Methods / Chapter 2.1 --- Introduction --- p.26 / Chapter 2.2 --- Extraction of eyestalk RNA from the shrimp --- p.27 / Chapter 2.3 --- Construction of the eyestalk cDNA library --- p.28 / Chapter 2.3.1 --- First-strand cDNA synthesis --- p.28 / Chapter 2.3.2 --- cDNA amplification by LD PCR --- p.29 / Chapter 2.3.3 --- Proteinase K digestion --- p.29 / Chapter 2.3.4 --- Sfi I digestion --- p.30 / Chapter 2.3.5 --- cDNA size fractionation --- p.30 / Chapter 2.3.6 --- Ligation of cDNA to λ TriplEx2 Vector --- p.31 / Chapter 2.3.7 --- Packaging --- p.31 / Chapter 2.3.8 --- Titering the unamplified library --- p.32 / Chapter 2.3.9 --- Library amplification --- p.33 / Chapter 2.3.10 --- Titering the amplified library --- p.34 / Chapter 2.4 --- Mass Excision of the eyestalk cDNA library --- p.34 / Chapter 2.5 --- PCR screening of inserted sequence --- p.35 / Chapter 2.6 --- RNA arbitrarily primed polymerase chain reaction (RAP-PCR) --- p.35 / Chapter 2.7 --- Dot blot hybridization --- p.37 / Chapter 2.7.1 --- Probe Labelling --- p.37 / Chapter 2.7.2 --- Dotting of membrane --- p.38 / Chapter 2.7.3 --- Hybridization --- p.38 / Chapter 2.7.4 --- Washing and chemiluminescent detection --- p.39 / Chapter 2.7.5 --- Probe stripping for re-hybridization --- p.40 / Chapter 2.8 --- Sequencing --- p.40 / Chapter 2.9 --- BLAST search --- p.41 / Chapter 2.10 --- Northern blot analysis --- p.41 / Chapter 2.10.1 --- Probe labelling --- p.41 / Chapter 2.10.2 --- RNA formaldehyde denaturing gel electrophoresis --- p.41 / Chapter 2.10.3 --- Northern blot --- p.42 / Chapter 2.10.4 --- Pre-hybridization --- p.43 / Chapter 2.10.5 --- Hybridization --- p.43 / Chapter 2.10.6 --- Washing and chemiluminescent detection --- p.43 / Chapter 2.11 --- Real-time RT-PCR --- p.44 / Chapter 2.11.1 --- DNaseI treatment --- p.44 / Chapter 2.11.2 --- First strand synthesis --- p.44 / Chapter 2.11.3 --- Primer design and verification --- p.45 / Chapter 2.11.4 --- Real-time PCR --- p.45 / Chapter 2.11.5 --- Statistical analysis --- p.45 / Chapter Chapter 3 --- Results / Chapter 3.1 --- Experimental animals --- p.50 / Chapter 3.2 --- Total RNA extraction --- p.50 / Chapter 3.3 --- Library construction --- p.50 / Chapter 3.4 --- PCR screening of inserted sequences --- p.50 / Chapter 3.5 --- RNA arbitrarily primed polymerase chain reaction (RAP-PCR) --- p.51 / Chapter 3.6 --- Dot blot hybridization --- p.51 / Chapter 3.7 --- Sequencing and BLAST search --- p.52 / Chapter 3.8 --- Northern blot analysis --- p.53 / Chapter 3.8.1 --- Housekeeping gene - elongation factor la --- p.53 / Chapter 3.8.2 --- Insulin-like growth factor binding protein (IGFBP) --- p.54 / Chapter 3.8.3 --- Arrestin --- p.54 / Chapter 3.8.4 --- Opsin --- p.54 / Chapter 3.9 --- Real-time RT-PCR --- p.55 / Chapter 3.9.1 --- β-Actin --- p.55 / Chapter 3.9.2 --- Farnesoic acid O-methyltranferase (FAMeT) --- p.56 / Chapter Chapter 4 --- Discussion / Chapter 4.1 --- Application of RAP-PCR and dot blot analysis in identifying differential expressed genes --- p.79 / Chapter 4.1.1 --- Abundant genes --- p.79 / Chapter 4.1.2 --- Appearance of 16S rRNA in eyestalk cDNA library of M. ensis --- p.80 / Chapter 4.1.3 --- False positive --- p.80 / Chapter 4.2 --- Investigation on common housekeeping genes --- p.81 / Chapter 4.3 --- Potential functions of identified differential expressed genes in reproduction of shrimp --- p.82 / Chapter 4.3.1 --- Arrestin --- p.84 / Chapter 4.3.2 --- Opsin --- p.86 / Chapter 4.3.3 --- Insulin-like growth factor binding protein (IGFBP) --- p.88 / Chapter 4.3.4 --- β-Actin --- p.89 / Chapter 4.4 --- Investigation for farnesoic acid O-methyltransferase (FAMeT) in shrimp --- p.89 / Chapter Chapter 5 --- General Conclusion --- p.91 / References --- p.95 / Appendix --- p.116

Post-transcriptional control of gene expression is critical for normal cellular function and viability. Poly(A)-binding protein (PABP) 1 is the prototypical member of a family of RNA-binding proteins which are key post-transcriptional regulators. PABP1 is multifunctional, acting as a primary determinant of translation efficiency and mRNA stability, regulating the fate of specific mRNAs, and participating in microRNAmediated regulation and nonsense-mediated mRNA decay. As well as binding various mRNAs, PABP1 achieves its multifunctionality through protein-protein interactions with numerous PABP-interacting motif (PAM)-2 motif-containing protein partners. These have been identified to bind the same site within the C-terminal PABC domain, therefore it is unclear how different PABP1 functions are coordinated. Recently, PABP1 was found to exhibit extensive post-translational modification (PTM), including putative lysine acetylation/methylation switches, which were suggested as a potential mechanism by which interactions with different PAM2 motifcontaining proteins may be regulated. In particular, in silico molecular modelling of the acetylation or dimethylation of the position 606 lysine residue (Lys606) within the PABC domain, using available structures of PABC in complex with PAM2 peptides of eukaryotic release factor (eRF)-3a and PABP-interacting protein (Paip)-2, suggested that modification of this residue, which is critical in PABC-PAM2 interactions, may differentially affect these PABP1 interactions. To examine the role of the Lys606 modification as a molecular switch to dictate PABC-mediated protein-protein interactions, site-specifically acetylated recombinant PABC domain was generated using cutting–edge amber codon suppression recoding technology. Following sequential purification by affinity, ion exchange and size exclusion chromatography, recombinant PABC protein quality was analysed by biophysical approaches such as thermal denaturation assay (TDA), dynamic light scattering (DLS), circular dichroism (CD) and liquid chromatography mass spectrometry (LCMS). Biochemical and biophysical analysis of PABC-PAM2 interactions was subsequently undertaken using GST-pulldown analysis, with the well characterised Paip2 protein, and Surface Plasmon Resonance (SPR) using PAM2 peptides of eRF3, Paip2 and trinucleotide repeat-containing (Tnrc) 6C (or GW182) proteins. These revealed that PABC Lys606 acetylation significantly increased the affinity and increased the association rate for eRF3 peptide. In contrast, effects on Paip2 peptide binding were less suggestive. Furthermore, although approaches to decipher the biological relevance of Lys606 and its modifications within cells are in their infancy, they reflect the complexity of studying PTM function in vitro. Overall, these data provide support for the hypothesis that Lys606 modification status confers selectivity between PABP1 protein partners suggesting a potential mechanism for how its multi-functionality may be coordinated.