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This research is concerned with the application of neural network techniques to the problems of classifying images in a manner that is invariant to changes in position and scale. In addition to the goal of invariant classification, the network has to classify the objects in a hierarchical manner, in which complex features are constructed from simpler features, and use unsupervised learning. The resultant hierarchical structure should be able to classify the image by having an internal representation that models the structure of the image. After finding existing neural network techniques unsuitable, a new type of neural network was developed that differed from the conventional multi-layer perceptron type of architecture. This network was constructed from neurons that were grouped into feature detectors. These neurons were taught in an unsupervised manner that used a technique based on Kohonen learning. A number of novel techniques were developed to improve the learning and classification performance of the network. The network was able to retain the spatial relationship of the classified features; this inherent property resulted in the capability for position and scale invariant classification. As a consequence, an additional invariance filter was not required. In addition to achieving the invariance property, the developed techniques enabled multiple objects in an image to be classified. When the network had learned the spatial relationships between the lower level features, names could be assigned to the identified features. As part of the classification process, the system was able to identify the positions of the classified features in all layers of the network. A software model of an artificial retina was used to test the grey scale classification performance of the network and to assess the response of the retina to changes in brightness. Like the Neocognitron, the resulting network was developed solely for image classification. Although the Neocognitron is not designed for scale or position invariance, it was chosen for comparison purposes because it has structural similarities and the ability to accommodates light changes in the image. This type of network could be used as the basis for a 2D-scene analysis neural network, in which the inherent parallelism of the neural network would provide simultaneous classification of the objects in the image.

The current project is to develop a transient expression system for Chinese Hamster Ovary (CHO) cells based on autonomous replication and retention of plasmid DNA. The expression system, named Epi-CHO comprises (1) a recombinant CHO-K1 cell line encoding the Polyoma (Py) virus large T-Antigen (PyLT-Ag), and (2) a DNA expression vector, pPy/EBV encoding the Py Origin (PyOri) for autonomous replication and encoding the Epstein-Barr virus (EBV), Nuclear Antigen-1 (EBNA-1) and EBV Origin of replication (OriP) for plasmid retention. The CHO-K1 cell line expressing PyLT-Ag, named CHO-T was adapted to suspension growth in serum-free media (EXCELL-302) to facilitate large scale transient transfection and recombinant (r) protein production. PyLT-Ag-expressed in CHO-T supported replication of PyOri-containing plasmids and enhanced growth and r- protein production. A scalable cationic lipid based transfection was optimised for CHO-T cells using LipofectAMINE-2000??. Destabilised Enhanced Green Fluorescence Protein (D2EGFP) and Human Growth Hormone (HGH) were used as reporter proteins to demonstrate transgene expression and productivity. Transfection of CHO-T cells with the vector pPy/EBV encoding D2EGFP showed prolonged and enhanced EGFP expression, and transfection with pPy/EBV encoding HGH resulted in a final concentration of 75 mg/L of HGH in culture supernatant 11 days following transfection.

The current project is to develop a transient expression system for Chinese Hamster Ovary (CHO) cells based on autonomous replication and retention of plasmid DNA. The expression system, named Epi-CHO comprises (1) a recombinant CHO-K1 cell line encoding the Polyoma (Py) virus large T-Antigen (PyLT-Ag), and (2) a DNA expression vector, pPy/EBV encoding the Py Origin (PyOri) for autonomous replication and encoding the Epstein-Barr virus (EBV), Nuclear Antigen-1 (EBNA-1) and EBV Origin of replication (OriP) for plasmid retention. The CHO-K1 cell line expressing PyLT-Ag, named CHO-T was adapted to suspension growth in serum-free media (EXCELL-302) to facilitate large scale transient transfection and recombinant (r) protein production. PyLT-Ag-expressed in CHO-T supported replication of PyOri-containing plasmids and enhanced growth and r- protein production. A scalable cationic lipid based transfection was optimised for CHO-T cells using LipofectAMINE-2000??. Destabilised Enhanced Green Fluorescence Protein (D2EGFP) and Human Growth Hormone (HGH) were used as reporter proteins to demonstrate transgene expression and productivity. Transfection of CHO-T cells with the vector pPy/EBV encoding D2EGFP showed prolonged and enhanced EGFP expression, and transfection with pPy/EBV encoding HGH resulted in a final concentration of 75 mg/L of HGH in culture supernatant 11 days following transfection.

<p> The proper organization of receptors and signal transduction protein complexes of epithelial and neuronal cells is crucial in tissue formation, cellular differentiation and proper overall development and function. Scaffolding proteins are major components involved in protein targeting and protein complex assembly. MAGUK.s, a family of scaffolding proteins with multiple binding domains such as PDZ, SH3 and GUK, are important regulators of cellular polarity by recruiting and assembling signal and cytoskeletal components into large complexes. Cell polarity is established and maintained by the proper formation and placement of cellular junctions, which separate the plasma membrane into two distinct domains: apical and basolateral. Epithelial polarity determinants from the Bazooka, Crumbs and Scribble complexes establish the boundaries between the apical and basolateral membrane domains and situate the adherens junctions (AJ) at the interface between the two domains. In neuronal cells, the organization and polarization of the presynaptic and the postsynaptic membranes is organized by the CASKIVELIIMINTl/Xllalpha complex. Both CASK and VELI also play a role in epithelial cells. </p> <p> Two novel proteins, originally discovered by Far Western overlay assay in Mus musculus, have been identified as additional binding partners of VELI: PALS I and PALS2. Both proteins are MAGUK.s and are thought to compete with CASK for binding VELI via L27 domain dimerization. PALSl, a major component of the Crumbs complex, is essential for the formation of AJ and the establishment of cellular polarity. PALS2 has been shown to co localize with E-cadherin below tight junctions and directly associate with nectin-like molecule-2 (Necl-2) at extra junctional regions, however its function remains unknown. </p> <p> Using Drosophila melanogaster as a model organism, we have identified the potential Drosophila ortholog of P ALS2, termed dP ALS2, and found that it is conserved across other species. We have done extensive sequence analysis of dP ALS2 at the nucleotide and amino acid level and determined the RNA transcript distribution and protein localization. </p> <p> dP ALS2 expression begins around stage 13 in embryonic tissues in a transversestriped pattern in the epithelia and continues in this striped pattern until the end of stage 17. dP ALS2 is expressed in adult tissues but undetectable in larval tissues. Based on homology and the expression pattern, dP ALS2 may play a role in cell adhesion or cell polarity, similar to the mammalian orthologs. However the striped expression pattern of dPALS2 is similar to segment polarity proteins thus implying dPALS2 may play a role in segment polarity. </p> / Thesis / Master of Science (MSc)

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