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.

Master of Science / Department of Physics / Lado Samushia / Part 1: The redshift-space bispectrum (three point statistics) of galaxies can be used to measure key cosmological parameters. In a homogeneous Universe, the bispectrum is a function of five variables and unlike its two point statistics counterpart -- the power spectrum, which is a function of only two variables -- is difficult to analyse unless the information is somehow reduced. The most commonly considered reduction schemes rely on computing angular integrals over possible orientations of the bispectrum triangle thus reducing it to sets of functions of only three variables describing the triangle shape. We use Fisher information formalism to study the information loss associated with this angular integration. We find that most of the information is in the azimuthal averages of the first three even multipoles. This suggests that the bispectrum of every configuration can be reduced to just three numbers (instead of a 2D function) without significant loss of cosmologically relevant information. Part 2: One way of enhancing the cosmological information extracted from the clustering of galaxies is by weighting the galaxy field. The most widely used weighting schemes assign weights to galaxies based on the average local density in the region and their bias with respect to the dark matter field. They are designed to minimize the fractional variance of the galaxy power-spectrum. We demonstrate that the currently used bias dependent weighting scheme can be further optimized for specific cosmological parameters. Part 3: Choice of the box-size of a cosmological simulation involves a crucial trade-off between accuracy and complexity. We use Lagrangian perturbation theory to study the effects of box size on the predicted power spectrum and Baryon Acoustic Oscillation ruler. We find that although the optimal size depends on the final redshift of evolution, in general, the 2-point statistics of relevant scales is fairly accurate for a simulation box-size of length greater than 1000 Mpc.

In this era of high-precision cosmology we are able to measure and predict properties of the large-scale structure of our Universe to a fine degree. However we still lack a clear and tested understanding of the effects of the large-scale environments on galaxies and their host halos. This thesis focuses on bettering our understanding of this issue by investigating the dependence of galaxies and halos on their location within the cosmic web. An algorithm based on the tidal tensor prescription is developed and applied to the MDR1 1 (h-1Gpc)3 dark matter simulation to classify the geometric environment of every location in the simulated volume as one of the four components of the cosmic web; voids, sheets, filaments and knots. Conditional halo mass functions are extracted to investigate the influence of tidal forces on the abundances and mass distribution of dark matter halos. A theoretical framework based on Gaussian statistics is presented and used to derive predictions for halo abundances in different geometric environments. The Gaussian theory predicts no coupling of tidal forces and, hence, that the halo mass function is independent of geometric environment for a given local mass density. It is shown that the halo mass functions extracted from the simulation are fully consistent with this picture. It is then shown how this method of classifying geometric environments can be extended to observational datasets. The Galaxy And Mass Assembly (GAMA) spectroscopic redshift survey, with its wide field and high completeness, is excellently suited to this study. The geometric environments of the three equatorial GAMA fields are classified, following a thorough analysis of the additional uncertainties introduced when moving to observational datasets. Additionally, the geometric environments of the GAMA galaxies and groups are classified, allowing the influence of the cosmic web on large-scale structure to be investigated. Both the galaxy luminosity function and the group mass function within the observed cosmic web are studied and no evidence of a direct impact of the web is seen. It is found that all modulations can be fully attributed to the indirect dependence of these properties on the local matter overdensity. Whilst these results indicate that there is no strong dependence of the scalar properties of large-scale structure on geometric environment, the final investigation of this thesis presents an attempt to look in more detail at the environmental dependence of stellar properties by investigating stellar-formation histories within the cosmic web.

The Protist EST program (PEP) aims to explore the diversity of eukaryotic genomes, in a systematic and comprehensive way. A main element of the PEP initiative is to establish a database, the Protist EST Database (PEPdb), which is the centerpiece of the PEP collaboration. The major functions of the PEPdb are management of the data generated by PEP, analysis of these data, and to allow collected sequence information to be accessed via the Internet by PEP members or other users. In this project, a consistent and easy to use relational database was implemented. All information about PEP members, Publications, Libraries and ESTs can be stored in the database system. The operations are achieved by a friendly user interface. This database stores about 10000 records and is displayed on the web site "http://info.biology.mcmaster.ca/ling/estHome.html" for demonstration. An analysis of ESTs from the ciliated protozoan Tetrahymena thermophila was undertaken. A total of 3740 non-redundant gene assemblies and singletons from TIGR were analyzed. These sequences have been compared against the NCBI non-redundant protein and nucleotide databases using BLASTX and BLASTN to identify putative genes. Of 850 highly significant matches with an expect value cut-off of 10^-20 , 35.5% represent genes previously cloned from T. thermophila, and 64.5% had significant similarity to genes from other organisms deposited in the NCBI. There are 26 sequences (3.1%) that matched signal transduction proteins, including Rac, Ras, MAPK, ERK1, PKC, cAMP and 14-3-3 (a protein involved in signal transduction, exocytosis and cell cycle regulation). This result indicates that T. thermophila likely encodes the MAPK/ERK signaling pathway. About 53 sequences (6.2%) matched to cytoskeleton proteins which were divided into two groups. The first group matched genes coding for microtubules, especially to tubulin genes. The other group matched to microfilament genes including one actin, three actin-related and one profilin proteins. There were no sequences similar to intermediate filaments. Comparison of the EST counts from one gene provide absolute estimates of mRNA expression levels. The most abundant genes represented are enolase, SerH3 and Tubulin. Among 850 highly significant similarities, 196 were restricted to the ciliophora. GRL and SerH are ciliate-specific genes. There were 508 sequences that had highly significant matches (expect value < 10^-20) to human genes. Approximately 189 of them were present in humans but not found in the completely sequenced Saccharomyces cerevisiae. Based on Venn diagram analysis, T. thermophila contains abundant Eukaryotic specific proteins and many prokaryotic-like genes, and some metabolic enzymes in T. thermophila are also present in plants. These results support the fact that T. thermophila is an excellent unicellular model system for gene discovery and functional analysis. / Thesis / Master of Science (MS)

The main objectives of this dissertation are the following. The first objective is concerned with the stabilization of large-scale systems by a decentralized control. The fundamental idea behind this type of control is the stabilization of the isolated subsystems of a large-scale system in such a way that the global stability requirement is also satisfied. For this purpose, a new stability criterion is introduced to identify a class of interconnected systems that can be stabilized by local state feedback. In addition to this, two specific classes of interconnections are presented for which the overall system stability can be ensured by a decentralized approach. A new constructive procedure for the design of decentralized controllers for the identified classes of large-scale systems is discussed. The principal advantages of this design procedure are that it requires a minimal amount of computation and is a systematic procedure eliminating the trial and error arguments as in the earlier methods. The second objective of the dissertation is to investigate the problem of the stabilization of a class of large-scale systems which are composed of identical subsystems and identical interconnections. For this class of systems, certain significant theorems, concerning the qualitative properties are introduced. Following the guidelines set forth by the above theorems, a few different schemes for the decentralized and multilevel control of the aforementioned class of large-scale interconnected systems are presented. The third objective concerns the development of a few different schemes for the design of an asymptotic state estimator for large-scale systems described as interconnections of several low-order subsystems. The most attractive feature of the present schemes is that the majority of the necessary computations are performed at the subsystem level only, thereby leading to a simple and practicable estimator design. Finally, all the above results are illustrated by numerical examples. Further, a comparison study is conducted to show the advantages of the methods and the results in this dissertation in comparison with some results available in the literature.

In the design of large-scale systems the problem is often too large to be approached by a single group. Then the system design problem must be resolved into component subproblems with different groups assigned to work on each subproblem. A. Wayne Wymore's "Tricotyledon Theory of Systems Design" (T3SD) provides a general system theoretic framework for the statement of large-scale system design problems. In this paper some results are developed for the extension of T3SD to the problem of the resolution of system design problems into system component design problems. Initially resolutions with respect to I/O specifications and technologies are defined and examined. Following this, resolutions with respect to merit orderings in which the merit orderings on the component problems have a specified relation with the merit orderings on the original problem are discussed. Ideal, strong and perfect resolutions with respect to merit orderings are defined and relationships among these types of resolutions are discussed. It is shown that trivial strong and ideal resolutions can always be developed from simple resolutions. Perfect resolutions are always ideal resolutions and ideal resolutions are always strong resolutions. Finally it is shown that given a class of simple resolutions there always exists a maximal ideal resolution for that class.

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