Development of packaging cell lines for rescuing BAV2 viral vectors

The construction of adenovirus vectors for cloning and foreign gene expression requires packaging cell lines that can complement missing viral functions caused by sequence deletions and/or replacement with foreign DNA sequences. In this study, packaging cell lines were designed to provide in trans the missing bovine adenovirus functions, so that recombinant viruses could be generated. Fetal bovine kidney and lUng cells, acquired at the trimester term from a pregnant cow, were tranfected with both digested wild type BAV2 genomic DNA and pCMV-EI. The plasmid pCMV-EI was specifically constructed to express El of BAV2 under the control of the cytomegalovirus enhancer/promoter (CMV). Selection for "true" transformants by continuous passaging showed no success in isolating immortalised cells, since the cells underwent crisis resulting in complete cell death. Moreover, selection for G418 resistance, using the same cells, also did not result in the isolation of an immortalised cell line and the same culture-collapse event was observed. The lack of success in establishing an immortalised cell line from fetal tissue prompted us to transfect a pre-established cell line. We began by transfecting MDBK (Mardin-Dardy bovine kidney) cells with pCMV-El-neo, which contain the bacterial selectable marker neo gene. A series of MDBK-derived cell lines, that constitutively express bovine adenoviral (BAV) early region 1 (El), were then isolated. Cells selected for resistance to the drug G418 were isolated collectively for full characterisation to assess their suitability as packaging cell lines. Individual colonies were isolated by limiting dilution and further tested for El expression and efficiency of DNA uptake. Two cell lines, L-23 and L-24, out of 48 generated foci tested positive for £1 expression using Northern Blot analysis. DNA uptake studies, using both lipofectamine and calcium phosphate methods, were performed to compare these cells, their parental MDBK cells, 8 and the unrelated human 293 cells as a benchmark. The results revealed that the new MDBKderived clones were no more efficient than MDBK cells in the transient expression of transfected DNA and that they were inferior to 293 cells, when using lacZ as the reporter gene. In view of the inherently poor transfection efficiency of MDBK cells and their derivatives, a number of other bovine cells were investigated for their potential as packaging cells. The cell line CCL40 was chosen for its high efficiency in DNA uptake and subsequently transfected with the plasmid vector pCMV El-neo. By selection with the drug G418, two cell lines were isolated, ProCell 1 and ProCell 2. These cell lines were tested for El expression, permissivity to BAV2 and DNA uptake efficiency, revealing a DNA uptake efficiency of 37 % , comparable to that of CCL40. Attempts to rescue BAV2 mutants carrying the lacZ gene in place of £1 or £3 were carried out by co-transfecting wild type viral DNA with either the plasmid pdlElE-Z (which contains BAV2 sequences from 0% to 40.4% with the lacZ gene in place of the £1 region from 1.1% to 8.25%) or with the plasmid pdlE3-5-Z (which contains BAV2 sequences from 64.8% to 100% with the lacZ gene in place of the E3 region from 75.8% to 81.4%). These cotransfections did not result in the generation of a viral mutant. The lack of mutant generation was thought to be caused by the relative inefficiency ofDNA uptake. Consequently, cosBAV2, a cosmid vector carrying the BAV2 genome, was modified to carry the neo reporter gene in place of the £3 region from 75.8% to 81.4%. The use of a single cosmid vector earring the whole genome would eliminate the need for homologous recombination in order to generate a viral vector. Unfortunately, the transfection of cosBAV2- neo also did not result in the generation of a viral mutant. This may have been caused by the size of the £3 deletion, where excess sequences that are essential to the virus' survival might have been deleted. As an extension to this study, the spontaneous E3 deletion, accidently discovered in our viral stock, could be used as site of foreign gene insertion.

"Attitude is a little thing that makes a big difference" : further exploration of attitude alignment in the context of close relationship dyads /

One hundred and five primarily Caucasian undergraduate couples were tested to study the phenomenon of attitude alignment, or the way in which individuals change their opinions to achieve greater attitudinal congruence with their romantic partners, and the hypotheses that relationship closeness, affect, attachment, social desirability, and centrality are each related to attitude alignment for individuals in close couples. The couples filled out an attitude questionnaire consisting of 50 issues in which they were asked to give their attitude on a scale from 1 to 9 as well as to rate the centrality, or importance, of that issue on a scale from 1 to 9. Before discussing four of these issues with each other - 2 were more central to the man and less central, or peripheral, to the woman, whereas the other 2 were central to the woman and peripheral to the man - the individuals completed established measures of relationship closeness, affect, attachment, and social desirability. The couples then filled out the identical attitudes questionnaire again, and their answers on the four discussion issues were compared to their prediscussion answers to determine whether attitude alignment had occurred. There were two experimental groups: a social desirability group, where the couples were told it was natural and normal to disagree with their partners, and a control group. Results indicated that attitude alignment did significantly occur across all couples, but most other variables - including centrality, relationship closeness, and affect - did not predict attitude alignment behaviour. As well, the social desirability experimental groups did not significantly differ on attitude alignment behaviours, but higher scores on the social desirability scale, specifically self-deception, significantly predicted higher attitude alignment scores across all couples. Large differences between individuals' frequency.

Exploring multiracial/cultural identities through mothers' voices /

This study explored the experiences of mothers of multiracial/cultural children within the context of family, school, and community. Three categories of mothers of multiracial/cultural children were interviewed privately and then invited to meet as a group to explore some of their reflections and experiences. The categories consisted of 4 mothers with multiracial/cultural children presently attending elementary school, 2 mothers of multiracial/cultural children who are now adults and 3 mothers from my own multiracial/cultural family. The study explored the researcher's personal quest for a multiracial/cultural identity and combined interviews with her daughter, her sister, and her mother to reveal the multiracial/cultural experience from a personal perspective. Content analysis of the narratives revealed that multiracial/cultural children produce their own culture and establish new and personally relevant priorities as they develop their self-identities. Findings further indicated that present-day, mainstream mothers from the dominant majority group of Canadians, tell a different story than similar mothers of previous cohorts, and that although sociopolitical and economic changes have influenced the experiences ofthese women, their stories remain remarkably similar across racial and cultural lines. The findings from this study may promote the development of multicultural programs in Canada as they offer both prospects and challenges to multiracial/cultural children and multicultural educators. It is hoped that this study will provide a better understanding of multiculturalism and encourage educators to heighten their racial and cultural awareness as they strive to critically examine their own cultural stories and realign their praxis within the evolving Canadian mosaic.

Drift dispersal in central Southern Ontario /

Two tills are readily identi-f i able in central Southern Ontario, a very stony, loose deposit o-f variable matrix (Dummer till) and a moderately stony, fissile and compact deposit that is more homogeneous (drumlinized till). The quantity o-f Precambr i an, Paleozoic and Shadow Lake Formation (Paleozoic) rock types were determined and corresponding isopleth maps drawn. The changes in lithology content occurred in the direction o-f transport, there-fore, compositional isopleths o-f till may be considered equipotential lines for the reconstruction of glacier flow paths. Areal gradations of drift lithology indicated that the prime agents of dispersal were ice and glacial meltwaters. The down-ice abundance trend of till components indicated a dispersal pattern showing the concentration of a given lithology type peaking within a few kilometres of the source followed by a rapid decline and thereafter, a more gradual decrease with increasing distance. Within the esker deposits, igneous rocks may form the major component and can extend further onto the limestone plain than in the adjacent till. Evidence is presented that indicates the "style" of dispersal was one in which glacial ice may have been strongly influenced by local bedrock topography and the regional structural trends. The ice tended to follow pre-existing valleys and lows, depositing till composed mainly of local bedrock. Gradations in Paleozoic clast content showed that the local bedrock lithology became the primary till component within 3 km of down-ice transport. Evidence is presented that indicated the last glaciation may have occurred as a relatively thin ice mass, followed by stagnation and recession. No evidence of a lateglacial re-advance was found within the study area. Because of the lack of a contact between the Dummer and drumlinized till, and because of results showing gradation of the Dummer till into the drumlinized till (as indicated by lithology content and grain size), it is suggested that no re-advance occurred.

Metabolic, biochemical and molecular profiling of Catharanthus roseus flower cultivars and transformed hairy roots for monoterpenoid indole alkaloid accumulation /

Catharanthlls rosellS (L.) G Don is a commercially significant flower species and in addition is the only source of the monoterpenoid indole alkaloids (MIA) vinblastine and vincristine, which are key pharmaceutical compounds that are used to combat a number of different cancers. Therefore, procurement of the antineoplastic agents is difficult but essential procedure. Alternatively, CatharanthllS tissue cultures have been investigated as a source of these agents; however they do not produce vindoline, which is an obligate precursor to vinblastine and vincristine. The interest in developing high MIA cultivars of Catharantlws rosellS has prompted metabolic profiling studies to determine the variability of MIA accumulation of existing flowering cultivars, with particular focus on the vindoline component ofthe pathway. Metabolic profiling studies that used high performance liquid chromatography of MIAs from seedlings and young leaf extracts from 50 different flowering cultivars showed that, except for a single low vindoline cultivar (Vinca Mediterranean DP Orchid), they all accumulate similar levels of MIAs. Further enzymatic studies with extracts from young leaves and from developing seedlings showed that the low vindoline cultivar has a IO-fold lower tabersonine-16-hydroxylase activity than those of CatharanthllS rosellS cv Little Delicata. Additionally, studies aimed at metabolic engineering ofvindoline bios}l1thesis in Catharanthus rosellS hairy root cultures have been performed by expressing the last step in vindoline biosynthesis [Dcacetylvindoline-4-0- acetyltransferase (DAT)]. Enzymatic profiling studies with transformed hairy roots have confirmed that over-expressing DAT leads to lines with high levels of O-acetyltransferase activity when compared to non-expressing hairy roots. One particular DA T over111 expressing hairy root culture (line 7) contained 200 times the OAT activity than leaves of control lines. Additional MIA analyses revealed that DAT over-expressing hairy roots have an altered alkaloid profile with significant variation in the accumulation of h6rhammericine. Further analysis of transformed hairy root line 7 suggests a correlation between the expression of OAT activity and h6rhammericine accumulation with root maturation. These studies show that metabolic and selective enzymatic profiling can enhance our ability to search for relevant MIA pathway mutants and that genetic engineering with appropriate pathway genes shows promise as a tool to modify the MIA profile of Catharanthus roseus.

Engineering an improved adenovirus packaging cell line

Recombinant human adenovirus (Ad) vectors are being extensively explored for their use in gene therapy and recombinant vaccines. Ad vectors are attractive for many reasons, including the fact that (1) they are relatively safe, based on their use as live oral vaccines, (2) they can accept large transgene inserts, (3) they can infect dividing and postmitotic cells, and (4) they can be produced to high titers. However, there are also a number of major problems associated with Ad vectors, including transient foreign gene expression due to host cellular immune responses, problems with humoral immunity, and the creation of replication competent adenoviruses (RCA). Most Ad vectors contain deletions in the E1 region that allow for insertion of a transgene. However, the E1 gene products are required for replication and thus must be supplied in trans by a helper ceillille that will allow for the growth and packaging of the defective virus. For this purpose the 293 cell line (Graham et al., 1977) is used most often; however, homologous recombination between the vector and the cell line often results in the generation of RCA. The presence of RCA in batches of adenoviral vectors for clinical use is a safety risk because tlley . may result in the mobilization and spread of the replication-defective vector viruses, and in significant tissue damage and pathogenicity. The present research focused on the alteration of the 293 cell line such that RCA formation can be eliminated. The strategy to modify the 293 cells involved the removal of the first 380 bp of the adenovirus genome through the process of homologous recombination. The first step towards this goal involved identifying and cloning the left-end cellular-viral jUl1ction from 293 cells to assemble sequences required for homologous recombination. Polymerase chain reaction (PCR) was performed to clone the junction, and the clone was verified through sequencing. The plasn1id PAM2 was then constructed, which served as the targeting cassette used to modify the 293 cells. The cassette consisted of (1) the cellular-viral junction as the left-end region of homology, (2) the neo gene to use for positive selection upon tranfection into 293 cells, (3) the adenoviral genome from bp 380 to bp 3438 as the right-end region of homology, and (4) the HSV-tk gene to use for negative selection. The plasmid PAM2 was linearized to produce a double strand break outside the region of homology, and transfected into 293 cells using the calcium-phosphate technique. Cells were first selected for their resistance to the drug G418, and subsequently for their resistance to the drug Gancyclovir (GANC). From 17 transfections, 100 pools of G418f and GANCf cells were picked using cloning lings and expanded for screening. Genomic DNA was isolated from the pools and screened for the presence of the 380 bps using PCR. Ten of the most promising pools were diluted to single cells and expanded in order to isolate homogeneous cell lines. From these, an additional 100 G41Sf and GANef foci were screened. These preliminary screening results appear promising for the detection of the desired cell line. Future work would include further cloning and purification of the promising cell lines that have potentially undergone homologous recombination, in order to isolate a homogeneous cell line of interest.

Ethnic conflicts and international politics : an analysis of the communal regional, and global dimensions of the Cyprus problem

The thesis assesses the impact of international factors on relations between Greek and Turkish Cypriots during and after the Cold War. Through an analysis of the Cyprus problem it explores both why external actors intervene in communal conflicts and how they influence relations between ethnic groups in plural societies. The analytical framework employed throughout the study draws on contributions of International Relations theorists and students of ethnic conflict. The thesis argues that, as in the global political system, relations between ethnic groups in unranked communal systems are anarchic; that is, actors within the system do not recognize a sovereign political authority. In bipolar communal systems dominated by two relatively equal groups, the struggle for security and power often leads to appeals for assistance from external actors. The framework notes that neighboring states and Great Powers may heed calls for assistance, or intervene without a prior request, if it is in their interest to do so. The convergence of regional and global interests in communal affairs exacerbates ethnic conflicts and precludes the development of effective political institutions. The impact of external intervention in ethnic conflicts has the potential to alter the basis of communal relations. The Cyprus problem is examined both during and after the Cold War in order to gauge how global and regional actors and the structure of their respective systems have affected relations between ethnic groups in Cyprus. The thesis argues that Cyprus's descent into civil war in 1963 was due in part to the entrenchment of external interests in the Republic's constitution. The study also notes that power politics involving the United States, Soviet Union, Greece and Turkey continued to affect the development of communal relations throughout the 1960s, 70s, and, 80s. External intervention culminated in July and August 1974, after a Greek sponsored coup was answered by Turkey's invasion and partition of Cyprus. The forced expulsion of Greek Cypriots from the island's northern territories led to the establishment of ethnically homogeneous zones, thus altering the context of communal relations dramatically. The study also examines the role of the United Nations in Cyprus, noting that its failure to settle the dispute was due in large part to a lack of cooperation from Turkey, and the United States' and Soviet Union's acceptance of the status quo following the 1974 invasion and partition of the island. The thesis argues that the deterioration of Greek-Turkish relations in the post-Cold War era has made a solution to the dispute unlikely for the time being. Barring any dramatic changes in relations between communal and regional antagonists, relations between Greek and Turkish Cypriots will continue to develop along the lines established in July/August 1974. The thesis concludes by affirming the validity of its core hypotheses through a brief survey of recent works touching on international politics and ethnic conflict. Questions requiring further research are noted as are elements of the study that require further refinement.

The establishment of two novel bovine cell lines by transfection with the putative transforming region of bovine adenovirus type 3

Human adenoviruses (Ads), members of the family adenoviridae, are medium-sized DNA viruses which have been used as valuable research tools for the study of RNA processing, oncogenic transformation, and for the development of viral vectors for use in gene delivery and immunization technology. The left 12% of the linear Ad genollle codes for products which are necessary for the efficient replication of the virus, as well as being responsible for the forlllation of tumors in animallllodels. The establishlllent of the 293 cell line, by immortalization of human embryonic kidney cells with th~ E1 region of Ad type S (AdS), has facilitated extensive manipulation of the Ads and the development of recombinant Ad vectors. The study of bovine adenoviruses (BAVs), which cause mild respiratory and gastrointestinal infections in cattle has, on the other hand, been limited primarily to that of infectivity, immunology and clinicallllanifestations. As a result, any potential as gene delivery vehicles has not yet been realized. Continued research into the molecular biolo~gy of BAVs and the development of recolllbinant vectors would benefit from the development of a cell line analogous to that of the 293 cells. In an attelllpt to establish such a cell line, the recombinant plaslllid pKC-neo was constructed, containing the left 0-19.7% of the BAV type 3 (BAV3) genome, and the selectable marker for resistance to the aminoglycoside G418, a neomycin derivative. The plasmid construct was then used to transfect both the Madin-Darby bovine kidney (MDBK) -iicell line and primary bovine lung cells, after which G418-resistant foci were selected for analysis. Two cell lines, E61 (MDBK) and E24 (primary lung), were subsequently selected and analysed for DNA content, revealing the presence of the pKC-neo sequences in their respective genomes. In addition, BAV3 RNA transcripts were detected in the E61 cells. Although the presence of E1 products has yet to be confirmed in both cell lines, the E24 cells exhibit a phenotype characteristic of partial transformation by E1. The apparent immortalization of the primary lung cells will permit exploitation of their ability to take up exogenous DNA at high efficiency.

The role of learning and growth hormone-releasing factor in the control of protein intake in rats /

Both learning and basic biological mechanisms have been shown to play a role in the control of protein int^e. It has previously been shown that rats can adapt their dietary selection patterns successfully in the face of changing macronutrient requirements and availability. In particular, it has been demonstrated that when access to dietary protein is restricted for a period of time, rats selectively increase their consumption of a proteincontaining diet when it becomes available. Furthermore, it has been shown that animals are able to associate various orosensory cues with a food's nutrient content. In addition to the role that learning plays in food intake, there are also various biological mechanisms that have been shown to be involved in the control of feeding behaviour. Numerous studies have documented that various hormones and neurotransmitter substances mediate food intake. One such hormone is growth hormone-releasing factor (GRF), a peptide that induces the release of growth hormone (GH) from the anterior pituitary gland. Recent research by Vaccarino and Dickson ( 1 994) suggests that GRF may stimulate food intake by acting as a neurotransmitter in the suprachiasmatic nucleus (SCN) and the adjacent medial preoptic area (MPOA). In particular, when GRF is injected directly into the SCN/MPOA, it has been shown to selectively enhance the intake of protein in both fooddeprived and sated rats. Thus, GRF may play a role in activating protein consumption generally, and when animals have a need for protein, GRF may serve to trigger proteinseeking behaviour. Although researchers have separately examined the role of learning and the central mechanisms involved in the control of protein selection, no one has yet attempted to bring together these two lines of study. Thus, the purpose of this study is to join these two parallel lines of research in order to further our understanding of mechanisms controlling protein selection. In order to ascertain the combined effects that GRF and learning have on protein intake several hypothesis were examined. One major hypothesis was that rats would successfully alter their dietary selection patterns in response to protein restriction. It was speculated that rats kept on a nutritionally complete maintenance diet (NCMD) would consume equal amount of the intermittently presented high protein conditioning diet (HPCD) and protein-free conditioning diet (PFCD). However, it was hypothesized that rats kept on a protein-free maintenance diet (PFMD) would selectively increase their intake of the HPCD. Another hypothesis was that rats would learn to associate a distinct marker flavour with the nutritional content of the diets. If an animal is able to make the association between a marker flavour and the nutrient content of the food, then it is hypothesized that they will consume more of a mixed diet (equal portion HPCD and PFCD) with the marker flavour that was previously paired with the HPCD (Mixednp-f) when kept on the PFMD. In addition, it was hypothesized that intracranial injection of GRF into the SCN/MPOA would result in a selective increase in HPCD as well as Mixednp-t consumption. Results demonstrated that rats did in fact selectively increase their consumption of the flavoured HPCD and Mixednp-f when kept on the NCMD. These findings indicate that the rats successfully learned about the nutrient content of the conditioning diets and were able to associate a distinct marker flavour with the nutrient content of the diets. However, the results failed to support previous findings that GRF increases protein intake. In contrast, the administration of GRF significantly reduced consumption of HPCD during the first hour of testing as compared to the no injection condition. In addition, no differences in the intake of the HPCD were found between the GRF and vehicle condition. Because GRF did not selectively increase HPCD consumption, it was not surprising that GRF also did not increase MixedHP-rintake. What was interesting was that administration of GRF and vehicle did not reduc^Mixednp-f consumption as it had decreased HPCD consumption.

A Survey of hMLH1 mRNA Splicing Profiles in Human Cell Lines: Comparing Primary Cultured Fibroblasts Before and After Oxidative Stress and Transiently versus Stably Demethylated Colon Cancer Derived Cell Lines

Most human genes undergo alternative splicing and loss of splicing fidelity is associated with disease. Epigenetic silencing of hMLH 1 via promoter cytosine methylation is causally linked to a subset of sporadic non-polyposis colon cancer and is reversible by 5-aza-2' -deoxycytidine treatment. Here I investigated changes in hMLHI mRNA splicing profiles in normal fibroblasts and colon cancer-derived human cell lines. I established the types and frequencies of hMLHI mRNA transcripts generated under baseline conditions, after hydrogen peroxide induced oxidative stress, and in acutely 5-aza-2' -deoxycytidine-treated and stably derepressed cancer cell lines. I found that hMLHI is extensively spliced under all conditions including baseline (50% splice variants), the splice variant distribution changes in response to oxidative stress, and certain splice variants are sensitive to 5- aza-2' -deoxycytidine treatment: Splice variant diversity and frequency of exon 17 skipping correlates with the level of hMLHI promoter methylation suggesting a link between promoter methylation and mRNA splicing.

Canada Steamship Lines Ltd. Niagara to the Sea (1923)

Sailing schedule and trip guide for Canada Steamship Lines Limited for the year 1923

Canada Steamship Lines Limited Niagara to the Sea 1917

Sailing schedule and trip guide for Canada Steamship Lines Limited for the year 1917.

Canada Steamship Lines Limited Richelieu and Ontario Division Niagara to the Sea July 1916

Sailing schedule and trip guide for Canada Steamship Lines Limited for July 1916.

Canada Steamship Lines Limited Richelieu (1915)

Sailing schedule and trip guide for Canada Steamship Lines Limited for July 1915.