Construction of bovine adenovirus type 3 E1 and E3, substitution plasmids and the sequencing analysis of DNA pol and pTP /

The relative ease to concentrate and purify adenoviruses, their well characterized mid-sized genome, and the ability to delete non-essential regions from their genome to accommodate foreign gene, made adenoviruses a suitable candidate for the construction of vectors. The use of adenoviral vectors in gene therapy, vaccination, and as a general vector system for expressing foreign genes have been documented for some time. In this study, the objective was to rescue a BAV3 E1 or E3 recombinant vector carrying the kanamycin resistant gene, a dominant selectable marker with useful applications in studying vectored gene expression in mammalian cells. To accomplish the objective of this study, more information about BAV3 DNA sequences was required in order to make the manipulation of the virus genome accessible. Therefore, sequencing of the BAV3 genome from 1 1 .7% to 30.8% was carried out. Analysis of the determined sequences revealed the primary structure of important viral gene products coded by E2 including BAV3 DNA pol and precursor to terminal protein. Comparative analysis of these proteins with their counterparts from human and non human adenoviruses revealed important insights as to the evolutionary lineage of BAV3. In order to insert the kanamycin resistance gene in either E1 or E3, it was necessary to delete BAV3 sequences to accommodate the foreign gene so as not to exceed the limit of the packaging capacity of the virus. To construct a recombinant BAV3 in which a foreign gene was inserted in the deleted E1 region, an E1 shuttle vector was constructed. This involved the deletion from the viral sequences a region between 1.3% to 9% and inserting the kanamycin resistance gene to replace the deletion. The E1 shuttle vector contained the left (0%- 53.9%) segment of the genome and was expected to generate BAV3 recombinants that can be grown and propagated in cells that can complement the missing E1 functions. To construct a similar shuttle vector for E3 deletion, DNA sequences extending from 78.9% to 82.5% (1281 bp) were deleted from within the E3 region that had been cloned into a plasmid vector. The deleted region corresponds to those that have been shown to be non-essential for viral replication in cell culture. The resulting plasmid was used to construct another recombinant plasmid with BAV3 DNA sequences extending from 37.1% to 100% and with a deletion of E3 sequences that were replaced by kanamycin resistance gene. This shuttle plasmid was used in cotransfections with digested viral DNA in an attempt to rescue a recombinant BAV3 carrying the kanamycin resistance gene to replace the deleted E3. In spite of repeated attempts of transfection, El or E3 recombinant BAV3 were not isolated. It seems that other approaches should be applied to make a final conclusion on BAV3 infectivity.

An exploration of intergenerational exchanges between grandparents and their older grandcildren

Changes within the family unit have resulted in changes in interactions between grandparents and older grandchildren. Existing research indicates that these relationships can result in positive outcomes. A relevant task for researchers is to continue to explore th~se intergenerational relationships. This qualitative phenomenological study explores the question: What functional patterns exist when grandparents interact with older grandchildren? Six grandparent-older grandchild pairs agreed to be involved. Kennedy's (1992) formulation of grandparent-older grandchild activity clusters was reviewed and revised. Activities were clustered related to socialization, companionship, support, entertainment, and education. Findings unique to this study indicate that shared activities were mutually chosen with consideration of activity tolerance, and were consistently evaluated as enjoyable. Partners were chosen because of a comfortable relationship established through frequent past and present interactions, and not because of family lineage preferences. Both grandparents and older grandchildren stated a desire to have a generation peer share activities with them. Exploration of dimension concepts for the "McMaster Model of Family Functioning" indicated that these relationships have potential to contribute to healthy family functioning. The implications for practice, theory development and further research are suggested.

HUMAN GENOME VARIATIONS AND EVOLUTION WITH A FOCUS ON THE ANALYSIS OF TRANSPOSABLE ELEMENTS

Genome sequence varies in numerous ways among individuals although the gross architecture is fixed for all humans. Retrotransposons create one of the most abundant structural variants in the human genome and are divided in many families, with certain members in some families, e.g., L1, Alu, SVA, and HERV-K, remaining active for transposition. Along with other types of genomic variants, retrotransponson-derived variants contribute to the whole spectrum of genome variants in humans. With the advancement of sequencing techniques, many human genomes are being sequenced at the individual level, fueling the comparative research on these variants among individuals. In this thesis, the evolution and functional impact of structural variations is examined primarily focusing on retrotransposons in the context of human evolution. The thesis comprises of three different studies on the topics that are presented in three data chapters. First, the recent evolution of all human specific AluYb members, representing the second most active subfamily of Alus, was tracked to identify their source/master copy using a novel approach. All human-specific AluYb elements from the reference genome were extracted, aligned with one another to construct clusters of similar copies and each cluster was analyzed to generate the evolutionary relationship between the members of the cluster. The approach resulted in identification of one major driver copy of all human specific Yb8 and the source copy of the Yb9 lineage. Three new subfamilies within the AluYb family – Yb8a1, Yb10 and Yb11 were also identified, with Yb11 being the youngest and most polymorphic. Second, an attempt to construct a relation between transposable elements (TEs) and tandem repeats (TRs) was made at a genome-wide scale for the first time. Upon sequence comparison, positional cross-checking and other relevant analyses, it was observed that over 20% of all TRs are derived from TEs. This result established the first connection between these two types of repetitive elements, and extends our appreciation for the impact of TEs on genomes. Furthermore, only 6% of these TE-derived TRs follow the already postulated initiation and expansion mechanisms, suggesting that the others are likely to follow a yet-unidentified mechanism. Third, by taking a combination of multiple computational approaches involving all types of genetic variations published so far including transposable elements, the first whole genome sequence of the most recent common ancestor of all modern human populations that diverged into different populations around 125,000-100,000 years ago was constructed. The study shows that the current reference genome sequence is 8.89 million base pairs larger than our common ancestor’s genome, contributed by a whole spectrum of genetic mechanisms. The use of this ancestral reference genome to facilitate the analysis of personal genomes was demonstrated using an example genome and more insightful recent evolutionary analyses involving the Neanderthal genome. The three data chapters presented in this thesis conclude that the tandem repeats and transposable elements are not two entirely distinctly isolated elements as over 20% TRs are actually derived from TEs. Certain subfamilies of TEs themselves are still evolving with the generation of newer subfamilies. The evolutionary analyses of all TEs along with other genomic variants helped to construct the genome sequence of the most recent common ancestor to all modern human populations which provides a better alternative to human reference genome and can be a useful resource for the study of personal genomics, population genetics, human and primate evolution.

Identification of renin progenitors in the mouse bone marrow that give rise to B-cell leukaemia

cell of origin and triggering events for leukaemia are mostly unknown. Here we show that the bone marrow contains a progenitor that expresses renin throughout development and possesses a B-lymphocyte pedigree. This cell requires RBP-J to differentiate. Deletion of RBP-J in these renin-expressing progenitors enriches the precursor B-cell gene programme and constrains lymphocyte differentiation, facilitated by H3K4me3 activating marks in genes that control the pre-B stage. Mutant cells undergo neoplastic transformation, and mice develop a highly penetrant B-cell leukaemia with multi-organ infiltration and early death. These reninexpressing cells appear uniquely vulnerable as other conditional models of RBP-J deletion do not result in leukaemia. The discovery of these unique renin progenitors in the bone marrow and the model of leukaemia described herein may enhance our understanding of normal and neoplastic haematopoiesis.

Drawing and Explanation of the Cleveland Family Coat of Arms

Drawing and explanation of the Cleveland Family coat of arms. Pro Deo et Patria means For God and Country. The name is Saxon in origin and in 1403 the “de” was dropped from the name. The drawing and text are said to be from Cleveland Genealogy by J.B. Cleveland, 1881. It can also be found in An Account of the Lineage of General Moses Cleaveland (founder of the city of Cleveland, Ohio) of Canterbury, compiled by H.G. Cleveland, n.d.

Some Incidents and Circumstances

- The first part of the document traces Mr. Haile’s lineage. His father, James Haile was a farmer. His grandfather, Amos Haile was a sailor for the early part of his life. He was placed on a British man-of- war in about 1758. He escaped and settled in Putney. (p.1) - His father’s mother’s maiden name was Parker. His mother’s maiden name was Campbell. Her father was a captain in the Revolutionary Army. (p.2) - His earliest memories revolve around the death of his aunt and the funeral of General Washington (although he did not witness this). At the time, his father was a Lieutenant in a regiment militia of Light Dragoons who wore red coats. (p.3) - In 1804, an addition was added to the Haile house which necessitated that William was to stay home to help with the building. He continued to study and read on his own. He was particularly interested in Napoleon Bonaparte’s victories. In that same year he was sent to Fairfield Academy where Reverend Caleb Alexander was the principal. (p.4) - On June 1, 1812, William was appointed as an Ensign in the Infantry of the Army of the United States. He was put into the recruiting service at Nassau (20 miles east of Albany) where he remained until September. (p.4) - He was assigned to the 11th Regiment of the W.S. Infantry and directed to proceed to Plattsburgh to report to Colonel Isaac Clark. (p.7) - He was assigned to the company commanded by Captain Samuel H. Halley who was not in the best of health and often absent. For a good part of the time William was in charge of the company. (p.8) - The 11th Regiment was encamped beside the 15th Regiment commanded by Col. Zebulon Montgomery Pike [Pike’s Peak was named after him]. Col. Pike generously drilled and disciplined the 11th Regiment since their officers didn’t seem capable of doing so. (p.8) - The first brigade to which William’s regiment was attached to was commanded by Brigadier General Bloomfield of New Jersey. Brigadier Chandler of Maine commanded the second brigade. (p.9) - At the beginning of November, Major General Dearborn took command of the army. He had been a good officer in his time, but William refers to him as “old and inefficient” earning him the nickname “Granny Dearborn” (p.9) - On November 17th, 1812, General Dearborn moved north with his army. The troops ended up in Champlain. There was no fighting, only a skirmish between a party of men under Colonel Pike and a few British troops who he succeeded in capturing. (p.10) - The troops were moved to barracks for the winter. Colonel Pike’s troops were put into suitable barracks and kept healthy but another part of the army (including the 11th Regiment) were sent to a barracks of green lumber north of Burlington. Disease soon broke out in the damp barracks and the hundreds of deaths soon followed. One morning, William counted 22 bodies who had died the previous night. He puts a lot of this down to an inexperienced commanding officer, General Chandler. (p.11) - At the beginning of 1813, William was stationed as a recruiter on the shore of Shoreham across from Fort Ticonderoga. In February, he returned to Burlington with his recruits. In March he received an order from General Chandler to proceed to Whitehall and take charge of the stores and provisions. In April and May it was decided that his half of the regiment (the First Battalion) should march to Sackett’s Harbour, Lake Ontario. They arrived at Sackett’s Harbour about the 10th of June, a few days after the Battle of Sackett’s Harbour. (p.12) - He was camped near the site of Fort Oswego and got word to head back to Sackett’s Harbour. A storm overtook the schooner that he was on. (p.14) - William was involved in the Battle of Williamsburg (or Chrysler’s Farm) which he calls a “stupid and bungling affair on the part of our generals”.(p. 18) - General Covington was wounded and died a few days after the battle. (p.19) - William speaks of being ill. The troops were ordered to march to Buffalo, but he is able to go to his father’s house in Fairfield where his mother nursed him back to health (p.23) - Upon arrival at Buffalo, the “old fogy Generals” were replaced with younger, more efficient men. (p.25) - On page 27 he sums up a few facts: In 1812, the army was assembled on Lake Champlain with the intention of capturing Montreal, and then Quebec. That year, under General Dearborn the army marched as far as Champlain, then turned back and went into winter quarters. In 1813, the army was assembled at Sackett’s Harbour and that year the campaign ended at French Mills which was 70 or 80 miles from Montreal. In 1814, the army at Buffalo were some 400 miles from Montreal with still the same object in view. - He says that these facts make “a riddle – difficult to explain”. (p.27) - On the evening of July 2nd they embarked on the boats with the objective of capturing Fort Erie. The enemy were all made prisoners of war (p.27) - On July 4th they went to Street’s Creek, 2 miles above the Chippewa [Chippawa] River (p.28) - Page 29 is titled The Battle of Chippewa [Chippawa] - He speaks of 2 drummers who were fighting over the possession of a drum when a cannonball came along and took of both of their heads (p.29) - He proclaims that this was one of the “most brilliant battles of the war”. The battle was fought and won in less than an hour after they left their tents. He credits General Scott with this success and states that was due to his rapid orders and movements. (p.30) - The dead of the battle remained on the field during the night. He describes this as quite gloomy seeing friend and foe lying side by side. At daybreak they set to work digging trenches to bury the dead. (p.31) - Colonel Campbell was wounded and advised to have his leg amputated. He refused, and subsequently died. (p.32) - It is said that the British threw several of their dead into the river and they went over the Falls. (p.32) - His troops repaired the bridge over Chippawa which the enemy had partially destroyed and then pursued the British as far as Queenston Heights. (p.32) - On pages 33 and 34 he speaks about meeting an old friend of his, Philip Harter. - The account ends at Queenston Heights