The cloning and reconstitution of a bovine adenovirus type 2 E3 deletion mutant and the sequencing and analysis of the early 4 region

Recombinant Adenoviruses (Ads) have been shown to have potential applications in three areas: gene therapy, high level protein expression and recombinant vaccines.' At least three different locations within the Ad genome can be deleted and subsequently used for the insertion of foreign sequences. These include the Early 3 (E3), Early 1 (E1) and Early 4 (E4) regions. Viral vectors of this type have been well studied in Human Ads 2 and 5, however one has not yet been constructed for Bovine Adenovirus Type 2 (BAV2). The E3 region is located between 76.6 and 86 m.u. on the r-strand and is transcribed in a rightward direction. The gene products of the Early 3 region (E3) have been shown to be non-essential for viral replication, in vitro, but are required for host immunosurveillance. This study represents the cloning and reconstitution of a BAV2 E3 deletion mutant. A deletion of 1800bp was made within the E3 region of BAV2 and the thymidine kinase gene was subsequently inserted in the deleted area . . The plasmid pdlE3-4tk1 (23.4Kbp) was constructed and used to to facilitate homologous recombination with the wild type BAV2 to produce a mutant. Southern Blotting and Hybridization results suggest the presence of a BAV2 E3 deletion mutant with thymidine kinase sequences present. The E4 region of Human Adenovirus types 2 and 5 is located at the extreme right end of the genome (91.3 map units - 99.1 map units) and is transcribed in a leftward direction giving rise to a complicated set of differentially spliced mRNAs. Essentially there are 7 open reading frames (ORFs) encoding for at least 7 polypeptides. The gene products encoded by the E4 region have been shown to be essential for the expression of late viral genes, host cell shutoff and normal viral growth. We have cloned and sequenced the right end segment between 90.5 map units and 100 map units of the BAV2 genome. The results show several open reading frames which encode polypeptides exhibiting homology to three polypeptides encoded by the E4 region of human adenovirus type 2. These include the 14kDa protein encoded by ORF1, the 34kDa protein encoded by ORF6 and the 13kDa protein encoded by ORF3. The nucleotide sequence, restriction enzyme map, and ORF map of the E4 region could be very useful in future molecular manipulation of this region and could possibly explain the slow growth rate of BAV2 in MDBK cells.

Tacitus' Germania and the Jesuit Relations: Intertextuality in the Transatlantic World of the Early Jesuits in New France.

The first Europeans who wrote about the Indigenous people of the newly discovered Americas, not only used medieval, but also classical literature as a tool of reference to describe 'otherness.' As true humanists, the French Jesuits who arrived in the New World were deeply influenced by their classical education and, as claimed by Grafton, reverted to ancient ethnographic texts, like Tacitus' Germania, to support their analyse of the Indigenous people they encountered. Books talk to books. Inspired by Germania, the early French Jesuits managed to convey to their readers a subtle critique of their own civilization, enhancing, like Tacitus, the virtuous aspect of the so-called barbarians they described while illustrating the corruption of their respective civilized worlds. This thesis suggests that the essence of Tacitus' work is definitively present in Pierre Biard's letters and his Relation. His testimonies illustrate the connection the early French Jesuits had with the humanist thought of their time.

Development of techniques for testing the effect of the E1 region on adenovirus integration /

Adenoviral vectors are currently the most widely used gene therapeutic vectors, but their inability to integrate into host chromosomal DNA shortened their transgene expression and limited their use in clinical trials. In this project, we initially planned to develop a technique to test the effect of the early region 1 (E1) on adenovirus integration by comparing the integration efficiencies between an E1-deleted adenoviral vector (SubE1) and an Elcontaining vector (SubE3). However, we did not harvest any SubE3 virus, even if we repeated the transfection and successfully rescued the SubE1 virus (2/4 transfections generated viruses) and positive control virus (6/6). The failure of rescuing SubE3 could be caused by the instability of the genomic plasmid pFG173, as it had frequent intemal deletions when we were purifying It. Therefore, we developed techniques to test the effect of E1 on homologous recombination (HR) since literature suggested that adenovirus integration is initiated by HR. We attempted to silence the E1 in 293 cells by transfecting E1A/B-specific small interfering RNA (siRNA). However, no silenced phenotype was observed, even if we varied the concentrations of E1A/B siRNA (from 30 nM to 270 nM) and checked the silencing effects at different time points (48, 72, 96 h). One possible explanation would be that the E1A/B siRNA sequences are not potent enough to Induce the silenced phenotype. For evaluating HR efficiencies, an HR assay system based on bacterial transfonmatJon was designed. We constmcted two plasmids ( designated as pUC19-dl1 and pUC19-dl2) containing different defective lacZa cassettes (forming white colonies after transformation) that can generate a functional lacZa cassette (forming blue colonies) through HR after transfecting into 293 cells. The HR efficiencies would be expressed as the percentages of the blue colonies among all the colonies. Unfortunately, after transfonnation of plasmid isolated from 293 cells, no colony was found, even at a transformation efficiency of 1.8x10^ colonies/pg pUC19, suggesting the sensitivity of this system was low. To enhance the sensitivity, PCR was used. We designed a set of primers that can only amplify the recombinant plasmid fomied through HR. Therefore, the HR efficiencies among different treatments can be evaluated by the amplification results, and this system could be used to test the effect of E1 region on adenovirus integration. In addition, to our knowledge there was no previous studies using PCR/ Realtime PCR to evaluate HR efficiency, so this system also provides a PCR-based method to carry out the HR assays.

Locating and sequencing of the E3 and neighbouring regions in bovine advenovirus type 2

Adenoviruses are nonenveloped icosahedral shaped particles. The double stranded DNA viral genome is divided into 5 major early transcription units, designated E1 A, E1 B, and E2 to E4, which are expressed in a regulated manner soon after infection. The gene products of the early region 3 (E3), shown to be nonessential for viral replication in vitro, are believed to be involved in counteracting host immunosurveillance. In order to sequence the E3 region of Bovine adenovirus type 2 (BAV2) it was necessary to determine the restriction map for the plasmid pEA48. A physical restriction endonuclease map for BamHl, Clal, Eco RI, Hindlll, Kpnl, Pstt, Sail, and Xbal was constructed. The DNA insert in pEA48 was determined to be viral in origin using Southern hybridization. A human adenovirus type 5 recombinant plasmid, containing partial DNA fragments of the two transcription units L4 and L5 that lie just outside the E3, was used to localize this region. The recombinant plasmid pEA was subcloned to facilitate sequencing. The DNA sequences between 74.8 and 90.5 map units containing the E3, the hexon associated protein (pVIII), and the fibre gene were determined. Homology comparison revealed that the genes for the hexon associated pV11I and the fibre protein are conserved. The last 70 amino acids of the BAV2 pV11I were the most conserved, showing a similarity of 87 percent with Ad2 pV1I1. A comparison between the predicted amino acid sequences of BAV2 and Ad40, Ad41 , Ad2 and AdS, revealed that they have an identical secondary structure consisting of a tail, a shaft and a knob. The shaft is composed of 22, 15 amino acid motifs, with periodic glycines and hydrophobic residues. The E3 region was found to consist of about 2.3 Kbp and to encode four proteins that were greater than 60 amino acids. However, these four open reading frames did not show significant homology to any other known adenovirus DNA or protein sequence.

Electrophysiological constituents of the P100 and N170 ERP complex: De-constructing of face processing using independent component analysis and robust estimation.

The initial timing of face-specific effects in event-related potentials (ERPs) is a point of contention in face processing research. Although effects during the time of the N170 are robust in the literature, inconsistent effects during the time of the P100 challenge the interpretation of the N170 as being the initial face-specific ERP effect. The interpretation of the early P100 effects are often attributed to low-level differences between face stimuli and a host of other image categories. Research using sophisticated controls for low-level stimulus characteristics (Rousselet, Husk, Bennett, & Sekuler, 2008) report robust face effects starting at around 130 ms following stimulus onset. The present study examines the independent components (ICs) of the P100 and N170 complex in the context of a minimally controlled low-level stimulus set and a clear P100 effect for faces versus houses at the scalp. Results indicate that four ICs account for the ERPs to faces and houses in the first 200ms following stimulus onset. The IC that accounts for the majority of the scalp N170 (icNla) begins dissociating stimulus conditions at approximately 130 ms, closely replicating the scalp results of Rousselet et al. (2008). The scalp effects at the time of the P100 are accounted for by two constituent ICs (icP1a and icP1b). The IC that projects the greatest voltage at the scalp during the P100 (icP1a) shows a face-minus-house effect over the period of the P100 that is less robust than the N 170 effect of icN 1 a when measured as the average of single subject differential activation robustness. The second constituent process of the P100 (icP1b), although projecting a smaller voltage to the scalp than icP1a, shows a more robust effect for the face-minus-house contrast starting prior to 100 ms following stimulus onset. Further, the effect expressed by icP1 b takes the form of a larger negative projection to medial occipital sites for houses over faces partially canceling the larger projection of icP1a, thereby enhancing the face positivity at this time. These findings have three main implications for ERP research on face processing: First, the ICs that constitute the face-minus-house P100 effect are independent from the ICs that constitute the N170 effect. This suggests that the P100 effect and the N170 effect are anatomically independent. Second, the timing of the N170 effect can be recovered from scalp ERPs that have spatio-temporally overlapping effects possibly associated with low-level stimulus characteristics. This unmixing of the EEG signals may reduce the need for highly constrained stimulus sets, a characteristic that is not always desirable for a topic that is highly coupled to ecological validity. Third, by unmixing the constituent processes of the EEG signals new analysis strategies are made available. In particular the exploration of the relationship between cortical processes over the period of the P100 and N170 ERP complex (and beyond) may provide previously unaccessible answers to questions such as: Is the face effect a special relationship between low-level and high-level processes along the visual stream?

Collective violence : a study of the gendered and socio-economic factors behind early modern Italian and English witch hunts

In this study, I build upon my previous research in which I focus on religious doctrine as a gendered disciplinary apparatus, and examine the witch trials in early modem England and Italy in light of socio-economic issues relating to gender and class. This project examines the witch hunts/trials and early modem visual representations of witches, and what I suggest is an attempt to create docile bodies out of members of society who are deemed unruly, problematic and otherwise 'undesirable'; it is the witch's body that is deemed counternormative. This study demonstrates that it is neighbours and other acquaintances of accused witches that take on the role of the invisible guard of Bantham's Panoptic on. As someone who is trained in the study of English literature and literary theory, my approach is one that is informed by this methodology. It is my specialization in early modem British literature that first exposed me to witch-hunting manuals and tales of the supernatural, and it is for this reason that my research commences with a study of representations of witches and witchcraft in early modem England. From my initial exposure to such materials I proceed to examine the similarities and the differences of the cultural significance of the supernatural vis-a.-vis women's activities in early modem Italy. The subsequent discussion of visual representations of witches involves a predominance of Germanic artists, as the seminal work on the discernment of witches and the application of punishment known as the Malleus Meleficarum, was written in Germany circa 1486. Textual accounts of witch trials such as: "A Pitiless Mother (1616)," "The Wonderful Discovery of the Witchcrafts of Margaret and Philippa Flower (1619)," "Magic and Poison: The Trial ofChiaretta and Fedele (circa 1550)", and the "The Case of Benvegnuda Pincinella: Medicine Woman or Witch (1518),"and witchhunting manuals such as the Malleus Melejicarum and Strix will be put in direct dialogue with visual representations of witches in light of historical discourses pertaining to gender performance and gendered expectations. Issues relating to class will be examined as they pertain to the material conditions of presumed witches. The dominant group in any temporal or geographic location possesses the tools of representation. Therefore, it is not surprising that the physical characteristics, sexual habits and social material conditions that are attributed to suspected witches are attributes that can be deemed deviant by the ruling class. The research will juxtapose the social material conditions of suspected witches with the guilt, anxiety, and projection of fear that the dominant groups experienced in light of the changing economic landscape of the Renaissance. The shift from feudalism to primitive accumulation, and capitalism saw a rise in people living in poverty and therefore an increased dependence upon the good will of others. I will discuss the social material conditions of accused witches as informed by what Robyn Wiegman terms a "minoritizing discourse" (210). People of higher economic standing often blamed their social, medical, and/or economic difficulties on the less fortunate, resulting in accusations of witchcraft.

In danger of jammed minds: A deconstructive discourse analysis of the criteria for early onset schizophrenia and imagination in children's literature

The purpose of this research is to expose and complicate those discourses of childhood imagination as demonstrated in the diagnostic criteria for early onset schizophrenia by using an antipsychiatry perspective. This will be done by evaluating those discourses alongside those found in popular children’s literature, specifically, Harry Potter and The Philosopher’s Stone, Bridge to Terabithia, and A Wrinkle in Time. Once uncovered, the underlying power discourses were then exposed. This research will then employ a minor reading as provided by Deleuze and Guattari’s (1987) approach to minor literature to demonstrate the ways in which the child can subvert those dominant discourses. The potential of literature is evaluated for its ability to provide alternative modes of experience and lines of flight for the child subjected to the diagnostic criteria of schizophrenia.

Letter to S.D. Woodruff regarding resolution #5 of the Board of Directors. Mr. Woodruff is requested to take steps to ensure the early completion of the Port Dalhousie and Thorold macadamized road

Letter to S.D. Woodruff regarding resolution #5 of the Board of Directors. Mr. Woodruff is requested to take steps to ensure the early completion of the Port Dalhousie and Thorold macadamized road. This is signed by James McCoppen, president. The letter is stained and worn torn in spots. This does not affect the text, July 11, 1857.