Isolation of DNA sequences with homology to a degenerate FaRP oligonucleotide from the crayfish Procambarus clarkii

The neuropeptide Th1RFamide with the sequence Phe-Met-Arg-Phe-amide was originally isolated in the clam Macrocallista nimbosa (price and Greenberg, 1977). Since its discovery, a large family ofFl\1RFamide-related peptides termed FaRPs have been found to be present in all major animal phyla with functions ranging from modulation of neuronal activity to alteration of muscular contractions. However, little is known about the genetics encoding these peptides, especially in invertebrates. As FaRP-encoding genes have yet to be investigated in the invertebrate Malacostracean subphylum, the isolation and characterization ofFaRP-encoding DNA and mRNA was pursued in this project. The immediate aims of this thesis were: (1) to amplify mRNA sequences of Procambarus clarkii using a degenerate oligonucleotide primer deduced from the common amino acid sequence ofisolated Procambarus FaRPS, (2) to determine if these amplification products encode FaRP gene sequences, and (3) to create a selective cDNA library of sequences recognized by the degenerate oligonucleotide primer. The polymerase chain reaction - rapid amplification of cDNA ends (PCR-RACE) is a procedure in which a single gene-specific primer is used in conjunction with a generalized 3' or 5' primer to amplify copies ofthe region between a single point in the transcript and the 3' or 5' end of cDNA of interest (Frohman et aI., 1988). PCRRACE reactions were optimized with respect to primers used, buffer composition, cycle number, nature ofgenetic substrate to be amplified, annealing, extension and denaturation temperatures and times, and use of reamplification procedures. Amplification products were cloned into plasmid vectors and recombinant products were isolated, as were the recombinant plaques formed in the selective cDNA library. Labeled amplification products were hybridized to recombinant bacteriophage to determine ligated amplification product presence. When sequenced, the five isolated PCR-RACE amplification products were determined not to possess FaRP-encoding sequences. The 200bp, 450bp, and 1500bp sequences showed homology to the Caenorhabditis elegans cosmid K09A11, which encodes for cytochrome P450; transfer-RNA; transposase; and tRNA-Tyr, while the 500bp and 750bp sequences showed homology with the complete genome of the Vaccinia virus. Under the employed amplification conditions the degenerate oligonucleotide primer was observed to bind to and to amplify sequences with either 9 or 10bp of 17bp identity. The selective cDNA library was obselVed to be of extremely low titre. When library titre was increased, white. plaques were isolated. Amplification analysis of eight isolated Agt11 sequences from these plaques indicated an absence of an insertion sequence. The degenerate 17 base oligonucleotide primer synthesized from the common amino acid sequence ofisolated Procambarus FaRPs was thus determined to be non-specific in its binding under the conditions required for its use, and to be insufficient for the isolation and identification ofFaRP-encoding sequences. A more specific primer oflonger sequence, lower degeneracy, and higher melting temperature (TJ is recommended for further investigation into the FaRP-encoding genes of Procambarlls clarkii.

Oral French Communication in French Immersion Canadian and World Studies Classrooms: A Primer for Educators

This project presents a primer for secondary French Immersion teachers that facilitates the use of French oral communicative activities in secondary Canadian and World Studies courses. The primer supports collaborative and inclusive teaching strategies that invite students to speak and develop their oral French communication skills. The primer is divided into 2 main components: (a) Rationale for the Primer, and (b) the Strategies themselves, comprising succinct descriptions as well as potential uses and suggestions. A critical content analysis of various Ontario Ministry of Education documents was undertaken in order to explore the importance of oral communication in second-language learning in Ontario secondary schools. Furthermore, holistic and invitational education perspectives were examined in order to define the advantages of collaborative learning. Moreover, research in the stream of French Immersion studies was also referenced to frame the relevance of second-language learning and the significant role the French Immersion teacher plays. The aforementioned research contributes to the advancement of theory and practice regarding the importance of opportunities for oral French communication in secondary Canadian and World Studies courses.

Resilience in a Watershed Governance Context: A Primer

Watersheds are complex systems involving social, economic, and ecological dimensions that are constantly interacting and influencing each other, and governance of these systems involve a large and diverse cast of actors that add to the complexity and difficulty in deciding what is best for the watershed and people. Resilience thinking offers a way to understand and navigate the uncertainty, dynamics and complexity of watershed governance. This primer describes key ideas associated with resilience: more inclusive participation; building a shared understanding; inclusion of ecosystem services and functions in long-term planning; strong leadership; institutional and decision making flexibility; and, a decentralized system. This primer is an initial effort to translate the scholarly understanding of these key ideas and initiate a dialogue about their application in the context of watershed governance.

List of Employees to be involved in the extension of the Port Dalhousie and Thorold Railway

List of Employees to be involved in the extension of the Port Dalhousie and Thorold Railway (1 page, handwritten). This is signed by S.D. Woodruff, Nov. 25, 1856.

Letter of estimate sent to S.D. Woodruff for the total cost of construction and equipment of the extension of the line to Port Colborne

Letter of estimate sent to S.D. Woodruff for the total cost of construction and equipment of the extension of the line to Port Colborne [this is unsigned]. There is an envelope with this letter that suggests that it is from Mr. Shanly, Mar. 12, 1857.

Chart of estimate of cost of Port Dalhousie and Thorold Railway extension Line no. 1

Chart of estimate of cost of Port Dalhousie and Thorold Railway extension Line no. 1 signed by Mr. Shanly, Mar. 12, 1857.

Report sent to the President and Director of the Port Dalhousie and Thorold Railway stating that the location of the line extension of the railway is completed

Report sent to the President and Director of the Port Dalhousie and Thorold Railway stating that the location of the line extension of the railway is completed. The distance from Thorold Station to Port Colborne is 20 miles. The estimate provides for construction of a permanent structure across the Chippewa and Welland River. Estimates for building a first class road, culverts and bridges will be of permanent and durable masonry. This includes estimates for various station buildings such as a warehouse in Port Colborne and a warehouse in Port Dalhousie. Surveys and plans are ready and will be registered this week. This is signed by S.D. Woodruff (2 copies) [one appears to be a rough copy] (The rough copy is 5 pages, handwritten and the other copy is 6 pages, handwritten), Apr. 8, 1857.

Voucher #2 from the Engineer Department of Port Dalhousie and Thorold Railway Extension paid to Daniel Plumsteel, Dec. 31, 1856

Voucher #2 from the Engineer Department of Port Dalhousie and Thorold Railway Extension for surveys, paid to Daniel Plumsteel, Dec. 31, 1856.

Voucher #3 from the Engineer Department of Port Dalhousie and Thorold Railway Extension paid to Thomas Woods, Dec. 31, 1856

Voucher #3 from the Engineer Department of Port Dalhousie and Thorold Railway Extension for sinking test pits, paid to Thomas Woods, Dec. 31, 1856.

Voucher #5 from the Engineer Department of Port Dalhousie and Thorold Railway Extension to George Forbes for surveys, Jan. 31, 1857

Voucher #5 from the Engineer Department of Port Dalhousie and Thorold Railway Extension to George Forbes for surveys, Jan. 31, 1857.

Voucher #6 from the Engineer Department of Port Dalhousie and Thorold Railway Extension to Thomas Woods for surveys, Jan. 31, 1857

Voucher #6 from the Engineer Department of Port Dalhousie and Thorold Railway Extension to Thomas Woods for surveys, Jan. 31, 1857.

Voucher #7 from the Engineer Department of Port Dalhousie and Thorold Railway Extension to W.G. Thompson accompanied by an abstract of vouchers and summaries of account paid by W.G. Thompson for surveys, Jan. 31, 1857

Voucher #7 from the Engineer Department of Port Dalhousie and Thorold Railway Extension to W.G. Thompson accompanied by an abstract of vouchers and summaries of account paid by W.G. Thompson for surveys, Jan. 31, 1857.

Pay roll voucher #8 from the Engineer Department of Port Dalhousie and Thorold Railway Extension for surveys in the month of February, Feb. 28, 1857

Pay roll voucher #8 from the Engineer Department of Port Dalhousie and Thorold Railway Extension for surveys in the month of February, Feb. 28, 1857.

Voucher #9 from the Engineer Department of Port Dalhousie and Thorold Railway Extension to W. G. Thompson accompanied by an abstract of vouchers for surveys, Feb. 28, 1857

Voucher #9 from the Engineer Department of Port Dalhousie and Thorold Railway Extension to W. G. Thompson accompanied by an abstract of vouchers for surveys, Feb. 28, 1857

Pay roll voucher #10 from the Engineer Department of Port Dalhousie and Thorold Railway Extension for the engineer corps. , Mar. 31, 1857

Pay roll voucher #10 from the Engineer Department of Port Dalhousie and Thorold Railway Extension for the engineer corps. , Mar. 31, 1857.