Extracting Ramachandran torsional angle distributions from 2D NMR data using Tikhonov regularization /

Solid state nuclear magnetic resonance (NMR) spectroscopy is a powerful technique for studying structural and dynamical properties of disordered and partially ordered materials, such as glasses, polymers, liquid crystals, and biological materials. In particular, twodimensional( 2D) NMR methods such as ^^C-^^C correlation spectroscopy under the magicangle- spinning (MAS) conditions have been used to measure structural constraints on the secondary structure of proteins and polypeptides. Amyloid fibrils implicated in a broad class of diseases such as Alzheimer's are known to contain a particular repeating structural motif, called a /5-sheet. However, the details of such structures are poorly understood, primarily because the structural constraints extracted from the 2D NMR data in the form of the so-called Ramachandran (backbone torsion) angle distributions, g{^,'4)), are strongly model-dependent. Inverse theory methods are used to extract Ramachandran angle distributions from a set of 2D MAS and constant-time double-quantum-filtered dipolar recoupling (CTDQFD) data. This is a vastly underdetermined problem, and the stability of the inverse mapping is problematic. Tikhonov regularization is a well-known method of improving the stability of the inverse; in this work it is extended to use a new regularization functional based on the Laplacian rather than on the norm of the function itself. In this way, one makes use of the inherently two-dimensional nature of the underlying Ramachandran maps. In addition, a modification of the existing numerical procedure is performed, as appropriate for an underdetermined inverse problem. Stability of the algorithm with respect to the signal-to-noise (S/N) ratio is examined using a simulated data set. The results show excellent convergence to the true angle distribution function g{(j),ii) for the S/N ratio above 100.

Isolation and characterization of genomic DNA sequences that enhance the stability of plasmid DNA in mammalian cells /

The ease of production and manipulation has made plasmid DNA a prime target for its use in gene transfer technologies such as gene therapy and DNA vaccines. The major drawback of plasmid however is its stability within mammalian cells. Plasmid DNA is usually lost by cellular mechanisms or as a result of mitosis by simple dilution. This study set out to search for mammalian genomic DNA sequences that would enhance the stability of plasmid DNA in mammalian cells.Creating a plasmid based genomic DNA library, we were able to screen the human genome by transfecting the library into Human Embryonic Kidney (HEK 293) Cells. Cells that contained plasmid DNA were selected, using G418 for 14 days. The resulting population was then screened for the presence of biologically active plasmid DNA using the process of transformation as a detector.A commercially available plasmid DNA isolation kit was modified to extract plasmid DNA from mammalian cells. The standardized protocol had a detection limit of -0.6 plasmids per cell in one million cells. This allowed for the detection of 45 plasmids that were maintained for 32 days in the HEK 293 cells. Sequencing of selected inserts revealed a significantly higher thymine content in comparison to the human genome. Sequences with high A/T content have been associated with Scaffold/Matrix Attachment Region (S/MAR) sequences in mammalian cells. Therefore, association with the nuclear matrix might be required for the stability of plasmids in mammalian cells.

Second Welland Canal - Book 1, Survey Map 1 - Entrance, Lock 1 and Port Dalhousie

Survey map of the Second Welland Canal created by the Welland Canal Company showing the areas in and around Port Dalhousie and Grantham Township. Identified structures associated with the Canal include Lock 1, Lighthouse, Lighthouse Keeper's House, East and West Piers, Harbour, Waste Weir, Store House, Collector's Office, Collector Assistant Office, Lock Tender's House and the new towing path. Features of the First Welland Canal are noted in red ink and includes the old Harbour, old Lock 1, old towing path and the original bed of the Twelve Mile Creek. The surveyors' measurements and notes can be seen in red and black ink and pencil. Local area landmarks and businesses are also identified and include streets and roads (ex. Lock Street and Colonel Clark's Cattle Road), Alex Muir's Dry Dock, RandJ Laurie Flouring Mill, R. Laurie and Company Grist Mill, A. Morrison Saw Mill, Johnson's Tavern, a store and a church. Properties and property owners of note are: Concession 1 Lots 21 and 22, John Christie, John Clark, N. Pawling, William Pawling, W. Carter, G.A. Clark, J. Maven, Mrs. Wood, James Drabble and J. Woodall.

Second Welland Canal - Book 1, Survey Map 2 - Lock 1 and Port Dalhousie

Survey map of the Second Welland Canal created by the Welland Canal Company showing the areas in and around Port Dalhousie and Grantham Township. Identified structures associated with the Canal include Lock 1, East and West Piers, Collector's Office, Lock Tender's House and the new towing path. The surveyors' measurements and notes can be seen in red and black ink and pencil. Local area landmarks and businesses are also identified and include streets and roads (ex. Road to St. Catharines, Side Line, Old Road to Port Dalhousie, Road to Niagara), the Welland Railway and its structures (ex. freight sheds, wood shed, raised platform, elevator, cranes, water tank, turn table, and passenger station), G. A. Clark's Wood Yard, Clark's Wood Office, Alex Muir's Dry Dock, Donald, Andrews and Ross' Dry Dock, RandJ Laurie Flouring Mill, R. Laurie and Company Grist Mill and A. Morrison Saw Mill. A New Road to St. Catharines is featured in red ink. Properties and property owners of note are: Concession 1 Lots 19, 20 and 21, John Christie, and John Clark.

Second Welland Canal - Book 1, Survey Map 3 - Old Lock 2 in Grantham Township

Survey map of the Second Welland Canal created by the Welland Canal Company showing a portion of the Grantham Township near Port Dalhousie. Identified structures associated with the Canal include the new towing path. The surveyors' measurements and notes can be seen in red and black ink and pencil. Features of the First Welland Canal are noted in red ink and include the old Lock 2, old towing path and the original bed of the Twelve Mile Creek. Local area landmarks are also identified and include streets and roads (ex. Side Line and Old Road), four unnamed bridges, and a tree stump along the old towing path. A New Road to Port Dalhousie is featured in red ink. Properties and property owners of note are: Concession 3 Lots 21, 22 and 23, Concession 4 Lots 21, 22 and 23, Jabez Johnson, Adam Gould, Peter Weaver and Samuel Wood.

Second Welland Canal - Book 1, Survey Map 9 - Lock 2 in Grantham Township

Survey map of the Second Welland Canal created by the Welland Canal Company showing a portion of the Grantham Township sometimes referred to as the Welland Vale. Identified structures associated with the Canal include Lock 2, several weirs, and the Lock Tender's House. The surveyors' measurements and notes can be seen in red and black ink and pencil. Features of the First Welland Canal are noted in red ink and include the old towing path and the Old Canal itself. Local area landmarks and businesses are also identified and include streets and roads (ex. Side Line and Old Road to Port Dalhousie), J. C. Clark's Ice House, J. L. Ranney Store House, a burnt mill, barrel shed, a building leased to Michael Kerrins, and a number of unidentified structures (possibly houses or cabins) belonging to D. Cain, R. Cain, W. Weaver and W. Huddy. A New Road to St. Catharines is featured in red ink. Properties and property owners of note are: Concession 5 Lots 20, 21 and 22, Concession 6 Lots 20 and 21, Thomas Adams, John Gould, George Rykert, Theophilus Mack, William H. Merritt, J. L. Ranney, and the Board of Works.

Second Welland Canal - Book 1, Survey Map 12 - St. Catharines and Lock 4

Survey map of the Second Welland Canal created by the Welland Canal Company showing the Town of St. Catharines. Identified structures associated with the Canal include Lock 4, Hydraulic Race, floating tow path, and the Canal waterway itself. The surveyors' measurements and notes can be seen in red and black ink and pencil. Local area landmarks are also identified and include streets and roads (ex. Geneva Street, St. Paul Street, Queenston Street, Niagara Street and Mill Street), C. Phelp's Grist Mill and Store House, Stinson's Distillery, and several unnamed bridges. Properties and property owners of note are: Concession 6 Lot 16, J. R. Benson, Calvin Phelps, J. Hudson, David Gray, A. Roberts, Mrs. McDonell, J. S. McDonell, T. B. Wragg, J. Donaldson, W. Barr Jr., C. L. Hall, G. Ward, Ridout Bros and Co., and the St. Catharines Grass Co.

Second Welland Canal - Book 1, Survey Map 14 - St. Catharines and Lock 5

Survey map of the Second Welland Canal created by the Welland Canal Company showing the Canal along the eastern edge of the Town of St. Catharines. Identified structures associated with the Canal include Lock 5 and the towing path. The surveyors' measurements and notes can be seen in red and black ink and pencil. Local area landmarks are also identified and include bridges, streets, and roads (ex. Queenston Street, St. Catharines Macdamized Road and Suspension Bridge), a Pond, a number of unnamed bridges, Stinson's Distillery, and R. Collier's Saw Mill. Properties and property owners of note are: Concession 6 Lots 14 and 15, R. Collier, W. Gillespie, Orson Phelps, W. Chase, M. Bryant, John Soper, Winsor Chace, John Berryman, John Boyle, J. Madigan, B. F. Reynolds, W. Smaill, F. Stinson, G. Ward and Mrs. Soper.

Second Welland Canal - Book 2, Survey Map 14 - Lock 26 and Allanburgh

Survey map of the Second Welland Canal created by the Welland Canal Company showing the canal as it passes through the Village of Allanburgh. Identified structures and features associated with the Canal include Lock 26, a Guard Lock, Lock Tender's House, the New Cut, waste wear, and the towing path. Parts of the old canal are indentified and include Old Lock 36 and 37, and the Old Cut. The surveyors' measurements and notes can be seen in red and black ink and pencil. Local area landmarks are also identified and include streets and roads (ex. Holland Road, Centre Street, Falls Street, Canal Street, and Clifton Street), Allanburgh Hotel, Rannies Store, Wright and Duncan Grist Mill, A. Vanderburgh Saw Mill, W. Pennock Shingle Factory, John Harper Tavern, a very delapitated Grist and Saw Mill, store house, a shanty, and a number of other structures - some of which are identified by their owners: A. Vanderburgh, W. Wright, C. Brent, and H. Mussen. Properties and property owners of note are: Lots 118 and 119, Captain Creighton, and William H. Merritt Jr. A number of small properties labeled 1 through 39 are present and of these 6 - 15 are reserved for a Mill Site and are outlined in red. Several other pieces of land are outlined in blue and belong to: W. B. Hendershot, P. Finlay, W. Wright, and L. Leslie. There is also a piece of land reserved for hydraulic purposes.

Second Welland Canal - Book 3, Survey Map 11 - Aqueduct Lock and Canal along Chippewa Creek in Thorold

Survey map of the Second Welland Canal created by the Welland Canal Company showing the canal as it crosses Chippewa Creek in the Thorold Township near Welland. Identified structures and features associated with the Canal include the towing path, the old canal, the aqueduct lock, the new aqueduct, and the waterway itself. The surveyors' measurements and notes can be seen in red and black ink and pencil. Local area landmarks are also identified and include roads (ex. Aqueduct Road, and Road to Weland), Chippewa Creek, the Spoil Bank, a house and a barn. Properties and property owners of note are: Lots 239, 247, and 248, Joseph Burgar, and Smith Shotwell.

Investigating the role of apoptosis regulator EndoG on exogenous DNA uptake, stability, replication and recombination

Endonuclease G (EndoG) is a well conserved mitochondrial nuclease with dual lethal and vital roles in the cell. It non-specifically cleaves endogenous DNA following apoptosis induction, but is also active in non-apoptotic cells for mitochondrial DNA (mtDNA) replication and may also be important for replication, repair and recombination of genomic DNA. The aim of our study was to examine whether EndoG exerts similar activities on exogenous DNA substrates such as plasmid DNA (pDNA) and viral DNA vectors, considering their importance in gene therapy applications. The effects of EndoG knockdown on pDNA stability and levels of encoded reporter gene expression were evaluated in the cervical carcinoma HeLa cells. Transfection of pDNA vectors encoding short-hairpin RNAs (shRNAs) reduced levels of EndoG mRNA and nuclease activity in HeLa cells. In physiological circumstances, EndoG knockdown did not have an effect on the stability of pDNA or the levels of encoded transgene expression as measured over a four day time-course. However, when endogenous expression of EndoG was induced by an extrinsic stimulus (a cationic liposome transfection reagent), targeting of EndoG by shRNA improved the perceived stability and transgene expression of pDNA vectors. Therefore, EndoG is not a mediator of exogenous DNA clearance, but in non-physiological circumstances it may non-specifically cleave intracellular DNA regardless of its origin. To investigate possible effects of EndoG on viral DNA vectors, we constructed and evaluated AdsiEndoG, a first generation adenovirus (Ad5 ΔE1) vector encoding a shRNA directed against EndoG mRNA, along with appropriate Ad5 ΔE1 controls. Infection of HeLa cells with AdsiEndoG at a multiplicity of infection (MOI) of 10 p.f.u./cell resulted in an early cell proliferation defect, absent from cells infected at equivalent MOI with control Ad5 ΔE1 vectors. Replication of Ad5 ΔE1 DNA was detected for all vectors, but AdsiEndoG DNA accumulated to levels that were 50 fold higher than initially, four days after infection, compared to 14 fold for the next highest control Ad5 ΔE1 vector. Deregulation of the cell cycle by EndoG depletion, which is characterized by an accumulation of cells in the G2/M transition, is the most likely reason for the observed cell proliferation defect. The enhanced replication of AdsiEndoG is consistent with this conclusion, as Ad5 ΔE1 DNA replication is intimately related to cell cycling and prolongation or delay in G2/M greatly enhances this process. Furthermore, infection of HeLa with AdsiEndoG at MOI of 50 p.f.u./cell resulted in an almost complete disappearance of viable, adherent tumour cells from culture, whereas almost a third of the cells were still adherent after infection with control Ad5 ΔE1 vectors, relative to the non-infected control. Therefore, targeting of EndoG by RNAi is a viable strategy for improving the oncolytic properties of first generation adenovirus vectors. In addition, AdsiEndoG-mediated knockdown of EndoG reduced homologous recombination between pDNA substrates in HeLa cells. The effect was modest but, nevertheless demonstrated that the proposed role of EndoG in homologous recombination of cellular DNA also extends to exogenous DNA substrates.

Sketch of Canal Lock 2

Sketch of canal lock 2 by S.D. Woodruff. This is a hand drawn sketch, n.d.

Clippings - Road Allowances, Lock 2 Bridge, Toronto Railway Company, St. Davids' History

Clipping regarding the original road allowance which was part of Mr. Woodruff’s property, n.d. Clipping regarding the Lock 2 Bridge. This is a letter to the editor of the Journal from Calvin Phelps of Lockport, Nov. 13, 1889. Clipping entitled “Settled beyond a Dispute”: a letter to the Editor of the Star from J. G. Currie regarding the road allowance in the 5th Concession of Grantham, Nov. 16, 1889. Clipping entitled “That Old Road Allowance” which is a rebuttal to Mr. Currie’s letter, [1889] 2 newspaper clippings regarding: “Reg. vs. Toronto Railway Company”. These 2 articles are glued to a piece of paper. Beneath the newspaper articles is the name M. Chambers, Nov. 18, 1898. Notes regarding the clippings are enclosed with the articles. These notes and written on the back of “Pattison, Collier and Shaw Barristers, Solicitors, Etc.” stationary (notes are 2 pages, handwritten), Nov. 18, 1898. Clipping entitled “History on the Site: St. Davids’ Students Learn Lessons where Events Occurred” (2 copies), n.d.

Lock of Hair - Charles Band

Lock of hair from Charles Band – an envelope within another envelope containing Charles Band’s hair. This is from a haircut for Granny’s anniversary. The writing of the envelope says “Pussy’s hair” Nov. 21, 1922.

Four Leaf Clover & Lock of Hair

Envelope containing a four leaf clover “for luck” and little Margaret’s hair (9 mos. old), Sept. 3, 1937.