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.

Orientation of Non-Native 2-Monosubstituted and 2,3-Disubstituted 1,4-Naphthoquinones in the A1 binding site of PSI and the effect on the rate of electron transfer : an electron paramagnetic resonance spectroscopy study

The proce-ss ofoxygenic photosynthesis is vital to life on Earth. the central event in photosynthesis is light induced electron transfer that converts light into energy for growth. Ofparticular significance is the membrane bound multisubunit protein known as Photosystem I (PSI). PSI is a reaction centre that is responsible for the transfer of electrons across the membrane to reduce NADP+ to NADPH. The recent publication ofa high resolution X-ray structure of PSI has shown new information about the structure, in particular the electron transfer cofactors, which allows us to study it in more detail. In PSI, the secondary acceptor is crucial for forward electron transfer. In this thesis, the effect of removing the native acceptor phylloquinone and replacing it with a series of structurally related quinones was investigated via transient electron paramagnetic resonance (EPR) experiments. The orientation of non native quinones in the binding site and their ability to function in the electron transfer process was determined. It was found that PSI will readily accept alkyl naphthoquinones and anthraquinone. Q band EPR experiments revealed that the non-native quinones are incorporated into the binding site with the same orientation of the headgroup as in the native system. X band EPR spectra and deuteration experiments indicate that monosubstituted naphthoquinones are bound to the Al site with their side group in the position occupied by the methyl group in native PSI (meta to the hydrogen bonded carbonyl oxygen). X band EPR experiments show that 2, 3- disubstituted methyl naphthoquinones are also incorporated into the Al site in the same orientation as phylloquinone, even with the presence of a halogen- or sulfur-containing side chain in the position normally occupied by the phytyl tail ofphylloquinone. The exception to this is 2-bromo-3-methyl --.- _. -. - -- - - 4 _._ _ _ - _ _ naphthoquinone which has a poorly resolved spectrum, making determination of the orientation difficuh. All of the non-native quinones studied act as efficient electron acceptors. However, forward electron transfer past the quinone could only be demonstrated for anthraquinone, which has a more negative midpoint potential than phylloquinone. In the case of anthraquinone, an increased rate of forward electron transfer compared to native PSI was found. From these results we can conclude that the rate ofelectron transfer from Al to Fx in native PSI lies in the normal region ofthe Marcus Curve.

Delineation of within-site terroir effects using soil and vine water measurement. Investigation of Cabernet Franc

. The influence of vine water status was studied in commercial vineyard blocks of Vilis vinifera L. cv. Cabernet Franc in Niagara Peninsula, Ontario from 2005 to 2007. Vine performance, fruit composition and vine size of non-irrigated grapevines were compared within ten vineyard blocks containing different soil and vine water status. Results showed that within each vineyard block water status zones could be identified on GIS-generated maps using leaf water potential and soil moisture measurements. Some yield and fruit composition variables correlated with the intensity of vine water status. Chemical and descriptive sensory analysis was performed on nine (2005) and eight (2006) pairs of experimental wines to illustrate differences between wines made from high and low water status winegrapes at each vineyard block. Twelve trained judges evaluated six aroma and flavor (red fruit, black cherry, black current, black pepper, bell pepper, and green bean), thr~e mouthfeel (astringency, bitterness and acidity) sensory attributes as well as color intensity. Each pair of high and low water status wine was compared using t-test. In 2005, low water status (L WS) wines from Buis, Harbour Estate, Henry of Pelham (HOP), and Vieni had higher color intensity; those form Chateau des Charmes (CDC) had high black cherry flavor; those at RiefEstates were high in red fruit flavor and at those from George site was high in red fruit aroma. In 2006, low water status (L WS) wines from George, Cave Spring and Morrison sites were high in color intensity. L WS wines from CDC, George and Morrison were more intense in black cherry aroma; LWS wines from Hernder site were high in red fruit aroma and flavor. No significant differences were found from one year to the next between the wines produced from the same vineyard, indicating that the attributes of these wines were maintained almost constant despite markedly different conditions in 2005 and 2006 vintages. Partial ii Least Square (PLS) analysis showed that leaf \}' was associated with red fruit aroma and flavor, berry and wine color intensity, total phenols, Brix and anthocyanins while soil moisture was explained with acidity, green bean aroma and flavor as well as bell pepper aroma and flavor. In another study chemical and descriptive sensory analysis was conducted on nine (2005) and eight (2006) medium water status (MWS) experimental wines to illustrate differences that might support the sub-appellation system in Niagara. The judges evaluated the same aroma, flavor, and mouthfeel sensory attributes as well as color intensity. Data were analyzed using analysis of variance (ANOVA), principal component analysis (PCA) and discriminate analysis (DA). ANOV A of sensory data showed regional differences for all sensory attributes. In 2005, wines from CDC, HOP, and Hemder sites showed highest. r ed fruit aroma and flavor. Lakeshore and Niagara River sites (Harbour, Reif, George, and Buis) wines showed higher bell pepper and green bean aroma and flavor due to proximity to the large bodies of water and less heat unit accumulation. In 2006, all sensory attributes except black pepper aroma were different. PCA revealed that wines from HOP and CDC sites were higher in red fruit, black currant and black cherry aroma and flavor as well as black pepper flavor, while wines from Hemder, Morrison and George sites were high in green bean aroma and flavor. ANOV A of chemical data in 2005 indicated that hue, color intensity, and titratable acidity (TA) were different across the sites, while in 2006, hue, color intensity and ethanol were different across the sites. These data indicate that there is the likelihood of substantial chemical and sensory differences between clusters of sub-appellations within the Niagara Peninsula iii

Niagara Parks Commission School of Gardening Graduation Program, 1956

The Niagara Parks Commission School of Gardening was organized in 1935 in order to help fill the Commission’s need for skilled gardeners to maintain the extensive parkland owned by the Commission. In 1959 the School was renamed the Niagara Parks Commission School of Horticulture. The name changed again in 1990 to the Niagara Parks Botanical Gardens and School of Horticulture to better reflect the development of the program.

Electron Transfer Involving the Phylloquinone (A1) Cofactor of Photosystem I Examined with Time Resolved Absorbance and Electron Paramagnetic Resonance Spectroscopy

The dependence of the electron transfer (ET) rate on the Photosystem I (PSI) cofactor phylloquinone (A1) is studied by time-resolved absorbance and electron paramagnetic resonance (EPR) spectroscopy. Two active branches (A and B) of electron transfer converge to the FX cofactor from the A1A and A1B quinone. The work described in Chapter 5 investigates the single hydrogen bond from the amino acid residue PsaA-L722 backbone nitrogen to A1A for its effect on the electron transfer rate to FX. Room temperature transient EPR measurements show an increase in the rate for the A1A- to FX for the PsaA-L722T mutant and an increased hyperfine coupling to the 2-methyl group of A1A when compared to wild type. The Arrhenius plot of the A1A- to FX ET in the PsaA-L722T mutant suggests that the increased rate is probably the result of a slight change in the electronic coupling between A1A- and FX. The reasons for the non-Arrhenius behavior are discussed. The work discussed in Chapter 6 investigates the directionality of ET at low temperature by blocking ET to the iron-sulfur clusters FX, FA and FB in the menB deletion mutant strain of Synechocyctis sp. PCC 6803, which is unable to synthesize phylloquinone, by incorporating the high midpoint potential (49 mV vs SHE) 2,3-dichloro-1,4-naphthoquinone (Cl2NQ) into the A1A and A1B binding sites. Various EPR spectroscopic techniques were implemented to differentiate between the spectral features created from A and B- branch electron transfer. The implications of this result for the directionality of electron transfer in PS I are discussed. The work discussed in Chapter 7 was done to study the dependence of the heterogeneous ET at low temperature on A1 midpoint potential. The menB PSI mutant contains plastiquinone-9 in the A1 binding site. The solution midpoint potential of the quinone measures 100 mV more positive then wild-type phylloquinone. The irreversible ET to the terminal acceptors FA and FB at low temperature is not controlled by the forward step from A1 to FX as expected due to the thermodynamic differences of the A1 cofactor in the two active branches A and B. Alternatives for the ET heterogeneity are discussed.

Letter to Robert Nelles from his son Henry Nelles

Letter to Robert Nelles from his son Henry Nelles (1 double-sided page, handwritten) regarding the shipping of walnut and cherry boards. The rest of the letter concerns farming and the price of ham and pork, July 7, 1819.