Investigation of single tryptophan proteins encapsulated in TEOS-derived sol-gel matrices by fluorescence spectroscopy /

In the work reported here, optically clear, ultrathin TEOS derived sol-gel slides which were suitable for studies of tryptophan (Trp) fluorescence from entrapped proteins were prepared by the sol-gel technique and characterized. The monitoring of intrinsic protein fluorescence provided information about the structure and environment of the entrapped protein, and about the kinetics of the interaction between the entrapped protein and extemal reagents. Initial studies concentrated on the single Trp protein monellin which was entrapped into the sol-gel matrices. Two types of sol-gel slides, termed "wet aged", in which the gels were aged in buffer and "dry-aged", in which the gels were aged in air , were studied in order to compare the effect of the sol-gel matrix on the structure of the protein at different aging stages. Fluorescence results suggested that the mobility of solvent inside the slides was substantially reduced. The interaction of the entrapped protein with both neutral and charged species was examined and indicated response times on the order of minutes. In the case of the neutral species the kinetics were diffusion limited in solution, but were best described by a sum of first order rate constants when the reactions occurred in the glass matrix. For charged species, interactions between the analytes and the negatively charged glass matrix caused the reaction kinetics to become complex, with the overall reaction rate depending on both the type of aging and the charge on the analyte. The stability and conformational flexibility of the entrapped monellin were also studied. These studies indicated that the encapsulation of monellin into dry-aged monoliths caused the thermal unfolding transition to broaden and shift upward by 14°C, and causedthe long-term stability to improve by 12-fold (compared to solution). Chemical stability studies also showed a broader transition for the unfolding of the protein in dry-aged monoliths, and suggested that the protein was present in a distribution of environments. Results indicated that the entrapped proteins had a smaller range of conformational motions compared to proteins in solution, and that entrapped proteins were not able to unfold completely. The restriction of conformational motion, along with the increased structural order of the internal environment of the gels, likely resulted in the improvements in themial and long-term stability that were observed. A second protein which was also studied in this work is the metal binding protein rat oncomodulin. Initially, the unfolding behavior of this protein in aqueous solution was examined. Several single tryptophan mutants of the metal-binding protein rat oncomodulin (OM) were examined; F102W, Y57W, Y65W and the engineered protein CDOM33 which had all 12 residues of the CD loop replaced with a higher affinity binding loop. Both the thermal and the chemical stability were improved upon binding of metal ions with the order apo < Ca^^ < Tb^"^. During thermal denaturation, the transition midpoints (Tun) of Y65W appeared to be the lowest, followed by Y57W and F102W. The placement of the Trp residue in the F-helix in F102W apparently made the protein slightly more thermostable, although the fluorescence response was readily affected by chemical denaturants, which probably acted through the disruption of hydrogen bonds at the Cterminal end of the F-helix. Under both thermal and chemical denaturation, the engineered protein showed the highest stability. This indicated that increasing the number of metal ligating oxygens in the binding site, either by using a metal ion with a higher coordinatenumber (i.e. Tb^*) which binds more carboxylate ligands, or by providing more ligating groups, as in the CDOM33 replacement, produces notable improvements in protein stability. Y57W and CE)OM33 OM were chosen for further studies when encapsulated into sol-gel derived matrices. The kinetics of interaction of terbium with the entrapped proteins, the ability of the entrapped protein to binding terbium, as well as thermal stability of these two entrapped protein were compared with different levels of Ca^"*^ present in the matrix and in solution. Results suggested that for both of the proteins, the response time and the ability to bind terbium could be adjusted by adding excess calcium to the matrix before gelation. However, the less stable protein Y57W only retained at most 45% of its binding ability in solution while the more stable protein CDOM33 was able to retain 100% binding ability. Themially induced denaturation also suggested that CDOM33 showed similar stability to the protein in solution while Y57W was destabilized. All these results suggested that "hard" proteins (i.e. very stable) can easily survive the sol-gel encapsulation process, but "soft" proteins with lower thermodynamic stability may not be able to withstand the sol-gel process. However, it is possible to control many parameters in order to successfully entrap biological molecules into the sol-gel matrices with maxunum retention of activity.

Anharmonic Helmholtz free energy to O(lambdaâ ´) in the non- leading term approximation /|nby Leslie Wilk. -- 260 St. Catharines, Ont. : [s. n.],

Expressions for the anharmonic Helmholtz free energy contributions up to o( f ) ,valid for all temperatures, have been obtained using perturbation theory for a c r ystal in which every atom is on a site of inversion symmetry. Numerical calculations have been carried out in the high temperature limit and in the non-leading term approximation for a monatomic facecentred cubic crystal with nearest neighbour c entralforce interactions. The numbers obtained were seen to vary by a s much as 47% from thos e obtai.ned in the leading term approximati.on,indicating that the latter approximati on is not in general very good. The convergence to oct) of the perturbation series in the high temperature limit appears satisfactory.

Fluorometric monitoring of the properties of proteins entrapped in sol-gel derived matrices

The successful development of stable biosensors incorporating entrapped proteins suffers from poor understanding of the properties of the entrapped biomolecules. This thesis reports on the use of fluorescence spectroscopy to investigate the properties of proteins entrapped in sol-gel processed silicate materials. Two different single tryptophan (Trp) proteins were investigated in this thesis, the Ca2 + binding protein cod III parvalbumin (C3P) and the salicylate binding protein human serum albumin (HSA). Furthermore, the reactive single cysteine (Cys) residue within C3P and HSA were labelled with the probes iodoacetoxynitrobenzoxadiazole (C3P) and acrylodan (C3P and HSA) to further examine the structure, stability and function of the free and entrapped proteins. The results show that both C3P and HSA can be successfully entrapped into sol-gelderived matrices with retention of function and conformational flexibility.

On the Ewald method and the quartic Helmholtz free energy of an anharmonic metallic crystal

Thesis (M. Sc.) - Brock University, 1978.

Free Niagara.--

6 leaves of advertisements at end.

How Niagara was made free : The passage of Niagara Reservation Act in 1885 /

Reprinted from the Buffalo Historical Society Publications.

An anharmonic contribution to the Helmholtz free energy O(lambda 6)

The anharmonic contributions of order A6 to the Helmholtz free energy for a crystal in which every atom is on a site of inversion symmetry, have been evaluated The cor~esponding diagrams in the various orders of the perturbation theory have been presented The validity of the expressions given is for high temperatures. Numerical calculations for the diagrams which contribute to the free energy have been worked out for a nearest-n~ighbour central-force model of a facecentered cubic lattice in the high-temperature limit and in the leading term and the Ludwig approximations. The accuracy of the Ludwig approximation in evaluating the Brillouin-zone sums has been investigated. Expansion for all diagrams in the high-temperature limit has been carried out The contribution to the specific heat involves a linear as well as cubic term~ We have applied Lennard-Jones, Morse and Exponential 6 types of potentials. A comparison between the contribution to the free energy of order A6 to that of order A4 has been made.

"Man of science, man of faith" : Lost, consumer agency and the fate/free will binary in the post-9/11 context

In 2004, Lost debuted on ABC and quickly became a cultural phenomenon. Its postmodem take on the classic Robinson Crusoe desert island scenario gestures to a variety of different issues circulating within the post-9II1 cultural consciousness, such as terrorism, leadership, anxieties involving air travel, torture, and globalization. Lost's complex interwoven flashback and flash-forward narrative structure encourages spectators to creatively hypothesize solutions to the central mysteries of the narrative, while also thematically addressing archetypal questions of freedom of choice versus fate. Through an examination of the narrative structure, the significance of technological shifts in television, and fan cultures in Lost, this thesis discusses the tenuous notion of consumer agency within the current cultural context. Furthermore, I also explore these issues in relation to the wider historical post-9/II context.

The true history regarding alleged connection of the order of ancient, free and accepted Masons: with the abduction and murder of William Morgan in western New York, in 1826... (1886)

Cover title: Masonic light on the abduction and murder of Wm. Morgan.

Youth, Global Citizenship and Help: A Critical Rhetorical Analysis of Free The Children

Since 1995 Free The Children (FTC) has grown to be one of the largest and most recognized youth-focused and youth-led non-governmental organizations (NGOs) in Canada. FTC has distinguished itself by developing slick marketing campaigns, promising youth that they will become agents of change who can make a significant contribution towards eradicating poverty and promoting global social justice. The organization has utilized the Internet, creating an engaging and dynamic web page used to promote its development initiatives and celebrate the altruistic actions of its young participants. FTC uses a variety of strategies including text, video and images to persuade the viewer to engage with and elicit support for the organization. FTC attracts viewers by highlighting the successes of its overseas initiatives and the contributions made by young Northern volunteers in the global South. The organization also uses celebrity ambassadors, and cultural events such as We Day to raise its profile. Using a critical rhetorical analysis, this thesis interrogates FTC’s online promotional materials, exploring how the organization uses rhetorical strategies to persuade young people to take an interest in social justice activities. More specifically, an examination of FTC web-based promotional materials identifies and problematizes the organization’s rhetorical emphasis on youth empowerment, global citizenship and direct forms of helping the global South. This thesis argues that FTC does not direct adequate attention to fostering critical awareness among it participants. Further, the organization fails to provide its online participants with the appropriate tools or opportunities to critically engage with the structural issues related to global inequities. This thesis also examines how the organization uses rhetoric that promotes simplistic, feel-good projects that avoid exposing young people to an analysis of global social injustices.

Totals of free goods up – class 3 and class 4

Totals of free goods up – class 3 and class 4 (1 page, handwritten), n.d.

Unsigned letter to A. Jeffrey written on an envelope stating that the glass was free from breakage, therefore there would be a deduction of 10 percent

Unsigned letter to A. Jeffrey written on an envelope stating that the glass was free from breakage, therefore there would be a deduction of 10 percent, June 28, 1875.

California : A history of Upper and Lower California from their first discovery to the present time, comprising an account of the climate, soil, natural productions, agriculture, commerce, Sc. A full view of the missionary establishments and condition of the free and domesticated indians. With an appendix relation to steam navigation in the Pacific. Illustrated with a new map, plans of the harbours, and numerous engravings

UANL