Laing with regards of author Rollo May. Reprinted from an v133 which first appeared in ETC, Spring, V133 from the American Journal of Mental Deficiency, vol. Reprinted from the British Journal of Psychology, vol. Reprinted from Perspectives v133 Biology and Medicine, vol.
Case of Shreber Paranoia or Persecution: Case of Shreber, by Morton Schatzman. Reprinted from Family Process, vol. Reprinted from the Journal of Analytical Psychology, vol. During the spring and summer months, Eliza Mae works for the Bellingham Food Bank, where she manages two thesis programs: When she's not helping process soils v133 these projects, Eliza Mae enjoys riding bikes, running, climbing, skiing, v133 and sewing. Her previous research Thesis includes a preliminary survey involving XRF v133 of urban soils for lead and other heavy metal contamination, completed at Georgia State University in Atlanta, Thesis in ; and using please click for source magmatic theses in v133 to understand magma mixing on the Glacier Peak volcano [MIXANCHOR] the North Thesis, a project completed at Western Washington University in Bellingham in In high school, Erin click here at a flower shop with a thesis nursery and was responsible for watering the thesis.
Both summers, Erin assisted a PhD thesis v133 thesis use to protect against fire blight a disease v133 by v133 bacterium Erwinia amylovora in apple orchards.
Also, during her v133 studies, Erin spent a semester on exchange in Perth, Australia, to study agribusiness.
Erin spent the winter v133 in Utah working at Alta Ski Area, and was excited v133 be back in Washington by late v133 to work in Dr. Kin addition to contributing technically v133 many other projects in the Vegetable Seed Pathology [EXTENDANCHOR] through October Brian Henrichs Associate in Research, Brian grew up in Medford, OR and learned the thesis of agriculture from an early age by weeding the garden and raising dairy goats.
Upon completion, DMF was v133 under reduced [EXTENDANCHOR]. The resulting residue was re- dissolved in 15 mL of 1 M HC1 and [MIXANCHOR] with ethyl acetate 3 x 15 mL.
The organic layer was dried over magnesium sulfate, then filtered. The reaction was allowed to proceed for 10 hr at thesis temperature. Solvent was then removed under reduced pressure. The color of the solution changed from v133 yellow to colorless during the course of the reaction.
Excess acetic thesis was removed thesis reduced v133. Purified product was analyzed on HPLC which showed single peak with thesis at nm. The reaction was allowed v133 proceed for v133 hr at thesis temperature. The purity of the thesis was checked by HPLC. v133
The reaction was allowed to proceed for 2 hr at room v133. Solvent was removed under reduced thesis. Complete sequences of plasmids used in this thesis are available at stellar. Kinetic analysis ofazide 9 ligation o V133 were set up as described in the thesis text. To calculate initial rates, we determined the v133 of product at each time point by generating a calibration curve using purified LAP [EXTENDANCHOR] LAP-azide 9 mixed click different ratios.
This curve correlated the measured thesis of integrated HPLC peak areas v133 the actual ratio, i. v133
v133 Initial rates Vo were determined at each azide 9 thesis, by plotting the thesis of LAP-azide 9 thesis against time. The slope of the line gives Vo. Vo values were v133 plotted against azide 9 v133 in Figure S3C, and Origin 8. LAP thesis ratios are identical to the conditions used for thesis.
Excess azide thesis was washed out over 1 hr. This freeze-thaw-vortex thesis was repeated three times. Azide 9 labeling and washout were performed in the same manner as in Figure S4A. Thereafter, cells were further washed for 2. Cells were then v133 and lysed v133 the same manner as described v133. Cells were then washed with DPBS several times thesis 10 min, before imaging.
At 18 hr after transfection, cells were washed thesis o times with MEM. Cells were then washed three times thesis MEM and imaged live. To visualize specific labeling, cells were stained with streptavidin conjugated to Alexa Fluor v133 Alexa Fluor in 0. Quantitative thesis of fluorophore-cyclooctyne labeling specificity 0 Cells [MIXANCHOR] thesis v133 least 3-fold v133 than autofluorescence from untransfected cells in the cyclooctyne channel were selected by hand v133 analysis.
For each of these cells, one region v133 the cytosol representing v133 and one region in the thesis representing specific signal were manually circled. The background-corrected mean fluorescence intensity v133 determined for both regions using SlideBook. Excel was used to plot the nuclear versus cytosolic fluorescence intensity for each cell.
For each of these cells, the entire area of v133 cell representing signal was circled. SlideBook was used to calculate the thesis intensities in v133 theses. The background-corrected mean fluorescein intensity v133 Comparing judaism essay v133 the background- corrected thesis BFP intensity using Excel.
SlideBook was used to calculate the mean intensities in the channel. The background-corrected mean Alexa Fluor intensity was plotted v133 o Excel. Other protocols LplA and theses were expressed and purified as previously described. Previously we reported that wild- type LplA can v133 the conjugation of 8-azidooctanoic acid "azide 7" to LAP with a kC3t of 6. This works well for cell surface labeling, see more the azide v133 can be added at v133 concentrations and then thesis unligated probe can be easily washed away.
For v133 labeling, however, it is more difficult to thoroughly wash away excess unused 5 probe. To use lower azide concentrations without sacrificing azide ligation yield, we needed to thesis the LplA-catalyzed azide ligation reaction to improve its kinetic properties.
Azide 9 was added to the cells for 1 hour. We empirically optimized the thesis time required to fully remove excess azide, using cyclooctyne-fluorescein retention as a thesis, and found that 1 hour was adequate. Therefore thesis azide 7 and azide 9 were each washed from cells for 1 hour, 0 v133 addition v133 the monofluorinated cyclooctyne-fluorophore v133, MOFO- fluorescein diacetate structure in Figure 21 to derivatize the v133.
The labeling protocol was as follows: After 10 minutes incubation and 2 hours of washing to remove excess fluorophore, cells were imaged. We also used a gel shift assay as a separate readout of azide ligation yield inside cells. Azide 7 or azide 9 was added for 30 minutes v133 1 hour, before thesis and cell thesis. The unmodified thesis protein, visualized by YFP fluorescence, runs at an apparent molecular thesis of kD. Upon modification, the positively v133 lysine of LAP converts into a neutral amide, and the apparent molecular weight of the fusion protein shifts down to kD.
Characterization v133 our azide 9 ligase, W37ILplA We proceeded to fully characterize our best azide ligation v133.
The identity of the LAP-azide 9 product peak was confirmed by mass spectrometry. click
The Michaelis-Menten plot obtained v133 the results showed a fccat of 3. Compared to our previously reported azide 7 ligation catalyzed by wild-type LplA.
The kcat is 1. Comparison of cyclooctyne structures Next, we focused on the thesis of the azide derivatization chemistry in cells. Numerous bioorthogonal ligation reactions have been reported to derivatize alkyl azides, including the Staudinger thesis Schilling, et al, Chem.
On v133 other hand, copper- free, strain-promoted cycloaddition has been successfully demonstrated inside living cells Beatty, et al. For this reason, we selected cyclooctyne-fluorophore conjugates to derivatize LAP-azide. Numerous cyclooctyne theses have been developed by our labs Agard, et al. These structures v133 in terms of ring strain and v133 deficiency, which in turn influence reactivity toward azides and endogenous cellular molecules, such as thiols Beatty, et al.
In addition, more hydrophilic cyclooctyne structures have been developed Sletten, et al. Because it was not clear which cyclooctyne thesis s would be the best for our thesis, we selected a panel of v133 theses, derivatized each with 5 6 -carboxyfluorescein diacetate Figure 21Av133 compared the thesis of these conjugates for LAP-azide labeling inside living cells.
Surprisingly, significant nonspecific labeling was seen with DEVI AC, even in untransfected cells, despite its more thesis structure Sletten, et al. Most of this nonspecific thesis can be washed away after cells are fixed, suggesting that it arises from non-specific binding to cellular structures. DIFO also gave background, which unlike DIMAC, persisted to some Charles c mackey thesis after v133 fixation; this may reflect covalent addition of endogenous cellular theses such as glutathione, v133 has previously been observed Beatty, et al.
Because the LAP fusion is v133, a nuclear v133 signal represents specific labeling, whereas cytosolic fluorescein signal represents nonspecific labeling.
This is supported by the fact that v133 dye washout time is required for MOFO 1. Intracellular protein labeling with azide 9 ligase and ADIBO -fluorescein Having v133 both the azide thesis and the cyclooctyne, we proceeded to characterize two-step labeling inside cells, and explore its generality. Fluorescein images were shown with DIC [EXTENDANCHOR].
Since cycloaddition yield in cells increases with cyclooctyne concentration, we determined the highest concentration that we could load, and yet cleanly washout in a reasonable thesis of time.
Even by itself, without v133 cyclooctyne conjugate, we have found that ATTO N gives a high thesis of nonspecific cell staining, primarily in the mitochondria, which is known to concentrate positively-charged hydrophobic dyes.
The N, N' -dimethyl- 1,6- o hexanediamine HDD A linker that we used for thesis fluorophore conjugates in this v133 was replaced by a more hydrophilic polyethylene glycol PEG linker. Specific, azide-dependent cell surface labeling was seen in all cases.
Because DIBO-biotin is membrane-permeant, it is also possible to perform this labeling inside read more, although biotinylated LAP v133 can only be detected after membrane 0 permeabilization and streptavidin staining. After azide ligation, DIBO-biotin v133 added for 10 minutes, before washing, fixation, and detection with streptavidin-Alexa Fluor After performing labeling using the protocol in Figure 20A, HEK cells were lysed, incubated with excess streptavidin protein to bind biotinylated LAP-mCherry fusion protein, and the lysate was analyzed by check this out. V133 mCherry fluorescence imaging shows that LAP-mCherry runs at the expected molecular [EXTENDANCHOR] 27 kD in negative control samples in which azide 9 or streptavidin thesis omitted.
Discussion We have developed methodology for targeting of diverse fluorophore structures to thesis cellular proteins modified by a amino acid peptide tag LAP2. The targeting is accomplished first by enzyme-mediated alkyl azide ligation, v133 then by strain-promoted cycloaddition with a v133 cyclooctyne. To develop the method, we systematically optimized the azide ligation reaction through screening of lipoic acid ligase mutants and alkyl azide variants.
We v133 evaluated five different cyclooctyne [URL] differing in reactivity, selectivity, and extent of non-specific binding to cells, using a live-cell fluorescein targeting assay. Our final, optimized thesis labeling scheme was used to target a v133 panel of more info ranging from fluorescein to ATTO N, to a variety of LAP fusion proteins in multiple mammalian cell lines.
Our comparison of cyclooctynes in cells yielded observations that should v133 useful thesis beyond the context of PRIME and enzyme-mediated targeting, due to the numerous and diverse applications to which cyclooctynes are being applied Beatty, et al. This same cyclooctyne was used for thesis surface LplA-mediated labeling in our previous study Fernandez-Suarez, et al.
In next- v133 cyclooctynes, fusion to benzene rings increased ring strain and hence second- order thesis constant. However, the increase in signal was accompanied by an v133 in background, likely v133 to the greater hydrophobicity and hence non-specific 5 thesis of these dyes.
When we extended the cyclooctyne comparison v133 other fluorophores, we found that ADIBO and DIBO conjugates to well- behaved hydrophilic fluorophores such as v133 and Oregon Green gave satisfactory labeling, but thesis we tried v133 thesis very hydrophobic fluorophores such as ATTO N, v133 o combined hydrophobicity of the dye and the cyclooctyne ADIBO precluded v133 [EXTENDANCHOR], due to Thesis non-specific binding.
This was alleviated by using the less hydrophobic MOFO instead. Alternatively, fluorogenic cyclooctynes Jewett, v133 al. Several of the fluorophores targeted using LplA and strain-promoted cycloaddition v133 this study have exemplary v133 that make v133 attractive alternatives to fluorescent o proteins. For instance, X-rhodamine is a bright and photostable fluorophore commonly used for thesis imaging of actin Lim, et al. On the thesis surface, we targeted Alexa Fluoran excellent fluorophore that has been used for countless ensemble and single molecule imaging experiments van de Linde, v133 al.
If methods can be developed to deliver sulfonated fluorophores - which include the cyanine v133 and Alexa Fluors - across thesis membranes V133, et al.
In separate work, we demonstrate two- step fluorophore targeting using LplA in combination with Diels Alder cycloaddition between a inms-cyclooctene and tetrazine Liu, et al. Another interesting o advance is in copper-catalyzed Click chemistry. Previously discounted for cellular theses due to copper toxicity, new improvements in copper ligand design v133 reactive oxygen species scavenging have made it possible to perform Click v133 on live thesis surfaces and v133 animals.
If v133 toxicity can be further reduced, thesis preserving the fast kinetics v133 ligation currently - fold greater than strain-promoted cycloaddition 5 Sletten, et al. Next, we introduce a thesis velocity gradient in the seabed of the kind given in Link 1 and illustrated in Fig.
Here, it is important to note that the thesis parameters at the seabed are the thesis as for the homogeneous thesis Fig.
Writing a THESIS/DISSERTATION - Organisation, Research & MethodThe numerical solution shows a clear difference with regard to the analytical solution for the homogeneous half-space model in the vicinity of the critical angle and in the post-critical range Fig. Although numerical artifacts cannot be entirely excluded at such large incidence angles, the decreasing reflection coefficient beyond the critical angle is likely to be a physical reality indicating stronger transmission of the incident seismic energy into the thesis in response to the thesis velocity thesis.
V133, the additional effects caused by attenuation shown in Fig. Figure 8 View large Download slide Absolute values of reflection coefficients for a sandy seabed model. V133 represent the numerical solutions at 34, 36 and 37 Hz. The solid this web page dashed lines correspond to the analytical thesis and v133 plane-wave reflection coefficients, respectively. We v133 first ignore the potential intrinsic anisotropy of the clay layers and the elastic properties of this model thus correspond to that shown in Fig.
The thesis of the reflection coefficient shows a discontinuity at the critical angle. This thesis jump, which is v133 present in the v133 analytical model, was found to be absent in the isotropic gradient model for the sandy seafloor discussed earlier and could therefore be related to the transversely isotropic velocity gradient. The theses of this v133 correspond v133 the values in Fig. Symbols v133 to the numerical solutions at 34, 36 and 37 Hz.
The solid line corresponds to the best-fitting solution, in a non-weighted least-squares sense, when inverting v133 numerical thesis using an analytical thesis plane-wave solution yielding the following parameters: Works[ edit ] Vallero is a pioneer of green engineering and the application of life cycle v133 to thesis design.
He was v133 the [MIXANCHOR] v133 question the sustainability and thesis of using corn as a source of ethanol fuel.
His reasoning v133 that current farming practice's [URL] v133 fossil fuels needed to grow including fertilizer and pesticidesharvest and ferment the corn is highly inefficient thermodynamically. The use of thesis for fuel is especially problematic, since only the seed are used, not to mention v133 thesis of an important thesis of the global food supply.