Gallium Nitride quantum dot layer formation

Thesis (S.B.)--Massachusetts Institute of Technology, Dept. of Materials Science and Engineering, 2006.Includes bibliographical references (p. 19).by Louise R. Giam.S.B

Correction: Extended Synaptotagmin (ESyt) Triple Knock-Out Mice Are Viable and Fertile without Obvious Endoplasmic Reticulum Dysfunction.

[This corrects the article DOI: 10.1371/journal.pone.0158295.]

Extended Synaptotagmin (ESyt) Triple Knock-Out Mice Are Viable and Fertile without Obvious Endoplasmic Reticulum Dysfunction.

Extended synaptotagmins (ESyts) are endoplasmic reticulum (ER) proteins composed of an N-terminal transmembrane region, a central SMP-domain, and five (ESyt1) or three C-terminal cytoplasmic C2-domains (ESyt2 and ESyt3). ESyts bind phospholipids in a Ca2+-dependent manner via their C2-domains, are localized to ER-plasma membrane contact sites, and may catalyze lipid exchange between the plasma membrane and the ER via their SMP-domains. However, the overall function of ESyts has remained enigmatic. Here, we generated triple constitutive and conditional knock-out mice that lack all three ESyt isoforms; in addition, we produced knock-in mice that express mutant ESyt1 or ESyt2 carrying inactivating substitutions in the Ca2+-binding sites of their C2A-domains. Strikingly, all ESyt mutant mice, even those lacking all ESyts, were apparently normal and survived and bred in a manner indistinguishable from control mice. ESyt mutant mice displayed no major changes in brain morphology or synaptic protein composition, and exhibited no large alterations in stress responses. Thus, in mice ESyts do not perform an essential role in basic cellular functions, suggesting that these highly conserved proteins may perform a specialized role that may manifest only during specific, as yet untested challenges

Polymer pen lithography

We report a low-cost, high-throughput scanning probe lithography method that uses a soft elastomeric tip array, rather than tips mounted on individual cantilevers, to deliver inks to a surface in a “direct write” manner. Polymer pen lithography merges the feature size control of dip-pen nanolithography with the large-area capability of contact printing. Because ink delivery is time and force dependent, features on the nanometer, micrometer, and macroscopic length scales can be formed with the same tip array. Arrays with as many as about 11 million pyramid-shaped pens can be brought into contact with substrates and readily leveled optically to ensure uniform pattern development.Accepted versio

Loss of ESyts does not affect the level of synaptic and ER markers in the brain.

<p>(A and B) Immunoblots and relative quantifications of major pre-synaptic proteins in WT and constitutive ESyt123 triple KO mice. Data are shown as means ± SEM, Student’s t-test, p>0.05, n = 4. (C and D) Western blot and relative quantification of ER proteins, showing no differences between WT and 123EC KO mice. Data are shown as means ± SEM, Student’s t-test, p>0.05, n = 4.</p

Constitutive ESyt123 triple KO mice are viable, fertile, develop apparently normally, and do not show major phenotypic changes.

<p>(A) Graph showing the distribution of the offspring obtained by crossing heterozygous 123EC KO mice. Black bars represent the expected ratio considering a Mendelian segregation of the alleles. Blue bars show the observed distribution. Chi-square test, p = 0.9432, n = 158. (B) Survival plot showing that homozygous 123EC KO mice are viable and have a normal lifespan (n = 8). (C) Both 123EC KO male and female mice do not show alterations in body weight if compared to WT mice. Data are shown as mean± SEM, One way ANOVA for repetitive measurements, p>0.05, n = 13 WT, n = 12 123EC. (D) Dapi staining of brain sections (top) confirming that 123EC KO mice do not show major alterations in the brain architecture. Magnification of the hippocampus (left) and cerebellum (right) confirming normal organizations of these structures in ESyt123 KO mice. Abbreviation: Cx, cortex; Hp, hippocampus; Cb, cerebellum; Bs, brainstem; St, striatum; Th, thalamus; DG, dentate gyrus; GCL, granule cell layer; WM, white matter.</p

Nanoreactors for Studying Single Nanoparticle Coarsening

The ability to observe intermediate structures as part of coarsening processes that lead to the formation of single nanoparticles (NPs) is important in gaining fundamental insight pertaining to nanostructure growth. Here, we use scanning probe block copolymer lithography (SPBCL) to create “nanoreactors” having attoliter volumes, which confine Au NP nucleation and growth to features having diameters <150 nm on a substrate. With this technique, one can use in situ TEM to directly observe and study NP coarsening and differentiate Ostwald ripening from coalescence processes. Importantly, the number of metal atoms that can engage in coarsening can be controlled with this technique, and TEM “snapshots” of particle growth can be taken. The size of the resulting nanostructures can be controlled in the 2–10 nm regime

Triple ESyt123 KO does not increase the susceptibility of neurons to stress, induce obvious changes in the ER, or affect calcium dynamics in neurons.

<p>(A) Hippocampal cultures obtained from ESyt123 conditional KO mice and infected with lentiviruses expressing GFP-Cre and GFP-ΔCre. Images show GFP fluorescence merged with brightfield signal to illustrate the high efficiency of infection of the virus for both Cre and ΔCre conditions. (B) Neuronal cell death in response to mild stress (DTT, Tunicamycin, Thapsigargin and Paraquat) was assessed by the MTT assay. The stress conditions induced partial neuronal cell death, which was unchanged in neurons infected with Cre or ΔCre, suggesting that loss of ESyts does not increase susceptibility to toxic agents. Data were normalized to the control ΔCre condition, and are expressed as means ± SEM. Two-way ANOVA, Bonferroni post-hoc test, *p<0.05 vs CTR, **p<0.01 vs CTR, ***p<0.001 vs control, n = 8. (C) Confocal images showing hippocampal neurons transfected with Sec61β-EGFP in green and mCherry signal in red. Insert shows a magnification of the soma to better visualize the ER associated with the nucleus. (D) Quantification of the total neuronal area (mCherry signal) and the fraction of the nuclear-associated ER (Sec61β area normalized to mCherry area) showing no difference between ΔCre and Cre treated neurons. Data are expressed as means ± SEM. Student t-test, p>0.05, n = 18 ΔCre, n = 15 Cre. (E) Representative images of Sec61β-EGFP signal (green) acquired at higher exposure, showing that cytoplasmic ER is present in dendrites as well as in the neck of a subset of spines (see arrowheads) in both ΔCre and Cre treated neurons. The mCherry signal (red) allows visualization of dendrites and dendritic spines. (F) Representative images of GCaMP6M-expressing hippocampal neurons at resting (left panel-absence of Ca2+-signal) or during network activity (right panel-presence of a Ca2+-signal) for ΔCre (top panel) and Cre (low panel) treated neurons. Calcium activity is present in both ΔCre and Cre treated neurons. (G) Quantification of the number of calcium peaks, the integrated peak area, as well as the rise time and decay time kinetics, during 5min recording of neuronal activity. No significant differences has been observed between ΔCre and Cre treated neurons. Data are expressed as means ± SEM. Student t-test, p>0.05, n = 5 pups (total of analyzed neurons = 184) ΔCre, n = 5 pups (total of analyzed neurons = 129) Cre.</p

Nanoreactors for Studying Single Nanoparticle Coarsening

The ability to observe intermediate structures as part of coarsening processes that lead to the formation of single nanoparticles (NPs) is important in gaining fundamental insight pertaining to nanostructure growth. Here, we use scanning probe block copolymer lithography (SPBCL) to create “nanoreactors” having attoliter volumes, which confine Au NP nucleation and growth to features having diameters <150 nm on a substrate. With this technique, one can use in situ TEM to directly observe and study NP coarsening and differentiate Ostwald ripening from coalescence processes. Importantly, the number of metal atoms that can engage in coarsening can be controlled with this technique, and TEM “snapshots” of particle growth can be taken. The size of the resulting nanostructures can be controlled in the 2–10 nm regime