Rescue of Synthetic Genomic RNA Analogs of Rabies Virus by Plasmid-Encoded Proteins

Proteins eolirely expressed from cDNA wen used to rescue synthetic RNA genome analogs into infectious defective particles or rabies virus (RV). Synthetic negative-stranded RNAs coßtalning 3' · and S'-terminal RV sequences and tnlßscriptional signal sequences wen transcribed (rom plasmids transfeded into cells expressing 1'7 RNA polymerase (rom recombinant vaccinia virus. After simultaneous expression or RV N, P, and L proteiDS (rom plasmids containing a T7 RNA polymerase promoter, tbe synthetic genomes wen encapsidated. replicated, and transcribed by tbe RV polymerase proteiDS. Insertion or the bac1erial chloramphenicol acetyUransferase gene or l3·galactosidase (IacZ) gene between the 3 ' and 5 ' termini containing transcriptional signal sequenees resulted in transcription of mRNAs and expression of ehloramphenlco

VITAMIN D WORKS THROUGH THE LIPID DROPLET PROTEIN PLIN2 TO AUGMENT MITOCHONDRIAL FUNCTION IN SKELETAL MUSCLE

Vitamin D has been connected with increased intramyocellular lipid (IMCL) mitochondrial function in skeletal muscle. It is also shown to prevent lipotoxicity in several tissues, but this has not yet been examined in skeletal muscle. Perilipin 2 (PLIN2), a lipid droplet protein upregulated with vitamin D treatment, is integral to managing IMCL capacity and lipid oxidation in skeletal muscle. Increased lipid storage and oxidation is associated with increased tolerance to a hyperlipidic environment and resistance to lipotoxicity. Therefore, I hypothesized that vitamin D increases β-oxidation and lipid turnover though a PLIN2 mediated mechanism, thereby preventing lipotoxicity. This hypothesis was divided into two specific aims: 1) Characterize the effect of vitamin D and PLIN2 on lipid turnover and β-oxidation in mature myotubes, and 2) Determine the role of vitamin D and PLIN2 in regulating key markers of lipotoxicity. To address these aims, cells were treated with or without vitamin D, palmitate, and PLIN2 siRNA in an eight group, 2x2x2 design. Key experiments included quantitative real time polymerase chain reaction for markers of lipid accumulation, lipolysis, and lipotoxicity; Seahorse oxygen consumption assay; 14C-palmitate oxidation assay; and analyses of lipid accumulation and profile. Failure of the palmitate treatment to produce a reliable model for lipotoxicity resulted in negative data for Aim 2 of this dissertation and a focus on vitamin D and PLIN2 knockdown treatments as a four group, 2x2 model. Aim 1 showed that vitamin D reliably increases markers of lipolysis and lipid accumulation. Most of these markers were in turn decreased after PLIN2 knockdown, and DGAT2 exhibited an interaction effect between the two treatments. Contrary to our hypothesis and some published research, PLIN2 knockdown did not prevent lipid accumulation. Vitamin D increased oxygen consumption, especially consumption driven by mitochondrial complex II. PLIN2 knockdown decreased oxygen consumption and demonstrated an interaction effect specific to mitochondrial complex II. Data in this dissertation show that vitamin D increases mitochondrial function, and these effects are at least in part accomplished through a PLIN2 mediated mechanism. However, this work lacks the data required to make specific claims regarding β-oxidation and lipid turnover. This research is some of the first to show that PLIN2 knockdown carries negative impacts for skeletal muscle mitochondria and makes valuable contributions to general knowledge of how vitamin D and lipid storage impact muscle health and function. This ultimately provides additional evidence to advocate for vitamin D supplementation as a means of improving musculoskeletal health and function. Future research should investigate how vitamin D and PLIN2 impact markers of lipotoxicity in skeletal muscle

The Relationship between Parental Involvement and Reading Achievement

This five week investigation determined a relationship exists between parental involvement and reading achievement. Students were separated into two groups, Group A and Group B. Group A contained students who received below the average amount of parental involvement points obtained and Group B contained students who received above the average amount of parental involvement points obtained. Students in Group B made a 1.14 text level gain above students in Group A. Group B also made a 6.34 sight word gain over Group A as well as a 5.57 oral reading fluency gain over students in Group A. The study included first and second grade Reading Recovery™ and Title I reading students. Students’ families differed in marital status, socioeconomic status, and ethnicities. Baseline data and gains in text level, oral reading fluency, and sight word knowledge were measured using: Fountas and Pinnell’s Benchmark Asssessment™, aimsweb™Plus Oral Reading Fluency, and Slossan™ Oral Reading Test. Parental involvement was measured using daily book log signatures, completion of cut-up sentences, parent communication with reading teacher via phone and/or email, SeeSaw™ views, attendance of Title I family night, attendance of student/teacher conferences, and observation of a reading lesson

Reliability and Stability of the Standard Fear of Crime Indicator in a National Panel Over 14 Years

Reducing citizens’ fear of crime is a salient policy topic in Western societies. For the measurement of fear of crime, variations of a single item measure (Is there any area near where you live that is, within a mile where you would be afraid to walk alone at night?) have been used in hundreds of surveys for half a century. Despite a long and ongoing discussion on the doubtful usefulness of this standard indicator, no estimate for the reliability of this item is available. Using panel data from the British Household Panel Survey in combination with the UK Household Longitudinal Survey, reliability and stability of the standard fear of crime indicator are estimated using quasi-Markov simplex models for the rst time. The model shows estimated reliabilities of about .67 for the standard indicator. This result is compared with results for other single item measures using the same data set. Finally, comparisons with reliability recommendations and recent reviews of survey measures are reported

Unconventional superconducting pairing symmetry induced by phonons

The possibility of non-s-wave superconductivity induced by phonons is investigated using a simple model that is inspired by Sr$_2$RuO$_4$. The model assumes a two-dimensional electronic structure, a two-dimensional spin-fluctuation spectrum, and three-dimensional electron-phonon coupling. Taken separately, each interaction favors formation of spin-singlet pairs (of s symmetry for the phonon interaction and d$_{x^2-y^2}$ symmetry for the spin interaction), but in combination, a variety of more unusual singlet and triplet states are found, depending on the interaction parameters. This may have important implications for Sr$_2$RuO$_4$, providing a plausible explanation of how the observed spin fluctuations, which clearly favor d$_{x^2-y^2}$ pairing, may still be instrumental in creating a superconducting state with a different (e.g., p-wave) symmetry. It also suggests an interpretation of the large isotope effect observed in Sr$_2$RuO$_4$. These results indicate that phonons could play a key role in establishing the order-parameter symmetry in Sr$_2$RuO$_4$, and possibly in other unconventional superconductors.Comment: 6 pages, 5 figures, submitted to Phys. Rev.

Mechanistic analysis of ammonium inhibition of atmospheric methane consumption in forest soils

Methane consumption by forest soil was studied in situ and in vitro with respect to responses to nitrogen additions at atmospheric and elevated methane concentrations. Methane concentrations in intact soil decreased continuously from atmospheric levels at the surface to 0.5 ppm at a depth of 14 cm. The consumption rate of atmospheric methane in soils, however, was highest in the 4- to 8-cm depth interval (2.9 nmol per g of dry soil per day), with much lower activities below and above this zone. In contrast, extractable ammonium and nitrate concentrations were highest in the surface layer (0 to 2 cm; 22 and 1.6 μmol per g of dry soil, respectively), as was potential ammonium-oxidizing activity (19 nmol per g of dry soil per day). The difference in zonation between ammonium oxidation and methane consumption suggested that ammonia-oxidizing bacteria did not contribute significantly to atmospheric methane consumption. Exogenous ammonium inhibited methane consumption in situ and in vitro, but the pattern of inhibition did not conform to expectations based on simple competition between ammonia and methane for methane monooxygenase. The extent of ammonium inhibition increased with increasing methane concentration. Inhibition by a single ammonium addition remained constant over a period of 39 days. In addition, nitrite, the end product of methanotrophic ammonia oxidation, was a more effective inhibitor of methane consumption than ammonium. Factors that stimulated ammonium oxidation in soil, e.g., elevated methane concentrations and the availability of cosubstrates such as formate, methanol, or β- hydroxybutyrate, enhanced ammonium inhibition of methane oxidation, probably as a result of enhanced nitrite production

Ammonium and nitrite inhibition of methane oxidation by Methylobacter albus BG8 and Methylosinus trichosporium OB3b at low methane concentrations

Methane oxidation by pure cultures of the methanotrophs Methylobacter albus BG8 and Methylosinus trichosporium OB3b was inhibited by ammonium choride and sodium nitrite relative to that in cultures assayed in either nitrate-containing or nitrate-free medium. M. albus was generally more sensitive to ammonium and nitrite than M. trichosporium. Both species produced nitrite from ammonium; the concentrations of nitrite produced increased with increasing methane concentrations in the culture headspaces. Inhibition of methane oxidation by nitrite was inversely proportional to headspace methane concentrations, with only minimal effects observed at concentrations of \u3e500 ppm in the presence of 250 μM nitrite. Inhibition increased with increasing ammonium at methane concentrations of 100 ppm. In the presence of 500 μM ammonium, inhibition increased initially with increasing methane concentrations from 1.7 to 100 ppm; the extent of inhibition decreased with methane concentrations of \u3e100 ppm. The results of this study provide new insights that explain some of the previously observed interactions among ammonium, nitrite, methane, and methane oxidation in soils and aquatic systems