Literacy, Autism, and Inclusion

Young children with significant developmental disabilities can be taught sophisticated literacy skills and many interventions have been shown effective in the research. However, there is a lack of research that looks at the effect of inclusion on literacy with respect to a single student. This study will investigate the influence of an inclusive, general education classroom on the literacy learning of a student with autism. A participant observer approach will be employed in order to collect the following data: in depth interviews and observations of a single individual over a fifteen hour period, and a qualitative case study design will be used to analyze the data collected

Total internal reflection fluorescence microscopy for characterizing biochemical interactions

Total internal reflection fluorescence (TIRF) microscopy is a widely used technique for the study of biologically relevant processes both in vitro and in vivo. In TIRF, an incident light beam propagates through a transparent solid and encounters a solid/liquid interface at an angle sufficient enough such that the light undergoes total internal reflection, which generates an evanescent field that decays exponentially with distance from the surface. Thus, the main advantage of TIRF is that it selectively excites fluorescent molecules at or near the solid-solution interface. TIRF is used to study the thermodynamic and kinetic properties of three systems in this dissertation. Pregnane X receptor (PXR) is a member of the nuclear receptor family of ligand activated transcriptional factors that regulate gene expression. Using TIRF, the ligand-dependent binding of PXR to the steroid receptor coactivator-1 (SRC-1) is studied in the presence of the known PXR activator, rifampicin. In this system, the ligand binding domain of PXR is immobilized at the solid-solution interface, and fluorescently-labeled SRC-1 peptide in solution is used to mimic the full length SRC receptor. By measuring the fluorescence intensity as a function of the solution concentration of fluorescently-labeled SRC-1 at constant, increasing concentrations of rifampicin, four equilibrium binding constants are obtained, which illustrate the cyclic nature of these interactions. Discoidin domain receptor 2 (DDR2) is a receptor tyrosine kinase that is activated by fibrillar collagens in the extracellular matrix. Upon activation through collagen binding, DDR2 regulates cell adhesion, proliferation, and extracellular matrix remodeling. To study the binding of the extracellular domain of DDR2 to collagen, methods to deposit thin films of aligned or unaligned collagen I on fused silica slides were developed. Additionally, to characterize the effect of oligomerization of DDR2 on collagen binding, methods to separate monomeric, dimeric, and aggregates of DDR2 were also explored. This work represents the preliminary steps necessary to study the binding of surface immobilized collagen to fluorescently-labeled DDR2 using TIRF. A new method using through-prism total internal reflection fluorescence microscopy with continuous photobleaching (TIR-CP) is presented. In this method, small structures, such as prokaryotic cells or isolated eukaryotic organelles, containing fluorescent molecules are adhered to a surface. This surface is continuously illuminated by an evanescent wave created by total internal reflection. The characteristic length describing the decay of the evanescent intensity with distance from the surface is smaller than the structures. The fluorescence decay rate resulting from continuous evanescent illumination is monitored as a function of the excitation intensity. The data at higher excitation intensities provide apparent translational diffusion coefficients for the fluorescent molecules within the structures because the decay results from two competing processes (the intrinsic photobleaching propensity and diffusion in the small structures). The theoretical basis for the technique is presented and the applicability of the technique is demonstrated by measuring the diffusion coefficient, 6.3 ± 1.1 µm2/sec, of green fluorescent protein (GFP) in Escherichia coli cells

Activity of Genes with Functions in Human Williams-Beuren Syndrome Is Impacted by Mobile Element Insertions in the Gray Wolf Genome.

In canines, transposon dynamics have been associated with a hyper-social behavioral syndrome, although the functional mechanism has yet to be described. We investigate the epigenetic and transcriptional consequences of these behavior-associated mobile element insertions (MEIs) in dogs and Yellowstone gray wolves. We posit that the transposons themselves may not be the causative feature; rather, their transcriptional regulation may exert the functional impact. We survey four outlier transposons associated with hyper-sociability, with the expectation that they are targeted for epigenetic silencing. We predict hyper-methylation of MEIs, suggestive that the epigenetic silencing of and not the MEIs themselves may be driving dysregulation of nearby genes. We found that transposon-derived sequences are significantly hyper-methylated, regardless of their copy number or species. Further, we have assessed transcriptome sequence data and found evidence that MEIs impact the expression levels of six genes (WBSCR17, LIMK1, GTF2I, WBSCR27, BAZ1B, and BCL7B), all of which have known roles in human Williams-Beuren syndrome due to changes in copy number, typically hemizygosity. Although further evidence is needed, our results suggest that a few insertions alter local expression at multiple genes, likely through a cis-regulatory mechanism that excludes proximal methylation

Primer and database choice affect fungal functional but not biological diversity findings in a national soil survey

The internal transcribed spacer (ITS) region is the accepted DNA barcode of fungi. Its use has led to a step-change in the assessment and characterisation of fungal communities from environmental samples by precluding the need to isolate, culture, and identify individuals. However, certain functionally important groups, such as the arbuscular mycorrhizas (Glomeromycetes), are better characterised by alternative markers such as the 18S rRNA region. Previous use of an ITS primer set in a nationwide metabarcoding soil biodiversity survey revealed that fungal richness declined along a gradient of productivity and management intensity. Here, we wanted to discern whether this trend was also present in data generated from universal 18S primers. Furthermore, we wanted to extend this comparison to include measures of functional diversity and establish trends with soil types and soil organic matter (SOM) content. Over the 413 individual sites examined (arable, grassland, woodland, moorland, heathland), we found congruent trends of total fungal richness and β-diversity across land uses, SOM class, and soil type with both ITS and 18S primer sets. A total of 24 fungal classes were shared between datasets, in addition to 15 unique to ITS1 and 12 unique to 18S. However, using FUNGUILD, divergent trends of functional group richness became apparent, especially for symbiotrophic fungi, likely driven by an increased detection rate of Glomeromycetes in the 18S dataset. The disparate trends were also apparent when richness and β-diversity were compared to soil properties. Additionally, we found SOM class to be a more meaningful variable than soil type biodiversity for predicting biodiversity analyses because organic matter was calculated for each sample whereas soil type was assigned from a national soil map. We advocate that a combination of fungal primers should be used in large-scale soil biodiversity surveys to capture important groups that can be underrepresented by universal barcodes. Utilising such an approach can prevent the oversight of ubiquitous but poorly described species as well as critically important functional groups

Polyphenism in social insects: insights from a transcriptome-wide analysis of gene expression in the life stages of the key pollinator, Bombus terrestris

Background: Understanding polyphenism, the ability of a single genome to express multiple morphologically and behaviourally distinct phenotypes, is an important goal for evolutionary and developmental biology. Polyphenism has been key to the evolution of the Hymenoptera, and particularly the social Hymenoptera where the genome of a single species regulates distinct larval stages, sexual dimorphism and physical castes within the female sex. Transcriptomic analyses of social Hymenoptera will therefore provide unique insights into how changes in gene expression underlie such complexity. Here we describe gene expression in individual specimens of the pre-adult stages, sexes and castes of the key pollinator, the buff-tailed bumblebee Bombus terrestris. Results: cDNA was prepared from mRNA from five life cycle stages (one larva, one pupa, one male, one gyne and two workers) and a total of 1,610,742 expressed sequence tags (ESTs) were generated using Roche 454 technology, substantially increasing the sequence data available for this important species. Overlapping ESTs were assembled into 36,354 B. terrestris putative transcripts, and functionally annotated. A preliminary assessment of differences in gene expression across non-replicated specimens from the pre-adult stages, castes and sexes was performed using R-STAT analysis. Individual samples from the life cycle stages of the bumblebee differed in the expression of a wide array of genes, including genes involved in amino acid storage, metabolism, immunity and olfaction. Conclusions: Detailed analyses of immune and olfaction gene expression across phenotypes demonstrated how transcriptomic analyses can inform our understanding of processes central to the biology of B. terrestris and the social Hymenoptera in general. For example, examination of immunity-related genes identified high conservation of important immunity pathway components across individual specimens from the life cycle stages while olfactory-related genes exhibited differential expression with a wider repertoire of gene expression within adults, especially sexuals, in comparison to immature stages. As there is an absence of replication across the samples, the results of this study are preliminary but provide a number of candidate genes which may be related to distinct phenotypic stage expression. This comprehensive transcriptome catalogue will provide an important gene discovery resource for directed programmes in ecology, evolution and conservation of a key pollinator

Rifampicin-Independent Interactions between the Pregnane X Receptor Ligand Binding Domain and Peptide Fragments of Coactivator and Corepressor Proteins

The pregnane X receptor (PXR), a member of the nuclear receptor superfamily, regulates the expression of drug-metabolizing enzymes in a ligand-dependent manner. The conventional view of nuclear receptor action is that ligand binding enhances the receptor's affinity for coactivator proteins, while decreasing its affinity for corepressors. To date, however, no known rigorous biophysical studies have been conducted to investigate the interaction among PXR, its coregulators, and ligands. In this work, steady-state total internal reflection fluorescence microscopy (TIRFM) and total internal reflection with fluorescence recovery after photobleaching were used to measure the thermodynamics and kinetics of the interaction between the PXR ligand binding domain and a peptide fragment of the steroid receptor coactivator-1 (SRC-1) in the presence and absence of the established PXR agonist, rifampicin. Equilibrium dissociation and dissociation rate constants of ~5 μM and ~2 s(-1), respectively, were obtained in the presence and absence of rifampicin, indicating that the ligand does not enhance the affinity of the PXR and SRC-1 fragments. Additionally, TIRFM was used to examine the interaction between PXR and a peptide fragment of the corepressor protein, the silencing mediator for retinoid and thyroid receptors (SMRT). An equilibrium dissociation constant of ~70 μM was obtained for SMRT in the presence and absence of rifampicin. These results strongly suggest that the mechanism of ligand-dependent activation in PXR differs significantly from that seen in many other nuclear receptors

Quantifying GFP Diffusion in Escherichia coli by Using Continuous Photobleaching with Evanescent Illumination

Fluorescence recovery after photobleaching and fluorescence correlation spectroscopy are the primary means for studying translational diffusion in biological systems. Both techniques, however, present numerous obstacles for measuring translational mobility in structures only slightly larger than optical resolution. We report a new method using through-prism total internal reflection fluorescence microscopy with continuous photobleaching (TIR-CP) to overcome these obstacles. Small structures, such as prokaryotic cells or isolated eukaryotic organelles, containing fluorescent molecules are adhered to a surface. This surface is continuously illuminated by an evanescent wave created by total internal reflection. The characteristic length describing the decay of the evanescent intensity with distance from the surface is smaller than the structures. The fluorescence decay rate resulting from continuous evanescent illumination is monitored as a function of the excitation intensity. The data at higher excitation intensities provide apparent translational diffusion coefficients for the fluorescent molecules within the structures because the decay results from two competing processes (the intrinsic photobleaching propensity and diffusion in the small structures). We present the theoretical basis for the technique and demonstrate its applicability by measuring the diffusion coefficient, 6.3 ± 1.1 µm2/sec, of green fluorescent protein (GFP) in Escherichia coli cells

Global evaluation of taxonomic relationships and admixture within the Culex pipiens complex of mosquitoes

Within the Culex pipiens mosquito complex, there are six contemporarily recognized taxa: Cx. quinquefasciatus, Cx. pipiens f. pipiens, Cx. pipiens f. molestus, Cx. pipiens pallens, Cx. australicus and Cx. globocoxitus. Many phylogenetic aspects within this complex have eluded resolution, such as the relationship of the two Australian endemic taxa to the other four members, as well as the evolutionary origins and taxonomic status of Cx. pipiens pallens and Cx. pipiens f. molestus. Ultimately, insights into lineage relationships within the complex will facilitate a better understanding of differential disease transmission by these mosquitoes. To this end, we have combined publicly available data with our own sequencing efforts to examine these questions.https://doi.org/10.1186/s13071-020-3879-