The number of students identified as having autism increased by 500% in the past 10 years (United States Government Accountability Office, 2005). All students with disabilities are required to be placed in least restrictive environments and to be given access to the general curriculum in the major subjects of math, reading, writing, and science as mandated by federal legislation such as the Individuals with Disabilities Education Act (IDEA, 2004) and No Child Left Behind (NCLB, 2001). As a result of this legislation, an increasing number of students with autism are being educated in inclusive classrooms. Most studies on general education access and curriculum modifications and/or instructional accommodations center on students with intellectual disabilities (e.g. Soukup, Wehmeyer, Bashinski, & Boviard, 2007; Wehmeyer, Lattin, Lapp-Rincker, & Agran, 2003). Wehmeyer et al. (2003) and Soukup et al. (2007) found included students with intellectual disabilities had more access to the general curriculum than mostly self-contained students. This meant included students were more likely to be working on the general curriculum as mandated by NCLB than those in only self-contained classrooms. This study builds and expands the research of Wehmeyer et al., as well as Soukup et al., by examining how students with autism are given access to the general curriculum through curriculum modifications and instructional accommodations used by general education teachers in three schools. This investigation focused on nine inclusive classrooms for students with autism using a parallel mixed methods design (Newman, Newman, & Newman, 2011). Classroom observations using both an IEP related checklist and field notes, teacher interviews, an archival document review of the Individual Education Plan (IEP) for the selected students with autism were performed. Findings of this study were organized by interview questions and subsequent coding categories. Quantitative data were organized in a nominal scale. Participants asserted that their middle school students with autism functioned well in their classrooms, occasionally exhibiting behavioral differences. Most instructional accommodations on IEPs were being implemented by participants, and participants often provided additional instructional accommodations not mandated by the IEP. The majority of participants credited county workshops for their knowledge of instructional accommodations.

Lysine methylation is a post-translational modification that regulates a large array of functionally diverse processes that are vital for cellular function. The role of methylation is best characterized on histone proteins due to their high concentration in the cell, but alongside histone modifications, lower abundance non-histone methylation is emerging as a prevalent and functionally diverse regulator of cellular processes. The direct biological impact of non-histone lysine methylation is less well understood because they are difficult to detect. The dynamic concentration range of the proteome masks their signal during proteomic analysis which impedes the detection of these low abundance methylated proteins. Increasing the concentration of proteins bearing methylation is required for improved discovery. This requires enriching the post-translational modification with a capturing reagent prior to analysis. This thesis details an optimized method for using the supramolecular host p-sulfonatocalix[4]arene as a stationary phase methyllysine enrichment reagent for real-life cell-extracted proteins. Prior to the optimizations described in this thesis, cell-derived peptide extracts were not retained within an early generation upper-rim modified calixarene column. But with the new protocols detailed in this thesis, proteins extracted from both cultured prostate cancer cells and industrially sourced brewer’s yeast were successfully retained by a lower-rim modified calixarene column. Thousands of methylated proteins with diverse functions and cellular localization were discovered using this method. Detection of low abundance methylated proteins will aid our discovery of all cellular methylation marks, which in turn, will help delineate their biological functions. / Graduate / 2020-11-30

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The molecular target of epigenetic signaling is chromatin. Histones are extensively post-translationally modified (PTM), and many of these individual modifications have been studied in depth. As PTMs occur at multiple positions within histones, the degree to which these modifications might influence each other remains one of the major challenges of chromatin biology. Although major discoveries in understanding the complex repertoire of histone modifications were achieved using reductionist experimental systems with synthetic histone peptides, they do not explain the role of putative PTM cross-talks in a chromatin context. However, generating chromatin substrates of defined modification status has proved to be a technically challenging task. In this thesis, I first demonstrate our work on establishing a novel approach to produce libraries of modified nucleosomes. We employed protein trans-splicing and sortase-mediated ligation strategies to incorporate chemical modifications on histone tails of ‘ligation-ready’ nucleosomes. Subsequently, the ‘designer’ nucleosome libraries were used for testing the binding of heterochromatin protein 1 (HP1) and elucidated the previously uncharacterized crosstalk of H3K9me2 and S28ph marks. Further investigations explained the mechanism of this crosstalk and highlighted the importance of developing chemical biology tools for elucidating complex chromatin signaling. Second, I describe our reconstitution systems for the assembly of semisynthetic recombinant chromatin carrying methylation marks on DNA and distinct modifications on histones, e.g. H3K9me3. I aimed to understand the mechanisms of the interplay between chromatin and one of the DNA maintenance methylation factors, UHRF1. I showed that UHRF1 strongly interacts with nucleosomes containing linker DNA. However, it exerts only residual enzymatic activity in this context. Based on functional H3 ubiquitylation assays in vitro, I found that hemi-methylated nucleosomes stimulate enzymatic activity of UHRF1, suggesting that the protein’s chromatin targeting and activation are a two-step process. The positioning of hemi-methylated CpG on nucleosome regulates UHRF1 target selectivity. Further, mutational analysis revealed that the PHD domain of the factor is indispensable for H3 binding and that its SRA domain is required for catalytic activation. Overall, our work adds a new layer of positional complexity to the me½CpG-dependent regulation of UHRF1 and expands the current model of DNA methylation maintenance.

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