The Early Promise of TBRI Implementation in Schools

The program known as Trust Based Relational Intervention® (TBRI®) began as an exploration into the detrimental behaviors of foster and adopted children placed in homes with unsuspecting caregivers who assumed their living environment would result in positive results rather than fear based emotions and behaviors. The researchers at the Karyn Purvis Institute of Child Development (KPICD) at Texas Christian University held summer camps for adopted children and through that work developed an intervention to meet the needs of children who had experienced trauma. KPICD identifies these young people as “children from hard places” (Purvis & Cross, 2005). Copeland et al (2007) reported that an estimated 68% of children in the United States have experienced some sort of trauma. This astounding statistic holds great meaning for teachers and administrators, because these children from hard places routinely manifest aggressive and undesired behaviors due to an altering of their physiology. The literature on TBRI® at this point mostly has chronicled success with families, group homes and summer camps (McKenzie, Purvis, & Cross, 2014; Howard, Parris, Neilson, Lusk, Bush, Purvis & Cross, 2014; Purvis & Cross, 2006). TBRI® has only recently been implemented in school settings. This report provides an overview of the impacts of trauma, trauma related work in schools, and the four articles published to this point related to the use of TBRI® in schools

Development of Peptidase-Resistant Peptide Substrates for Measurement of Protein Kinase B and Bcr-Abl Kinase Activity

Synthetic peptides are widely used by the biomedical research community as kinase substrates for purified kinases, in cell lysates, and sometimes in intact single cells. Peptides are relatively straightforward to construct, they have a long shelf-life, and they are easily derivatized with labels to facilitate detection. However, despite the benefits of peptides, they suffer from susceptibility to degradation by intracellular peptidases. Peptidolysis is often extremely rapid, yielding peptide fragmentation within minutes of introduction into a cell lysate or single cell. While protease inhibitors can be used to slow degradation, they do not entirely eliminate protease activity and they are difficult to use with intact cells. In order to render peptides more suitable as substrates in in vivo settings, proteolytic degradation needs to be dramatically slowed or halted. The work described in this dissertation develops an iterative strategy to screen rationally designed peptides for their suitability as kinase substrates in cell lysates and single cells. Peptide bonds susceptible to peptidolysis were identified and stabilized by replacement of native residues with non-native residues, which are poor substrates for peptidases. Modified peptides were screened for substrate suitability as well as for resistance to degradation by peptidases. Substrates were designed for two kinases, protein kinase B and Bcr-Abl kinase, because of their roles in cancer. Protein kinase B is involved in regulation of multiple cellular functions, including stress response and programmed cell death, and is upregulated in many cancers, including pancreatic, breast, and prostate tumors. Bcr-Abl is the primary driver of chronic myelogenous leukemia. This dissertation outlines the development of peptidase-resistant substrate reporters for these two kinases. Initial characterization and design was performed in cytosolic lysates. Once suitably designed peptide substrates were synthesized, they were incubated in single cells to characterize substrate metabolism and phosphorylation in the intracellular environment. Finally, protein kinase B activity was measured in single primary cells from a human pancreatic cancer xenograft. The design strategy presented in this dissertation should be applicable for future design of peptidase-resistant peptide substrates for alternative kinases.Doctor of Philosoph

Co-fabrication of chitosan and epoxy photoresist to form microwell arrays with permeable hydrogel bottoms

Microfabrication technology offers the potential to create biological platforms with customizable patterns and surface chemistries, allowing precise control over the biochemical microenvironment to which a cell or group of cells is exposed. However, most microfabricated platforms grow cells on impermeable surfaces. This report describes the co-fabrication of a micropatterned epoxy photoresist film with a chitosan film to create a freestanding array of permeable, hydrogel-bottomed microwells. These films possess optical properties ideal for microscopy applications, and the chitosan layers are semi-permeable with a molecular exclusion of 9.9 ± 2.1 kDa. By seeding cells into the microwells, overlaying inert mineral oil, and supplying media via the bottom surface, this hybrid film permits cells to be physically isolated from one another but maintained in culture for at least 4 days. Arrays co-fabricated using these materials reduce both large-molecular-weight biochemical crosstalk between cells and mixing of different clonal populations, and will enable high-throughput studies of cellular heterogeneity with increased ability to customize dynamic interrogations compared to materials in currently available technologies

Development of a protease-resistant reporter to quantify BCR–ABL activity in intact cells

A peptidase-resistant ABL kinase substrate was developed by identifying protease-susceptible bonds on an ABL substrate peptide and replacing flanking amino acids with non-native amino acids

Prospectus, March 27, 1996

https://spark.parkland.edu/prospectus_1996/1009/thumbnail.jp

Metabolism of peptide reporters in cell lysates and single cells

The stability of an Abl kinase substrate peptide in a cytosolic lysate and in single cells was characterized. In the cytosolic lysate, the starting peptide was metabolized at an average initial rate of 1.7 ± 0.3 zmol pg−;1 s−;1 with a t1/2 of 1.3 min. Five different fragments formed over time; however, a dominant cleavage site was identified. Multiple rational design cycles were utilized to develop a lead peptide with a phenylalanine and alanine replaced by an (N-methyl)phenylalanine and isoleucine, respectively, to attain cytosolic peptidase resistance while maintaining Abl substrate efficacy. This lead peptide possessed a 15-fold greater lifetime in the cytosolic lysate while attaining a 7-fold improvement in kcat as an Abl kinase substrate compared to the starting peptide. However, when loaded into single cells, the starting peptide and lead peptide possessed nearly identical degradation rates and an altered pattern of fragmentation relative to that in cell lysates. Preferential accumulation of a fragment with cleavage at an Ala-Ala bond in single cells suggested that dissimilar peptidases act on the peptides in the lysate versus single cells. A design strategy for peptide stabilization, analogous to that demonstrated for the lysate, should be effective for stabilization in single cells

Development of a Peptidase-Resistant Substrate for Single-Cell Measurement of Protein Kinase B Activation

An iterative design strategy using three criteria was utilized to develop a peptidase-resistant substrate peptide for protein kinase B. Libraries of peptides possessing non-native amino acids were screened for time to 50% phosphorylation, degradation half-life within a lysate, and appearance of a dominant fragment. The lead peptide possessed a half-life of 92 ± 7 and 16 ± 2 min in HeLa and LNCaP cytosolic lysates, respectively, representing a 4.6- and 2.7-fold lifetime improvement over that of the starting peptide. The redesigned peptide possessed a 4.5-fold improvement in phosphorylation efficiency compared to the starting peptide. The same peptide fragments were formed when the lead peptide was incubated in a lysate or loaded into single cells although the fragments formed in significantly different ratios suggesting that distinct peptidases metabolized the peptide in the two preparations. The rate of peptide degradation and phosphorylation was on average 0.1 ± 0.2 zmol pg−1 s−1 and 0.04 ± 0.08 zmol pg−1 s−1, respectively, for single LNCaP cells loaded with 4 ± 8 μM of peptide. Peptidase-resistant kinase substrates should find wide-spread utility in both lysate-based and single-cell assays of kinase activity

Fluorous enzymatic synthesis of phosphatidylinositides

Fluorous enzymatic synthesis is used to synthesize multiple phosphatidylinositides, which are directly immobilized on a microarray to probe protein–lipid interactions