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Item Search Methods for Interventions to support gaining independent living skills for adults with serious mental illness: A Rapid Scoping Review(2025-08)To identify studies to include or consider for this rapid scoping review, the review team worked with a librarian (SB) to develop detailed search strategies for each database. The PRISMA-S extension was followed for search reporting. The librarian (SB) developed the search for PubMed and translated the search for every database searched. The PubMed search strategy was reviewed by the research team to check for accuracy and term relevancy. All final searches were peer-reviewed by another librarian (Travis Nace, MLIS) following the Peer Review of Electronic Search Strategies (PRESS checklist). The search included no major limits or date restrictions.Item ATF4 REGULATES AMINO ACID BIOSYNTHESIS AND TRNA CHARGING TO COORDINATE NUCLEOTIDE SYNTHESIS AND SELECTIVE PROTEIN TRANSLATION TO DRIVE THE G1/S TRANSITION(Temple University. Libraries, 2025-05)Metabolic programming is a common feature in many human cancers, including acute myeloid leukemia (AML). However, the upstream regulators of metabolic processes that are commandeered in AML remain largely unknown. We previously discovered that the Activating Transcription Factor 4 (ATF4) supports disease progression in experimental models of AML. We also observed that the expression of ATF4 is significantly elevated in numerous genetic subtypes of AML compared to healthy hematopoietic stem and progenitor cells. This suggests that ATF4 is broadly deregulated in AML and interventions targeting this pathway may be broadly applicable. To better understand the molecular role of ATF4 in AML cell biology, we engineered multiple human AML cells lines (NOMO-1, MV4-11, and OCIAML-3) to express control (shNT) or shRNAs targeting ATF4 under the regulation of a tetracycline-inducible promoter. Using these models, we found that ATF4 inhibition promotes AML cell cycle arrest, differentiation, and death suggesting that ATF4 supports the differentiation blockade. To identify downstream gene targets of ATF4 in AML, we carried out RNA-sequencing analysis in NOMO-1 cells at a time point where we observe maximal knockdown of ATF4 protein (24 hours post-doxycycline [DOX] treatment), but prior to the evolution of cellular changes (~60-72 hours post-DOX). An enrichment analysis of genes that were specifically downregulated by ATF4 inhibition uncovered that genes associated with amino acid metabolism and tRNA aminoacylation are regulated by ATF4 – these results were confirmed by qPCR in all of our human cell line models. Additionally, chromatin immunoprecipitation (ChIP) assays showed that ATF4 localized to the promoters of many of these genes, suggesting that they are direct transcriptional targets. Metabolomic assays were also conducted in NOMO-1 cells to further investigate how ATF4 regulates these pathways. A pathway enrichment analysis of total steady-state polar metabolites indicated that ATF4 inhibition reduces metabolites involved in the synthesis and catabolism of several amino acids, disrupts the de novo synthesis of nucleotides, and diminishes metabolites required for aminoacyl-tRNA biosynthesis. To further understand the impact ATF4 has on these amino acid metabolic pathways we conducted several stable-isotope tracing metabolomic assays. These analyses showed that ATF4 inhibition disrupts serine and cysteine metabolism, synthesis of TCA cycle derived amino acids, and nucleotide synthesis through reduced glucose and glutamine flux. Quantitative proteomics using stable isotope labeling by amino acids in cell culture (SILAC) was also utilized to understand the direct effects ATF4 regulation of tRNA aminoacylation has on changes in the proteome. This analysis showed immense proteomic and transcriptomic overlap of many proteins involved in amino acid metabolism and tRNA aminoacylation, however, only 56% of significantly decreased proteins could be explained by decreased RNA expression. Further analysis revealed that ATF4 inhibition affects the translation of selective proteins that are unaffected at the RNA level, fold enrichment and STRING analyses uncovered that these selective proteins are involved in the G1/S transition. From these data it is apparent that ATF4 acts as an upstream regulator to promote metabolic reprogramming in AML, specifically in amino acid metabolism and subsequent nucleotide synthesis, as well as linking these newly produced amino acids to protein synthesis through tRNA aminoacylation for selective protein translation to drive the G1/S transition. Our results have shown that ATF4 could be a potential broadly applicable therapeutic target in AML. Additionally, our findings will likely extend beyond AML as this pathway has been implicated in the pathogenesis of a variety of human cancers. Further investigation is needed to uncover the potential therapeutic effects of a pharmacological inhibitor that targets ATF4, as well as various components of the pathways ATF4 regulates, in combination with current standard of care therapies.Item LOCALIZING NEURONAL CIRCUITS OF COCAINE CONTEXTUAL MEMORIES(Temple University. Libraries, 2023-08)Drug craving triggered by cues that were once associated with drug intoxication is a main contributor to continued drug-seeking behaviors. Addictive drugs engage molecular pathways of associative learning in which reactivation of a memory is followed by its reconsolidation. Reactivated memories are vulnerable to interference. Here we examined the circuitry of cocaine contextual memory reconsolidation and explored neuroplasticity following memory reactivation. Mice underwent chemogenetic inhibition of either nucleus accumbens (NA) neurons or the glutamatergic projection neurons from the ventral hippocampus (vHPC) to NA using inhibitory designer receptors exclusively activated by designer drugs (iDREADD). Mice underwent cocaine conditioned place preference followed by reactivation of the cocaine contextual memory. Clozapine N-Oxide (CNO) was administered after memory reactivation to inhibit either NA neurons or the accumbens–projecting glutamatergic neurons from the vHPC during the reconsolidation period. When retested 3 days later, a significant reduction in the previously established preference for the cocaine context was found in both conditions, but not in control groups. FosTRAP2-Ai14 mice were used to identify neurons activated by cocaine memory recall and to evaluate plasticity in NA medium spiny neurons (MSN) upon recall of cocaine memories. Results indicate a significant increase in dendritic spine density in NA MSNs activated by cocaine memory recall. Of these active cells, 43% expressed D1 receptors compared to 34% expressing D2 receptors. These results implicate a circuit involving glutamatergic projections from the vHPC onto NA neurons which is necessary for the reconsolidation of cocaine memories. Attenuation of cocaine memory reconsolidation reduced drug-seeking behavior.Item Engineered dual antioxidant enzymes complexes targeting ICAM-1 on brain endothelium reduces brain injury associated neuroinflammation.(Temple University. Libraries, 2024-08)The neuroinflammatory cascade triggered by traumatic brain injury represents a clinically important point for therapeutic intervention. Neuroinflammation generates oxidative stress in the form of high-energy reactive oxygen and nitrogen species, which are key mediators of TBI pathology. The role of the blood-brain barrier is essential for proper neuronal function and is vulnerable to oxidative stress. Results herein explores the notion that attenuating oxidative stress at the vasculature after TBI may result in improved BBB integrity and neuroprotection. Utilizing amino-chemistry, a biological construct (designated as dual-conjugate for short) was generated by covalently binding two antioxidant enzymes (superoxide dismutase 1 and catalase) to antibodies specific for ICAM-1. Bioengineering of the conjugate preserved its targeting and enzymatic functions as evaluated by real-time bioenergetic measurements (via the Seahorse-XF platform) in brain endothelial cells exposed to increasing concentrations of hydrogen peroxide or a superoxide anion donor. Results showed that the dual-conjugate effectively mitigated the mitochondrial stress due to oxidative damage. Furthermore, dual-conjugate administration also improved BBB and endothelial protection under oxidative insult in an in-vitro model of TBI utilizing a software controlled stretching device that induces a 20% in mechanical strain on the endothelial cells. Additionally, the dual-conjugate was also effective in reducing indices of neuroinflammation in the controlled cortical impact CCI-TBI animal model. Thus, these studies provide proof-of-concept that targeted dual antioxidant biologicals may offer a means to regulate oxidative stress associated cellular damage during neurotrauma.Item Rescuing the Function of Mutant Von Hippel-Lindau Tumor Suppressor Protein using Stabilizing Small Molecules(Temple University. Libraries, 2025-05)The Von Hippel-Lindau tumor suppressor gene, VHL, encodes for the pVHL tumor suppressor protein. While pVHL plays a role in many cellular activities, it is most well-known for its role in the oxygen sensing pathway. In this context, pVHL forms part of an E3 ubiquitin ligase complex that polyubiquitinates Hypoxia Inducible Factor α (HIF-α) for proteasomal degradation under normoxic conditions. Unsurprisingly, germline and somatic mutations in the VHL gene are strongly linked to several forms of cancer. These include the hereditary cancer syndrome, Von Hippel-Lindau Disease (VHLD), and sporadic clear cell renal cell carcinoma (ccRCC). I hypothesize that many clinically relevant missense mutations in the VHL gene thermodynamically destabilize the pVHL protein, resulting in a loss of protein foldedness and function. The central aim of this work is to identify small molecules that bind to and stabilize these pVHL mutants in the properly folded (native) conformation, thus restoring function.To that end, I utilized in silico methods to generate an ensemble of low energy, pocket containing conformations of pVHL (in the setting of the pVHL / Elongin C / Elongin B protein complex – hereby referred to as the “VCB” complex) and ranked these pockets in terms of druggability as predicted by a previously developed machine learning model. Notably, these computational studies identified the solvent filled pocket (and immediately adjacent areas) around Pro192, Gly127, and Asp197 as the most druggable site on the protein. The highly druggable pockets corresponding to this site were subsequently used as the basis for a virtual screen of an ultra-large virtual compound library to identify suitably complementary ligands capable of binding to these pockets. In the end, a series of 18 make on demand small molecules (known as “CP1-18”) were purchased for experimental validation. The initial set of CP1-18 were screened for potential binding activity to purified VCB protein complex in pools of 4-5 compounds using Saturation Transfer Difference (STD) NMR techniques. One such hit, CP4, was selected for cellular testing by Dr. Mariam Fouad and demonstrated the ability to enhance thermal stability of wild type pVHL (via CETSA) and increase the cellular stability (via cycloheximide pulse chase assay) the missense mutant pVHL P86S. However, preliminary experiments failed to show robust ability to reduce HIF-2α protein levels in RCC-MF cells. To begin optimizing this CP4 for the ability to reduce HIF-2α, an initial round of SAR by catalog was conducted to identify a set of 32 CP4 derivatives from the Enamine REAL Space. Screening these compounds for the ability to decrease HIF-2α levels yielded CP4.29 that showed a marked improvement in terms of potency. Additionally, CP4.29 demonstrated the ability to reduce additional pVHL targets including ZHX2 and AURKA. Mechanistic studies involving co-treatments with a proteasome inhibitor, a NEDD8 activating enzyme inhibitor, and a prolyl hydroxylase inhibitor showed that CP4.29’s mechanism of action is consistent with direct pVHL reactivation. In hopes of further increasing potency, a series of rationally designed CP4.29 derivatives were synthesized but failed to demonstrate enhanced potency compared to CP4.29. Having established CP4 and CP4.29 as the most promising compounds, rigorous confirmation of a direct interaction between CP4 and the VCB protein complex was conducted using a variety of orthogonal NMR based approaches. Having demonstrated a direct interaction between CP4 and VCB, attempts were made to determine the binding pose of CP4 using X-Ray crystallography. Unfortunately, these experiments were not successful. To determine the binding site of CP4, a point mutation was generated at the intended binding pocket (D197K) on pVHL and the same NMR experiments were repeated. These results showed a significant difference in binding kinetics between the wild type and pocket mutant constructs corroborating the computational models. These results, taken together, lay the foundation for a) pVHL reactivating small molecules as a novel potential therapeutic strategy for the treatment of ccRCC and VHLD and b) computational based screening approaches that may be generalized for the discovery of small molecules capable of reactivating other destabilized mutant proteins.