How Did I Get Here? A Capstone Review

In this thesis, I highlight my personal and professional growth as a college student. I use structured reflection and meta-analysis to uncover insights about how I learn and develop new skills. Over the past five years, I have built a toolbox of skills, including teamwork, public speaking, navigating uncertainty, strategic thinking, and leadership. I trace the development of each of these skills back to my coursework as a business student, my role in PSU’s student-run advertising agency, and my professional experience. I show how these skills culminated in my business capstone project. My communication and leadership skills allowed me to serve my team by stepping into a role as a facilitator. However, the fear of overstepping my authority prevented me from proactively establishing a stronger team dynamic and pressing the client for greater clarity on deliverables. My public speaking skills helped my team communicate our strategic recommendations effectively and back them up with research. My experience navigating uncertainty and thinking strategically helped us uncover underlying business problems and make valuable suggestions. By systematically reflecting on my influences, I highlight pivotal moments of learning and transformation. I adopt a metacognitive approach revealing patterns in how I learn new skills. This process highlights how discomfort and uncertainty become catalysts for growth; I learn by oscillating between structured theory, safe experimentation, and high-stakes stress testing; I use mentoring as a tool to reinforce my own learning; and I am constantly looking for ways to bolster the skills I am weakest at. Ultimately, this thesis demonstrates my strengths and identifies areas where further growth is needed. This project has made me aware that I must learn to manage action bias and balance generalized development with specialization. Taking the opportunity to reflect on my academic journey has given me greater clarity on my professional ambitions. I have concluded that my skills and instincts align strongly with a future career in consulting.By systematically reflecting on my influences, I highlight pivotal moments of learning and transformation. I adopt a metacognitive approach emphasizing how discomfort and uncertainty became catalysts for growth. Meta-analysis reveals that I learn by oscillating between structured theory, safe experimentation, and high-stakes stress testing. I use mentoring as a tool to reinforce my own learning, and I am constantly looking for ways to bolster the skills I am weakest at. Ultimately, this paper demonstrates my strengths and identifies areas where further growth is needed. This project has made me aware that I must learn to manage action bias and balance generalized development with specialization. Taking the opportunity to reflect on my academic journey has given me greater clarity on my professional ambitions. I have concluded that my skills and instincts align strongly with a future career in consulting

MYC phase separation selectively modulates the transcriptome.

Dysregulation and enhanced expression of MYC transcription factors (TFs) including MYC and MYCN contribute to the majority of human cancers. For example, MYCN is amplified up to several hundredfold in high-risk neuroblastoma. The resulting overexpression of N-myc aberrantly activates genes that are not activated at low N-myc levels and drives cell proliferation. Whether increasing N-myc levels simply mediates binding to lower-affinity binding sites in the genome or fundamentally changes the activation process remains unclear. One such activation mechanism that could become important above threshold levels of N-myc is the formation of aberrant transcriptional condensates through phase separation. Phase separation has recently been linked to transcriptional regulation, but the extent to which it contributes to gene activation remains an open question. Here we characterized the phase behavior of N-myc and showed that it can form dynamic condensates that have transcriptional hallmarks. We tested the role of phase separation in N-myc-regulated transcription by using a chemogenetic tool that allowed us to compare non-phase-separated and phase-separated conditions at equivalent N-myc levels, both of which showed a strong impact on gene expression compared to no N-myc expression. Interestingly, we discovered that only a small percentage (<3%) of N-myc-regulated genes is further modulated by phase separation but that these events include the activation of key oncogenes and the repression of tumor suppressors. Indeed, phase separation increases cell proliferation, corroborating the biological effects of the transcriptional changes. However, our results also show that >97% of N-myc-regulated genes are not affected by N-myc phase separation, demonstrating that soluble complexes of TFs with the transcriptional machinery are sufficient to activate transcription

TRIM16 Acts as an E3 Ubiquitin Ligase and Can Heterodimerize with Other TRIM Family Members

The TRIM family of proteins is distinguished by its tripartite motif (TRIM). Typically, TRIM proteins contain a RING finger domain, one or two B-box domains, a coiled-coil domain and the more variable C-terminal domains. TRIM16 does not have a RING domain but does harbour two B-box domains. Here we showed that TRIM16 homodimerized through its coiled-coil domain and heterodimerized with other TRIM family members; TRIM24, Promyelocytic leukaemia (PML) protein and Midline-1 (MID1). Although, TRIM16 has no classic RING domain, three-dimensional modelling of TRIM16 suggested that its B-box domains adopts RING-like folds leading to the hypothesis that TRIM16 acts as an ubiquitin ligase. Consistent with this hypothesis, we demonstrated that TRIM16, devoid of a classical RING domain had auto-polyubiquitination activity and acted as an E3 ubiquitin ligase in vivo and in vitro assays. Thus via its unique structure, TRIM16 possesses both heterodimerization function with other TRIM proteins and also has E3 ubiquitin ligase activity

MYCN promotes neuroblastoma malignancy by establishing a regulatory circuit with transcription factor AP4

Amplification of the MYCN oncogene, a member of the MYC family of transcriptional regulators, is one of the most powerful prognostic markers identified for poor outcome in neuroblastoma, the most common extracranial solid cancer in childhood. While MYCN has been established as a key driver of malignancy in neuroblastoma, the underlying molecular mechanisms are poorly understood. Transcription factor activating enhancer binding protein-4 (TFAP4) has been reported to be a direct transcriptional target of MYC. We show for the first time that high expression of TFAP4 in primary neuroblastoma patients is associated with poor clinical outcome. siRNA-mediated suppression of TFAP4 in MYCN-expressing neuroblastoma cells led to inhibition of cell proliferation and migration. Chromatin immunoprecipitation assay demonstrated that TFAP4 expression is positively regulated by MYCN. Microarray analysis identified genes regulated by both MYCN and TFAP4 in neuroblastoma cells, including Phosphoribosyl-pyrophosphate synthetase-2 (PRPS2) and Syndecan-1 (SDC1), which are involved in cancer cell proliferation and metastasis. Overall this study suggests a regulatory circuit in which MYCN by elevating TFAP4 expression, cooperates with it to control a specific set of genes involved in tumor progression. These findings highlight the existence of a MYCN-TFAP4 axis in MYCN-driven neuroblastoma as well as identifying potential therapeutic targets for aggressive forms of this disease

ATP7A is a novel target of retinoic acid receptor β2 in neuroblastoma cells

Increased retinoic acid receptor β (RARβ2) gene expression is a hallmark of cancer cell responsiveness to retinoid anticancer effects. Moreover, low basal or induced RARβ2 expression is a common feature of many human cancers, suggesting that RARβ2 may act as a tumour suppressor gene in the absence of supplemented retinoid. We have previously shown that low RARβ2 expression is a feature of advanced neuroblastoma. Here, we demonstrate that the ABC domain of the RARβ2 protein alone was sufficient for the growth inhibitory effects of RARβ2 on neuroblastoma cells. ATP7A, the copper efflux pump, is a retinoid-responsive gene, was upregulated by ectopic overexpression of RARβ2. The ectopic overexpression of the RARβ2 ABC domain was sufficient to induce ATP7A expression, whereas, RARβ2 siRNA blocked the induction of ATP7A expression in retinoid-treated neuroblastoma cells. Forced downregulation of ATP7A reduced copper efflux and increased viability of retinoid-treated neuroblastoma cells. Copper supplementation enhanced cell growth and reduced retinoid-responsiveness, whereas copper chelation reduced the viability and proliferative capacity. Taken together, our data demonstrates ATP7A expression is regulated by retinoic acid receptor β and it has effects on intracellular copper levels, revealing a link between the anticancer action of retinoids and copper metabolism

TRIM16 acts as a tumour suppressor by inhibitory effects on cytoplasmic vimentin and nuclear E2F1 in neuroblastoma cells

The family of tripartite-motif (TRIM) proteins are involved in diverse cellular processes, but are often characterized by critical protein–protein interactions necessary for their function. TRIM16 is induced in different cancer types, when the cancer cell is forced to proceed down a differentiation pathway. We have identified TRIM16 as a DNA-binding protein with histone acetylase activity, which is required for the retinoic acid receptor β2 transcriptional response in retinoid-treated cancer cells. In this study, we show that overexpressed TRIM16 reduced neuroblastoma cell growth, enhanced retinoid-induced differentiation and reduced tumourigenicity in vivo. TRIM16 was only expressed in the differentiated ganglion cell component of primary human neuroblastoma tumour tissues. TRIM16 bound directly to cytoplasmic vimentin and nuclear E2F1 in neuroblastoma cells. TRIM16 reduced cell motility and this required downregulation of vimentin. Retinoid treatment and enforced overexpression caused TRIM16 to translocate to the nucleus, and bind to and downregulate nuclear E2F1, required for cell replication. This study, for the first time, demonstrates that TRIM16 acts as a tumour suppressor, affecting neuritic differentiation, cell migration and replication through interactions with cytoplasmic vimentin and nuclear E2F1 in neuroblastoma cells

Preliminary Investigation into the Frequency of Unequal Crossovers within the 21-Hydroxylase Genes, CYP21A and CYP21B

21-hydroxylase deficiency is the major cause of congenital adrenal hyperplasia (CAH), a disorder that results in impaired Cortisol biosynthesis. It is caused by mutations in the 21-hydroxylase gene that codes for the steroid 21-hydroxylase enzyme. The majority of these mutations have arisen as a result of recombination events between the active gene (CYP21B) and a highly homologous pseudogene (CYP21A). Gene deletions, caused by misalignment and unequal crossing over between the CYP21A and CYP21B genes, account for 20% of these mutations while the remainder are apparent gene conversions. Previous studies have indicated that the unequal crossovers occur predominately in meiotic cells with a frequency of approximately 1 in 10 5 – 10 6 genomes, and that gene conversions occur in both meiotic and mitotic cells at a frequency of 1 in 10 3 – 10 5. The product of an unequal crossover is a chimeric gene (CYP21A/CYP21B) in which the 5' region corresponds to CYP21A and the 3' region corresponds to CYP21B. This research uses gene-specific PCR to analyse the frequency of unequal crossing over within the CYP21 gene in genomic DNA enriched by restriction enzyme digestion of genomic DNA and agarose gel electrophoresis, followed by excision of the appropriate CYP21A fragment (3.2 kb). This approach minimises artefacts caused by in vitro recombination during the PCR. Estimation of the number of chimeric genes in an enriched sample was by serial dilution to a null point (the point at which there is no amplifiable copy). The PCR assay was sensitive enough to detect a single copy of chimeric gene. Detection of rare chimeric genes in a high background of CYP21A and CYP21B required high numbers of PCR cycles, however, this generated artefacts caused by in vitro recombination. The enrichment for CYP21A and CYP21B solved this problem by eliminating non-specific target genes, thus minimising non-specific amplification. In this study, no de novo recombinations were detected using 10 7 copies of matched sperm or leukocyte DNA, suggesting that data from previous studies were likely to be erroneous due to PCR artefacts