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Age-related macular degeneration (AMD) is a leading cause of irreversible blindness in the Western world. Although, the majority of stem cell research to date has focused on production of RPE and photoreceptor cells for the purpose of evaluating disease pathophysiology and cell replacement, there is strong evidence that the choroidal endothelial cells (CECs) that form the choriocapillaris vessels are the first to be affected in this disease. As such, to accurately evaluate disease pathophysiology and develop an effective treatment, production of patient-specific stem cell-derived CECs will be required. During the first stage of my Ph.D work, represented in Chapter 1 of this dissertation, I developed a co-culture system to differentiate mouse stem cells into CECs. I reprogrammed dermal fibroblasts from the Tie2-GFP mouse into two independent iPSC lines. TheTie2-GFP iPSCs were differentiated into CECs using a co-culture method with either the monkey RF/6A CEC line or primary mouse CECs. IPSC-derived CECs were characterized via rt-PCR and immunocytochemistry (ICC) for EC- and CEC-specific markers. The mouse iPSC-derived CECs described in Chapter 1 expressed the CEC-specific marker carbonic anhydrase IV (CA4), eNOS, FOXA2, PLVAP, CD31, CD34, ICAM-1, Tie2, TTR, VE-cadherin, and vWF. These Tie2-GFP iPSC-derived CECs paved the way for the rest of my Ph.D, in which I transitioned into using human iPSCs to generate patient-specific CECs. During the second phase of my graduate work, presented in Chapter 3, I developed a novel stepwise differentiation protocol suitable for generating human iPSC-derived CECs. I used previously published RNA-seq data of the monkey CEC line, RF/6A and two statistical screens to develop media comprised of various protein combinations. In both screens, I identified connective tissue growth factor (CTGF) as the key component required for driving CEC development. I also found that a second factor, called TWEAKR, promoted iPSC to CEC differentiation by inducing endogenous CTGF secretion. CTGF-driven iPSC-derived CECs formed capillary tube-like vascular networks, and expressed the EC-specific markers CD31, ICAM1, PLVAP, vWF, and the CEC-restricted marker CA4. These patient-specific iPSC-derived CECs made it possible for me to proceed into the next phase of my Ph.D work, in which I started working with AMD patient-specific iPSC-derived CECs to evaluate AMD pathophysiology. In the final stage of my Ph.D, represented in Chapter 4, I used the novel CEC differentiation method I developed to generate AMD iPSC-derived CECs and use these cells for AMD disease modeling. In line with previous studies that the membrane attack complex (MAC) forms in the AMD choriocapillaris, I showed that the AMD iPSC-derived CECs were much more susceptible to MAC formation and cell death when the cells were antagonized with complement components. I also demonstrated that, unlike the control CECs, the AMD CECs lost their capillary tube-like structures when the cells were cultured for over ten days, indicating that the AMD CECs may also exhibit other disease phenotypes other than susceptibility to MAC and cytolysis. Overall, the work I present in this dissertation will help push the AMD research field forward by providing a way to directly study AMD patient-specific iPSC-derived CECs and how they differ from healthy iPSC-derived CECs. In combination with RPE and photoreceptor cells, these patient-specific iPSC-CECs will make it possible to study AMD patient-specific CECs in vitro to better understand AMD pathogenesis and to develop autologous cell replacement therapies to replenish patients’ damaged choroids with healthy CECs.

Cancer is the second leading cause of death in the United States. The majority of cases are caused by sporadic somatic mutation, which leads to cellular transformation over time. Therefore, cancer gene identification is a major focus of current research efforts. Understanding how key driver mutations result in cancer could lead to the design of better targeted therapies. The Sleeping Beauty (SB) transposon system can be used to identify driver mutations in a variety of tumor types. SB mutagenesis mimics the sporadic accumulation of somatic mutations found in spontaneous human cancers. This system also has an additional benefit over chemical carcinogenesis models in that key cancer gene candidates are easily identified with high-throughput sequencing and subsequent bioinformatic analysis. Using SB, our lab recently identified a novel oncogene involved in non-melanoma skin cancer called Zmiz1, the biological function of which is not well studied. A major focus of my thesis work was to characterize Zmiz1 and its role in skin cancer. This gene encodes a protein with predicted E3 SUMO ligase activity. My work has provided the first evidence firmly establishing an oncogenic role for Zmiz1 in cutaneous malignancy, thereby generating a novel transgenic mouse model of skin carcinogenesis. Importantly, we observed tumor-specific overexpression of an endogenous ZMIZ1 isoform in human squamous cell carcinomas. Non-melanoma skin cancer is the most common malignancy worldwide, and it disproportionally affects immunosuppressed patients. One proposed explanation for this is the concept of tumor immunosurveillance, whereby the immune system suppresses tumor growth. When the immune system is compromised, transformed cells can develop into tumors. However, immunocompetent people also develop cancer, despite an intact immune system. It is thought that while the immune system is keeping transformed cells from forming a tumor, it simultaneously influences the acquisition of new mutations that eventually allow escape from immune detection and clearance. This process, called immunoediting, is widely believed to be dependent upon the adaptive immune system and another focus of my research was studying immunoediting mechanisms using SB mutagenesis. Subtle differences were observed in the SB-induced mutation spectra of tumors generated in immunocompetent mice and immunocompromised mice, suggesting that some level of lymphocyte-dependent immunoediting of tumors had occurred. However, the adaptive immune system was not effective in suppressing tumor formation, which is in contrast with previously published data. My work represents an independent and original assessment of the immunoediting process, and cautions against reliance on a single animal model to study this area of cancer biology.

Age-related macular degeneration (AMD) is a devastating disease causing vision loss in millions of people around the world. Loss of choroidal endothelial cells (CECs) is one of the earliest detectable events in AMD, and, because the outer retina relies on the choriocapillaris for metabolic support, this loss may be the trigger for progression to more advanced stages. A crucial event that occurs in the aging choriocapillaris is accumulation of the membrane attack complex (MAC), which may result in complement-mediated CEC lysis, and may be a primary cause for AMD-associated choriocapillaris degeneration. Previous studies have also shown the accumulation of C-reactive protein (CRP) in the choriocapillaris in eyes with AMD and those with the high-risk CFH genotype. While both CRP and the MAC have been implicated in AMD, the precise contribution of these molecules to disease pathophysiology has not been fully elucidated. Furthermore, there is a critical need to better understand the causes for pathologic changes to CECs during AMD and to establish methods for treatment in cases where CECs have already been lost. Therefore, the goals of this thesis are 1) to investigate the role of CRP and complement activation in AMD pathogenesis, and 2) to develop an in vitro method to study CEC replacement strategies. To address these questions, we first evaluated MAC levels in the choriocapillaris in comparison to 19 other tissues throughout the human body in order to determine in which tissues MAC accumulates with normal aging. Interestingly, we found that the choriocapillaris was the only tissue with high levels of the MAC, which was not detected in any of the other tissues. The restricted accumulation of MAC in the choriocapillaris may, in part, explain the specificity of AMD to the neural retina, RPE and choroid, and the relative absence of systemic pathology in this disease. We then studied genotyped human donor eyes and found that eyes homozygous for the high-risk CFH (Y402H) allele had elevated monomeric CRP (mCRP) within the choriocapillaris and Bruch's membrane, compared to those with the low-risk genotype. In order to assess the physiological effects mCRP has on CECs in vitro, CECs and organ cultures were treated with recombinant mCRP. Treatment of CECs with mCRP increased migration rate and monolayer permeability, while organ cultures treated with mCRP exhibited dramatically altered expression of inflammatory genes. Furthermore, in vitro complement activation assays suggest that complement activation on CECs can lead to the dissociation of pCRP into monomers on CECs. Our data indicate that 1) mCRP levels are elevated in individuals with the high-risk CFH genotype, 2) pro-inflammatory mCRP significantly affects endothelial cell phenotypes directly, both in vitro and ex vivo, and 3) MAC formation may be the driving force for accumulation of mCRP in the choriocapillaris. Altogether, this work suggests a role for mCRP in choroidal vascular dysfunction in AMD. Finally, we aimed to develop a reliable method for the production of human choroidal extracellular matrix (ECM) scaffolds to study CEC replacement strategies in an environment that closely resembles the native tissue. Human RPE/choroid tissue was treated sequentially with Triton X-100, SDS, and DNase to remove all native cells. While all cells were successfully removed from the tissue, collagen IV, elastin, and laminin remained, with preserved architecture of the acellular vascular tubes. The ECM scaffolds were then co-cultured with exogenous ECs to determine if the tissue can support cell growth and allow EC reintegration into the decellularized choroidal vasculature. Both monkey and human ECs took up residence in the choriocapillary tubes of the decellularized tissue. These data suggest that our decellularization methods are sufficient to remove all cellular material yet gentle enough to preserve tissue structure and allow for the optimization of cell replacement strategies. Together, these studies provide insight into the mechanism of AMD pathogenesis, suggest potential targets for drug therapies, and develop methods to study the replacement of CECs in more advanced cases of AMD.

Two of the most commonly diagnosed malignancies in men and women are cancers of the prostate and breast, respectively. Though many advances have been made in reducing the overall morbidity and mortality associated with these diseases, the high number of deaths that still occur emphasizes the need for safer and more effective therapeutic options. To this end, our lab was the first to describe the use of RNA aptamers to specifically deliver cytotoxic siRNAs to PSMA positive prostate cancer cells. This reagent, termed an aptamer-siRNA chimera, was shown to be an effective targeted cancer therapeutic upon intratumoral injection in a pre-clinical, xenograft, mouse model of prostate cancer. However, further work was needed to realize the full clinical potential of RNA aptamer-siRNA chimeras as a targeted therapeutic modality. The thesis laid out herein, describes work performed to optimize aptamer-siRNA technology in order to enable clinical translation and to increase the scope of this technology (i.e. increase the cancer types for which this technology can be used). We describe several improvements to our first generation PSMA aptamer-siRNA chimera which, include: decreasing the overall nucleotide content to aid in chemical synthesis, altering the siRNA structure to improve RNAi processing and addition of a 20kDa PEG moiety to increase pharmacokinetics/pharmacodynamics. All of these modifications lead to a more effective reagent at lower doses. Importantly, we demonstrate that our optimized reagent is now effective upon systemic administration in an in vivo mouse model of prostate cancer. In addition, we have also identified new aptamers to the receptor tyrosine kinase (RTK) EphA2. Given the broad expression of this RTK on various cancers, this work seeks to extend the scope of targeted aptamer therapeutics beyond that of prostate cancer. Finally, we demonstrate a novel aptamer selection methodology termed cell-internalization SELEX. This approach allowed us to select for aptamers that specifically targeted and internalize into HER2 expressing cells. This allowed us to readily translate all identified aptamers into aptamer-siRNA chimeras. We show that all chimeras tested were able to sensitize HER2+ breast cancer cells to low- dose cisplatin treatment. Taken together, the work described in this thesis significantly advances the field of targeted cancer therapeutics. Importantly, by demonstrating cancer cell-specific delivery of siRNA, our technology overcomes one of the most significant hurdles to the therapeutic use of siRNAs, delivery.

Protein aggregation has long been associated with disease states, such as Alzheimer’s, Huntington’s, and Parkinson’s diseases. This model is challenged by increasing recognition of functional protein aggregates. Our focus has been exploration of intra-aggregate organization, and investigation of the ability of ATP to mediate energy independent aggregate solubilization by acting as a hydrotrope. Furthering our understanding of the composition and regulation of these structures will provide insight into how their dysregulation gives rise to disease, and provide a template for the development of novel therapeutics. Sub-nuclear organelles are a class of non-membrane bound organelle. These compartments form through liquid-liquid phase separation of their components, resulting in a functional aggregate that demonstrates liquid properties. Many of the factors driving liquid-liquid phase separation also drive the formation of amyloid fibrils. In vitro studies have demonstrated that liquid-liquid phase separation can promote amyloid fibril formation, and that liquid-liquid phase separated droplets can mature through amyloid fibril formation. In vivo, functional amyloids are known to contribute to peptide hormone storage, melanin production, and maintenance of long-term memory. Despite these findings, there has been limited investigation into the relationship between liquid-liquid phase separated droplets and amyloid fibrils in vivo. Our exploration of intra-aggregate organization took advantage of the numerous protein aggregates found within Xenopus oocytes. Using a dye and antibodies specific to the defining cross-β structure of amyloid, we demonstrated that amyloids form as a normal part of Xenopus oogenesis. Amyloid was detected within nuclear and cytosolic aggregates. In the cytosol, amyloid was observed within yolk platelets and a population of cortical particles whose identity has yet to be determined. In the nucleus, multiple liquid-liquid phase separated sub-nuclear organelles, including those associated with RNA polymerase I, II and III transcription as well as RNA processing, were found to have an amyloid component. Proteomic analysis of aggregate enriched material revealed proteins associated with ribonucleoprotein complex biogenesis, DNA replication, RNA processing and DNA-templated transcription. Our analysis further demonstrated that nuclear amyloids were stable, remaining intact for hours post isolation, but rapidly destabilized following treatment with RNase. Recently, adenosine triphosphate (ATP) was demonstrated to act as a hydrotrope in vitro. Physiologic levels were shown to maintain protein solubility and solubilize recombinant liquid-liquid phase separated droplets, in the absence of ATP hydrolysis. These observations suggest that this previously unappreciated activity of ATP has a large influence on liquid-liquid phase separated droplet stability. However, these observations were made under conditions that diverge from those found in vivo. The liquid-liquid phase separated droplets tested were devoid of RNA and composed of a single protein. Amyloid containing aggregates required super physiological levels of ATP, suggesting the sub-nuclear organelles we previously described would be resistant to this activity. By observing nucleoli within isolated Xenopus oocyte nuclei, we demonstrate that ATP has the capacity to act as a hydrotrope in vivo. However, the hydrotropic action alone of ATP is not sufficient to solubilize nucleoli. Instead, solublization requires a sensitization step which is dependent on a soluble factor(s) and requires ATP hydrolysis. Our studies support an in vivo model of liquid-liquid phase separated aggregate solubilization where a soluble factor(s) sensitizes an aggregate in an energy dependent process. The susceptible aggregate is then solubilized by the hydrotropic action of ATP. We further speculate that liquid-liquid phase separated aggregate formation is a reversal of the above steps. These findings highlight a need to further understand the relationship between liquid-liquid phase separation and amyloid formation, and identify an urgent need to dissect the factors influencing intracellular ATP levels.

Proliferating cell nuclear antigen (PCNA) is a versatile protein involved in all pathways of DNA metabolism. It is best known as a processivity factor for classical polymerases, which synthesize DNA on non-damaged templates during DNA replication (ex: pol δ). Non-classical polymerases, on the other hand, are those that synthesize DNA on damaged templates (ex: pol η). PCNA also functions in repair, recombination, and most other DNA-dependent cellular processes. A number of separation of function mutant PCNA proteins have been identified, suggesting that PCNA could be a valuable target to manipulate DNA metabolism. This thesis focuses on the study of PCNA mutant proteins that affect translesion synthesis (TLS) and mismatch repair (MMR). During TLS, the process by which DNA polymerases replicate through DNA lesions, PCNA recruits and stabilizes polymerases at the replication fork. TLS requires the monoubiquitylation of PCNA, and PCNA and ubiquitin-modified PCNA (Ub-PCNA) stimulate TLS by classical and non-classical polymerases. Two mutant forms of yeast PCNA, one with an E113G substitution and one with a G178S substitution, support normal cell growth but inhibit TLS. To better understand the role of PCNA in TLS, I re-examined the structures of both mutant PCNA proteins and identified substantial disruptions of the subunit interface that forms the PCNA trimer. This resulted in reduced trimer stability in the mutant proteins. The mutant forms of PCNA and Ub-PCNA do not stimulate TLS of an abasic site by either classical pol δ or non-classical pol η. Normal replication by pol η was also impacted, but normal replication by pol δ was much less affected. These findings support a model in which reduced trimer stability causes these mutant PCNA proteins to occasionally undergo conformational changes that compromise their ability to stimulate TLS by both classical and non-classical polymerases. During MMR, PCNA recruits and coordinates proteins involved in the mismatch recognition, excision, and resynthesis steps. Previously, two mutant forms of PCNA were identified that cause defects in MMR with little if any other defects. These are the C22Y and C81R mutant PCNA proteins. In order to understand the structural and mechanistic basis by which these two substitutions in PCNA proteins block MMR, we solved the X-ray crystal structures of both mutant proteins and carried out further biochemical studies. I found that these amino acid substitutions lead to distinct structural changes in PCNA. The C22Y substitution alters the positions of the α-helices lining the central hole of the PCNA ring, whereas the C81R substitution creates a distortion in the β-sheet at the PCNA subunit interface. I conclude that the structural integrity of the α-helices lining the central hole and the β-sheet at the subunit interface are both necessary to form productive complexes with MutSα and mismatch-containing DNA. As described above, my studies focused on four amino acid substitutions in PCNA that disrupt TLS and MMR: the E113G and G178S substitutions cause defects in TLS while the C22Y and C81R substitutions cause defects in MMR. The structures of these mutant PCNA proteins revealed that three of the four substitutions caused disruptions near the subunit interface of PCNA. To further examine the importance of this region, we generated random mutations of the PCNA subunit interface and performed in vivo genetics assays and in vitro biochemical assays to examine their effects on TLS and MMR. We determined that the subunit interface of PCNA is very dynamic and that small changes at this interface can cause drastically different effects on TLS and MMR. Moreover, we suggest that the integrity of the subunit interface as well as the nearby β-strands in domain A are crucial for proper PCNA function in vivo and in vitro.

Hrr25 is a highly conserved serine/threonine protein kinase with diverse functions and it localizes to a number of intracellular sites. Hrr25 possesses an N-terminal kinase domain, a middle region, and a C-terminal proline/glutamine rich domain. In this thesis, we systematically characterized the roles of these three domains of Hrr25 on cell growth, cell morphology, and its cellular localizations. Additionally, we identified a novel target of Hrr25, Pin4. We show that Hrr25-dependent phosphorylation of Pin4 is required for cell wall integrity maintenance. We found that that Hrr25 and Pin4 physically interact in vivo and that the C-terminal domain of Hrr25 is required for this interaction and for maintaining cell wall integrity. Together, we identified a new target and a novel function for Hrr25 and provided insights into the structure-function relationship of Hrr25 domains.

Pdr5 and Yor1 are two ATP-binding cassette transporters regulated by the pleiotropic drug resistance (PDR) network in the yeast Saccharomyces cerevisiae. Recent work from another group demonstrated that a pdr5Δ yor1 strain confers remarkable resistance to a sphingolipid intermediate, phytosphingosine (PHS), which was surprising as loss of these transporters normally leads to elevated drug sensitivity. PHS is toxic to the cell at high levels due to mislocalization of nutrient permeases, such as the high affinity tryptophan transporter, Tat2. Although the above study suggested that this resistance was due to increased expression of Rsb1, a known mediator of PHS tolerance, this was not reproducible in our hands and we sought to identify other determinants for this phenotype. The work presented here demonstrates that the pdr5Δ yor1 strain exhibits delayed turnover of Tat2 and an increase in tryptophan uptake, which we postulate is due to changes membrane asymmetry resulting in decreased endocytosis. Conversely, cells lacking Rsb1 showed a decrease in tryptophan import and increased Tat2 turnover, independent of endogenous PHS levels. Rsb1 has a predicted 7 transmembrane (7TM) topology, which argues against the idea that Rsb1 functions directly in PHS transport, as there are currently no known transporters with this topology. These data suggest that Rsb1 and Pdr5/Yor1 function in regulation of endocytosis of Tat2, and possibly other membrane proteins. Ethyl methanesulfonate mutagenesis of the pdr5Δ yor1 strain and a candidate gene approach were alternative methods used to identify mediators of PHS tolerance in this strain. Inconsistent results from PHS selection led to the discovery that the pdr5Δ yor1 strain was also robustly resistant to the sphingolipid biosynthesis inhibitor, Aureobasidin A (AbA), which was subsequently used for analysis. These approaches revealed several genes, including Gda1, Mss4, and Ypk1 that are important for AbA tolerance in the pdr5Δ yor1 strain. Many of these determinants play a role in cell wall integrity, suggesting that loss of Pdr5 and Yor1 may lead to activated cell wall integrity pathways resulting in altered cell wall structure.