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No description available.

A nonsynonymous, loss of function variant (rs58542926, E167K) located in the gene encoding TM6SF2 was identified in multiple genetic association studies as significantly correlating with increased risk for non-alcoholic fatty liver disease (NAFLD) and decreased risk for hyperlipidemia. Given the pivotal role that lipoproteins play at the juncture of these two conditions, researchers hypothesize that the ER-membrane spanning TM6SF2 protein regulates the degree of lipidation of VLDL particles synthesized in the liver. However, not all published data supports this theory and contradictions regarding many aspects of the mechanistic function of TM6SF2 remain. The inconsistencies observed in the literature are in part due to drawbacks of the models used to study TM6SF2 activity; thus, there is an obvious need for an improved hepatocyte model to better understand how TM6SF2 impacts lipid metabolism.To address this need, we present an optimized protocol for the differentiation of inducible pluripotent stem cells (iPSCs) into hepatocyte-like cells (HLCs), created in collaboration with the Leong Lab. We provide extensive validation of HLC maturity and hepatic functionality, including prolonged albumin secretion, evidence of membrane polarity, and cytochrome P450 induction. We also demonstrate that HLCs express proteins essential for lipoprotein metabolism, secrete authentic VLDL particles, and respond to metabolic perturbations, supporting their value for modeling hepatosteatosis and VLDL metabolism in vitro. To investigate the effect of TM6SF2 variant expression on hepatic lipid metabolism, we produced HLCs derived from 4 homozygous TM6SF2-carrier individuals (KK) and 4 age- and sex-matched unaffected siblings (EE). We describe the variability in differentiation efficiency that we observed in our sibling-matched HLC model and present the gene editing strategy we developed using CRISPR/Cas9 technology and transgene-induced expression to create isogenic iPSCs differing only in their TM6SF2 genotype [EE, KK, or knockout (KO)]. After extensive confirmation of successful gene editing, we explore the effect of TM6SF2 on lipid metabolism in the edited iPSCs. RNA-sequencing and qPCR validation reveal that the Sterol Regulatory Element Binding Protein 2 (SREBP2)-mediated transcriptional program regulating cholesterol synthesis is significantly increased in TM6SF2 KO iPSCs. However, lipidomics analysis and de novo lipogenesis functional assays show that free cholesterol (FC) levels are unchanged. In TM6SF2 KO iPSCs, we further show a reduction in the activities of Acyl-Coenzyme A: Cholesterol Acyltransferase 1 (ACAT1) and Phosphatidylserine Synthase 1 (PSS1), two enzymes that display optimal function when specifically localized to cholesterol enriched ER lipid raft-like domains. Our findings suggest that TM6SF2 may impact cholesterol localization within ER subdomains, which regulate expression levels of cholesterol synthesis genes and activities of ER lipid-raft associated enzymes. In summary, we present here methodological approaches for generating multiple cell culture models in which to investigate the function of TM6SF2, as well as novel evidence supporting a role for TM6SF2 in iPSC cholesterol metabolism.

yes / The aim of the experiment reported here was to investigate the processes underlying the construction of truthful and deliberately fabricated memories. Properties of memories created to be intentionally false (fabricated memories) were compared to properties of memories believed to be true (true memories). Participants recalled and then wrote or spoke true memories and fabricated memories of everyday events. It was found that true memories were reliably more vivid than fabricated memories and were nearly always recalled from a first-person perspective. In contrast, fabricated differed from true memories in that they were judged to be reliably older, were more frequently recalled from a third-person perspective, and linguistic analysis revealed that they required more cognitive effort to generate. No notable differences were found across modality of reporting. Finally, it was found that intentionally fabricated memories were created by recalling and then “editing” true memories. Overall, these findings show that true and fabricated memories systematically differ, despite the fact that both are based on true memories.

The pig has similar features to the human in aspects such as physiology, immunology, and organ size. Because of these similarities, genetically modified pigs have been generated for xenotransplantation. Also, when using the pig as a model for human diseases (e.g. cystic fibrosis transmembrane conductance regulator), the pig exhibited similar symptoms to those that human patients present. The main goal of this work was to examine the efficacy of direct injection of the CRISPR/Cas9 system (clustered regularly interspaced short palindromic repeats/ CRISPR associated protein 9) in pigs and to overcome shortcomings that resulted after direct injection into the cytoplasm of developing zygotes. By using direct injection of CRISPR/Cas9 into developing zygotes, we successfully generated fetuses and piglets containing 9 different mutations. The total number of aborted fetuses was 20 and of live piglets was 55. Moreover, one issue that was encountered during the production of mutated pigs was that insertion or deletion (indel) mutations did not always introduce a premature stop codon because it did not interfere with the codon read. As a result of these triplet indel(s) mutations, a hypomorphic phenotype was presented; consequently, the mutated gene was partially functional. To prevent this hypomorphic phenotype, we introduced two sgRNAs to generate an intended deletion that would remove a DNA fragment on the genome by causing two double-strand breaks (DSB) during non-homologous end joining (NHEJ). The injection of two sgRNAs successfully generated the intended deletion on the targeted genes in embryos and live piglets. Results after using intended deletions, in IL2RG mutation pigs, did not show hypomorphic phenotypes even when a premature stop codon was not present. After using the intended deletion approach, function of the targeted genes was completely disrupted regardless of the presence or absence of a premature stop codon. Our next aim was to introduce (i.e. knock-in) a portion of exogenous (donor) DNA sequence into a specific locus by utilizing the homology direct repair (HDR) pathway. Because of the cytotoxicity of the linear form of the donor DNA, the concentration of the injected donor DNA was adjusted. After concentration optimization, four different donor DNA fragments targeting four different genes were injected into zygotes. Efficiency of knock-in was an average of 35%. Another donor DNA was used in this study which is IL2RG-IA donor DNA carried 3kb of exogenous cassette. It showed 15.6% of knock-in efficiency. IL2RG-IA Donor DNA injected embryos were transferred into surrogates, and a total of 7 pigs were born from one surrogate, but none of the 7 were positive for the knock-in. Future experiments need to be developed to optimize this approach. Overall, the direct injection of CRISPR/Cas9 is advantageous in cost, time, and efficiency for large animal production and for biomedical research. However, there are still unsolved challenges (off-targeting effects, low efficiency of knock-in, and monoallelic target mutation) that need to be elucidated for future application in humans and other species. / Doctor of Philosophy / The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9) system is commonly used to make genetically modified pigs. The CRISPR/Cas9 system can break the DNA on a desired gene region. During the DNA repair process, random DNA base pairs can be inserted or deleted on the broken regions, thus generating a mutation on the desired gene. Scientists have adopted new methods to disrupt genes in many species. One of these new methods is the direct injection of CRISPR/Cas9 into a fertilized oocyte. In our first project, we used direct injection of the CRISPR/Cas9 system into the fertilized one-cell embryo. A total of 55 live pigs and aborted 20 fetuses with specifically disrupted genes were produced for biomedical research model. During these studies, one critical drawback of the direct injection method was encountered. Partial function of the gene was possible. To prevent this problem, two DNA broken regions were generated by the CRISPR/Cas9 system to remove the middle part the DNA by two DNA breaking. This method successfully removed the middle portion of the DNA targeted region in the pig embryos. Embryos injected with the CRISPR/Cas9 system to cut the two specific DNA regions were transplanted into surrogate pigs, and a total of 15 piglets were produced. All 15 pigs confirmed that a specific part of the gene had been removed by two DNA breakage. Also, no function of the desired gene was found in the 15 pigs. The objective of the last experiment was to introduce a specific exogenous DNA sequences into specific region of DNA using the CRISPR/Cas9 system. For this study, four different exogenous DNA fragments were synthesized for four different genes. When injected, one exogenous DNA along with the CRISPR/Cas9 system, the average integration efficiency of the four exogenous DNA fragments was 35% in the embryo. Another exogenous DNA, which was longer than other four DNA fragments showed 15.6% integration efficiency. The embryos injected with the long exogenous DNA fragment, along with the CRISPR/Cas9 system, were transferred into surrogate pigs. The result was that a total of 7 piglets were born, but the exogenous DNA sequence was not found in none of the seven piglets. In conclusion, the CRISPR/Cas9 system showed effective removal of the entire gene function of specific genes in the pig. However, for future application in the human and other species, some problems (un-wanted region mutation and low efficiency of exogenous DNA integration) continue to emerge and need to be addressed in future experiments.

Dysfunction within complex I (CI) of the mitochondrial electron transport system has been implicated in a number of disease states ranging from cardiovascular diseases to neuro-ophthalmic indications. Herein, we provide three novel approaches to model and study the impacts of injury on the function of CI. Cardiovascular ischemia/reperfusion (I/R) injury has long been recognized as a leading contributor to CI dysfunction. Aside from the physical injury that occurs in the tissue during the ischemic period, the production of high levels of reactive oxygen species (ROS) upon reperfusion, led by reverse electron transport (RET) from CI, causes significant damage to the cell. With over 700,000 people in the US set to experience an ischemic cardiac event annually, the need for a pharmacological intervention is paramount. Unfortunately, current pharmacological approaches to treat I/R related injury are limited and the ones that have shown efficacy have often done so with mixed results. Among the current approaches to treat I/R injury antioxidants have shown some promise to help preserve mitochondrial function and assuage tissue death. The studies described herein have provided new, more physiologically matched, methods for assessing the impact of potential therapeutic interventions in I/R injury. With these methods we evaluated the efficacy of the coenzyme-Q derivative idebenone, a proposed antioxidant. Surprisingly, in both chemically induced models of I/R and I/R in the intact heart, we see no antioxidant-based mechanism for rescue. The mechanistic insight we gained from these models of I/R injury directed us to further examine CI dysfunction in greater detail. Through the use of two cutting edge genetic engineering approaches, CRISPR/Cas9 and Artificial Site-specific RNA Endonucleases (ASRE), we have been able to directly edit the mitochondria to accurately model CI dysfunction in disease. The use of these genetic engineering technologies have provided first in class methods for modeling three unique mitochondrial diseases. The culmination of these projects has provided tremendous insight into the role of CI in disease and have taken a significant step towards elucidating potential therapeutic avenues for targeting decrements in mitochondrial function. / Doctor of Philosophy / Within the mitochondria, “the powerhouse of the cell,” exists a series of five enzyme complexes that produce 90% of the energy for our cells need to function. The largest of these enzymes, complex I (CI), plays an important role in ensuring proper mitochondrial function. Injury to CI contributes to a number of diseases, but surprisingly few options exist to treat complex I. One of the most prevalent forms of CI dysfunction can be seen in ischemia/ reperfusion injury, a form of which is most commonly recognized as a heart attack. Surprisingly, the American Heart Association reports that in the next year over 700,000 people in the US will suffer from an ischemic event. With such a profound impact on the population, the need for new therapeutic developments is extremely high. Some current therapeutic approaches have been shown to be effective at treating cardiac dysfunction, but few address the dysfunction that occurs in the mitochondria. Here we test both a method for modeling these ischemia/reperfusion-based injuries and a potential therapeutic for treating these injuries within the context of CI dysfunction. We further evaluate CI dysfunction by using both established genetic engineering approaches as well as a completely new method to model CI disease. Through the use of two cutting edge genetic engineering approaches, we have been able to directly edit components of the mitochondria to accurately model CI dysfunction in disease. The use of these genetic engineering technologies have provided a first-in-class method for modeling three unique mitochondrial diseases. The culmination of these projects has provided tremendous insight into the role of CI in disease and have taken a significant step towards elucidating potential therapeutic avenues for targeting decrements in mitochondrial function.

<p dir="ltr">Citrus greening has severely impacted orange trees in America, causing significant damage to farmers and industry. Gene editing has been proposed as a potential solution, but understanding consumer acceptance of the resulting gene edited products is essential to evaluate the marketability of this potential solution.</p><p dir="ltr">We conducted a hypothetical discrete choice experiment to analyze consumer preferences for gene edited orange juice. We further explored how consumer willingness-to-pay (WTP) for the gene edited attribute varies depending on the total number of attributes presented in the experiment and how information about the benefits of the technology influences WTP across the different attribute scenarios.</p><p dir="ltr">We find that, on average, consumers devalue the gene edited attribute, but their WTP for the attribute increases when information is provided. Importantly, we also observe that the number of attributes significantly impacts the WTP estimates, but the effect is moderated when information is provided.</p>

Slo2, a large conductance potassium channel in the nervous system is important for regulating neuronal function and excitability. Mutations in the gene that encodes the Slo2 channel are associated with neurological disorders, including epilepsy and intellectual disabilities in humans. However, much remains unknown about the genes and proteins that regulate Slo2 channel activity in the physiological system. This study investigates regulation of SLO-2, a homologue of mammalian Slo2 in C. elegans, by thoc-7 in an RNA editing-dependent pathway. Prior research has shown that adr-1, the gene important for RNA editing, promotes SLO-2 function by RNA editing of scyl-1 that encodes a regulator of SLO-2. To gain a better understanding of the regulation of SLO-2, this study employed a forward genetic approach to screen for mutants with a specific phenotype. Through SNP mapping and whole genome sequencing, we identified the gene thoc-7, which is predicted to be involved in mRNA export from nucleus, from the isolated mutants. The identification was further confirmed by CRISPR/Cas9-mediated gene knock-out, which showed a similar phenotype to the mutant strain. Results of electrophysiological recordings suggest that thoc-7 likely contributes to SLO-2 function in a common pathway with scyl-1. A reporter gene revealed strong expression of thoc-7 in most of the cells of C. elegans, particularly muscular and digestive system. Translational fusion with GFP showed the primary localization of the THOC-7 protein in cytoplasm, with some weak expression in the nucleus. RT-qPCR analysis suggests that thoc-7 regulates scyl-1 by through a post-transcriptional mechanism, possibly involving the transport of mRNA from cytoplasm to nucleus. This study highlights thoc-7 as a potential key regulator recruited by adr-1 to control SLO-2 via scyl-1 expression.

Thesis advisor: Jia Niu / High-Throughput Particle Display Screening of RNA-Protein Interactions RNA-protein binding interactions have essential roles in many biological processes including transcriptional and translational control; thus, it is important to quantify the binding affinities of these biological complexes through functional binding assays. Although conventional binding assays have provided significant insight to these dynamic networks, they generally provide a relatively low throughput for a limited number of samples. To overcome the limitations of these conventional binding assays to study RNA-protein binding interactions, we propose to develop an in vitro, high-throughput particle display-based for RNA aptamer screening of RNA-protein complexes for the subsequent identification and characterization of novel RNA aptamers that influence protein binding. With this technique, we will be able to profile large numbers of binding events based on binding-induced fluorescence-enhancement for a more holistic understanding of the corresponding RNA-protein network. So far, we have confirmed that this particle display-based technique can be used to estimate the binding affinity of the well-characterized MS2-MCP model system, and plan to advance this technique to screen a library of MS2 variants for mutational analysis. Prime Editor-Mediated Programmable Insertion of UAAs into Endogenous Proteins The introduction of unnatural amino acids (UAAs) to endogenous cell surface proteins for site-specific bioconjugation reactions allows for the incorporation of clickable, fluorescent handles in vivo; however, the transient expression of proteins harboring UAAs is limited by its transfection efficiency. Thus, we propose to employ prime editors and tRNA/aminoacyl-tRNA synthetase technologies to introduce an UAA to endogenous proteins for downstream bioconjugation applications. Briefly, we propose to stably incorporate a stop codon into mammalian cells by prime editing which will be confirmed with a reporter system, such that this stop codon can mediate the introduction of an UAA through the associated tRNA/aminoacyl-tRNA synthetase technology. By permanently introducing a bioorthogonal, clickable handle onto an endogenous protein, its cellular signaling and localization patters can be monitored in vivo for further classification of the behaviors of these proteins. So far, we identified a promising fluorescent reporter construct to validate the introduction of a stop codon into the mammalian genome by prime editing. / Thesis (MS) — Boston College, 2021. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.