京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第22707号 / 農博第2423号 / 新制||農||1080(附属図書館) / 学位論文||R2||N5300(農学部図書室) / 京都大学大学院農学研究科地域環境科学専攻 / (主査)教授 本田 与一, 教授 田中 千尋, 教授 吉村 剛 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DGAM

Human immunodeficiency virus still remains the leading cause of death globally including women of child-bearing age. The rate of AIDS-related death has significantly declined since the introduction of antiretroviral treatment and other non-medical interventions such as the distribution and use of condoms. The introduction of antiretroviral treatment has however led to insulin resistance amongst users. Clustered regularly interspaced short palindromic repeats (CRISPR) CRISPR-associated nuclease 9 (Cas) has been used to knockout NFκB to understand the pathway at which antiretroviral treatment causes insulin resistance. Heteroduplex mobility assay has shown that CRISPR-Cas9 knock out the gene of interest. These results have played a foundation in understanding how CRISPR-Cas9 can be integrated and utilized in medical research. / Dissertation (MSc (Chemical Pathology))--University of Pretoria, 2020. / National Research Foundation (NRF) / Chemical Pathology / MSc (Chemical Pathology) / Restricted

Zebrafish is a frequently used model organism with an array of transgenic lines that have been used indevelopmental and physiological studies. We aim to generate novel transgenic zebrafish reporter lines to study subpopulations of spinal neurons in vivo. The gene editing system called CRISPR/Cas9 system was used to knock in reporter genes such as green fluorescent protein (GFP) or Gal4 transcription factor, to generate transgenic fish lines. Zebrafish embryos were injected with gRNAs targeting gabrb1 or nr4a2a and GFP or Gal4 plasmid, respectively. F0 larvae were screened, positive fish were raised until sexual maturity, and founders characterized to verify germline insertion. Three founders were found for gabrb1 and the location and the direction of the insert verified. The GFP expression was studied during development and differential expression patterns were identified whereas all founders had expression in brain and spinal cord. In parallel, positive fish from the Gal4 injections were raised and will be screened. Immunohistochemistry was performed to check if nr4a2a is expressed in the same cells as known neuronal markers. However, no co-localization was detected. The three gabrb1 founders identified in this study highlight the challenges into creating stable transgenic lines recapitulating true expression of the gene of interest. Sequencing, in-situ hybridization and immunohistochemistry should be performed to verify the line. A possible reason for the varying expression may be that through the knock-in we may interfere with regions regulating gene. The nr4a2a-Gal4 line will be used to perform functional studies. Those experiments will be performed using reporter genes, such as opsins or GCaMP, controlled by Upstream Activation Sequence (UAS). These transgenic lines will provide important insights regarding neuronal subpopulations that express gabrb1 and nr4a2a to unravelhow the locomotor network is formed.

Thesis advisor: Katie Rapier / Through the years, people began to breed their dogs and cats with the intention for the pets to be useful in a certain skill. However, that has shifted to focus on their looks rather than their skills thanks to kennel clubs imposing standards on each breed. This has led irresponsible breeding practices to occur which in turn caused breeds to evolve negatively as breeds began to suffer from preventable genetic disorders and negative physical changes. Genetically manipulating soon shifted from pets to humans with PGD/IVF and CRISPR-Cas9. At first, there was a focus to use these methods to help cure and prevent genetic disorders. That has since shifted to people wanting to create the perfect child. In this thesis I will argue that designer breeds help lead the way to designer babies, and that any genetic manipulation to embryos should only be done if a medical reason is present. / Thesis (BA) — Boston College, 2020. / Submitted to: Boston College. College of Arts and Sciences. / Discipline: Departmental Honors. / Discipline: Philosophy.

Gene editing with the CRISPR/Cas9 system is one of the options that sets a new trend in the development of gene therapy. The most commonly used delivery of DNA into the cells are via viruses. Nevertheless, they are often unable to take CRISPR/Cas9 system, which can be bigger than several kb. Nanoparticles (NPs), as non-viral transporters, seem to be a good alternative delivery system. For this work magnetic Fe3O4 NPs (MNPs) were selected, because of their excellent properties such as multifunctionality, biocompatibility, easy degradation and simple synthesis. The aim of this work was to synthesise MNPs and a complex of MNPs coated with PEI/CRISPR-Cas9 plasmid and to characterize them by physicochemical methods. The created complex MNPs/PEI/CRISPR-Cas9 was defined by exact parameters that are suitable for possible cell uptake. The hypothesis of stabilization of the MNPs/CRISPR-Cas9 plasmid complex by polyethylenimine (PEI), which can also protect plasmid DNA against restriction endonucleases, was verified. Next a stable modified cell line HEK293-TLR3, designed to evaluate the efficacy of double strand break (DSB) repair by nonhomologous end joining (NHEJ) or homologous recombination (HR) was, transfected with the synthesised MNPs/PEI/CRISPR-Cas9 complex. The results indicate a 25% transfection...

Parkinson’s disease (PD) is the second most prevalent neurodegenerative disease, featuring motor signs such as tremors, bradykinesia, and impaired gait that are often preceded by nonmotor symptoms such as anxiety/depression and olfactory dysfunction. Interestingly, significant olfactory loss was found to be manifested in the majority of PD patients and may precede motor symptoms by years, and thus can be used for the risk assessment of developing PD in asymptomatic individuals. The main pathological feature of PD is the progressive and irreversible loss of dopaminergic (DA) neurons in the substantia nigra pars compacta of the midbrain. Although the detailed etiology of PD remains unclear, most PD cases were found to be sporadic and can be associated with environmental factors. Only 5–10% of patients result from familial PD. With considerable effort in the past two decades, a number of genes associated with familial PD have been identified and interestingly, many of these genes are involved in regulating and maintaining mitochondrial function. The presenilin-associated rhomboid-like (PARL) gene was found to contribute to mitochondrial morphology and function and was linked to familial Parkinson’s disease (PD). The PARL gene product is a mitochondrial intramembrane cleaving protease that acts on a number of mitochondrial proteins involved in mitochondrial morphology, apoptosis, and mitophagy. To date, functional and genetic studies of PARL have been mainly performed in mammals. However, little is known about PARL function and its role in dopaminergic (DA) neuron development in vertebrates. The zebrafish genome comprises two PARL paralogs: parla and parlb. Here, we show novel information concerning the role of PARL in zebrafish by establishing a loss-of-function mutation in parla and parlb via CRISPR/Cas9- mediated mutagenesis. We examined DA neuron numbers in the adult brain and expression of genes associated with DA neuron function in larvae and adults. We show that loss of parla function, as well as loss of both parla and parlb function result in loss of DA neurons in the olfactory bulb and telencephalon of adult zebrafish brain. Changes in the levels of tyrosine hydroxylase transcripts supported this neuronal loss. Expression of fis1, a gene involved in mitochondrial fission, was increased in parla mutants and in fish with loss of parla and parlb function. Furthermore, we showed that loss of parla and/or parlb function translates into altered locomotion parameters and that loss of parla but not parlb function results in impaired olfaction. Finally, increased susceptibility to neurotoxin exposure was identified in mutants with loss of both parla and parlb function but not with loss of parla or parlb function. These results suggest an evident role for parla in the development and/or maintenance of DA neuron function in zebrafish and confirm the existence of redundant and non-redundant functions for the two paralogs, parla and parlb.

>Magister Scientiae - MSc / Saccharomyces cerevisiae is an important host in industrial biotechnology. This yeast is the host of choice for the first and second-generation biofuels for ethanol production. Genome modification in S. cerevisiae has been extremely successful largely due to this yeast’s highly efficient homology-directed DNA repair machinery. The advent of CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) genome editing technology has made multi-gene editing in yeast more accessible. In this study, we aimed at targeting the Cas9 to multiple genomic positions for integrating multiple genes at different sites. We have developed two CRISPR-Cas9 systems, based on published one- and two-plasmid systems, for application in S. cerevisiae strains. In this study, these CRISPR-Cas9 systems were used to transform fungal heterologous genes into yeast using the electroporation transformation method.

Replication Factor A (RFA) is an essential heterotrimeric single-stranded DNA (ssDNA) binding complex, comprised of Rfa1, Rfa2, and Rfa3 in Saccharomyces cerevisiae. RFA is required for DNA replication, repair, recombination, and cell cycle regulation. RFA acts as a sensor of ssDNA, a common intermediate of these processes, and coordinates these processes through recruitment of proteins. For example, during the DNA damage response (DDR), RFA-coated ssDNA is necessary for the recruitment and activation of the sensor kinase Mec1. Additional checkpoint proteins, also recruited by RFA, are necessary for the downstream recruitment and activation of the effector kinase Rad53 that ultimately leads to cell cycle arrest. Thus, RFA acts as a bridge to recruit the proteins required for checkpoint regulation in response to DNA damage. Importantly, cell cycle resumption is contingent on Rad53 deactivation. There are two known scenarios in which Rad53 is deactivated: (1) checkpoint recovery, in which cells resume the cell cycle after DNA repair or (2) checkpoint adaptation, in which cells proceed with the cell cycle despite the continued presence of irreparable DNA damage. Previous work has demonstrated that cells undergoing checkpoint adaptation display late Rfa2 N-terminal (NT) phosphorylation that is correlated with the inactivation (dephosphorylation) of Rad53. Additionally, the use of rfa2 NT mutations consistently demonstrate that a negatively charged NT promotes adaptation in all adaptation-deficient strain backgrounds investigated. Interestingly, Rfa2 NT phosphorylation also occurs early during meiosis. This work demonstrates that: (1) Rfa1-DBD-F participates in protein-protein interactions that are sensitive to DNA damage, (2) Rfa2 phosphorylation increases the DNA damage sensitivity of mutants with deficient DNA damage checkpoints, (3) the Rfa2 NT is required for proper progression through meiosis that appears to be unrelated to RFA functions in replication or DNA repair by homologous recombination (HR), and (4) Rfa2 phosphorylation may regulate Mec1 checkpoint signaling during the DDR to control checkpoint exit and cell cycle resumption. A mechanism is proposed that considers both Rfa1 DBD-F and the Rfa2 NT involvement to initiate HR repair that essentially allows for the continuation of the cell cycle by the delocalization of Mec1.

Permanent repair of the F9 gene is a significant goal to cure Hemophilia B disease. Advanced gene therapy using CRISPR/Cas9 system can increase circulation level of Factor IX proteins to a significant level without the need of demanding infusions of FIX concentrates. Induced pluripotent stem cells represent an ideal cell for gene therapy because patient-derived cells could be reprogrammed into iPSCs, genetically modified, selected, expanded and then induced to differentiate into fully functional hepatocytes in vitro. This study covered a portion of a 5-year project which ultimately aims at establishing therapeutic results in transgenic Hemophilia B mice by injecting genetically corrected iPSC-derived hepatocytes into the liver. The purpose of this thesis is to summarize what has been completed up to now: generation of the proper model of Hemophilia B human iPSCs using CRISPR/Cas9-mediated genome editing and differentiation of healthy and disease specific iPSCs into hepatocytes which will allow disease modelling to look for cell function, viability, homogeneity and drug screening. Further research will be done to effectively knock-in the F9 allele into liver safe harbor site of disease specific iPSCs, which will express FIX at a significant level to show therapeutic effects.

京都大学 / 0048 / 新制・課程博士 / 博士(医科学) / 甲第21689号 / 医科博第93号 / 新制||医||1036(附属図書館) / 京都大学大学院医学研究科医科学専攻 / (主査)教授 斎藤 通紀, 教授 中川 一路, 教授 竹内 理 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM