Cell migration is an important component of many physiological and pathological processes such as tissue and organ morphogenesis during development, wound healing, inflammatory immune response, and tumor metastasis. The actin cytoskeleton is the basic engine driving cell migration. In the present study, we elucidate the role of an important actin interacting proteins, Adducins, in motility of normal lung epithelium and lung cancer cells. Adducins are the family of cytoskeleton protein capping the fast growing end and facilitating the bundling of actin filaments. Adducins are encoded by the three closely related genes namely alpha (ADD1), beta (ADD2) and gamma (ADD3) Adducin. ADD1 and ADD3 are ubiquitously expressed, whereas ADD2 is most abundant in brain and erythrocytes. Adducins are also involved in recruiting spectrin to the actin filaments forming spectrin-actin membrane skeletal network. Its role in cell motility remains controversial. In this study, we observed that CRISPR/Cas9 mediated stable knockout of ADD1 and ADD3 in 16HBE normal lung epithelium cells significantly increases transfilter migration of cells. On the other hand, stable overexpression of ADD1 in H1299 Non-Small Cell lung cancer cells significantly decreases wound healing, transfilter migration and Matrigel invasion of the cells. Importantly, the effects of Adducin depletion and overexpression on cell motility were not due to altered cell proliferation. ADD1 overexpressed H1299 cells were characterized by the increased adhesion and spreading on the collagen matrix. Fluorescence microscopy revealed alterations in their cortical actin cytoskeleton that was manifested in the assembly of peripheral F-actin bundles and formation of filopodia-like protrusions. These findings suggest that Adducins are negative regulators of motility of normal lung epithelial and lung cancer cells that act by altering the architecture of submembranous actin cytoskeleton and modulating cell adhesion to the extracellular matrix.

The serine/arginine-rich (SR-) family proteins constitute a diverse group of pre-mRNA splicing factors that are essential for viability. They can be characterised based on the presence of one or two RRMs and an RS domain. A subset, of which SRSF1 is the prototype, is capable of nucleocytoplasmic shuttling; a process governed by continual cyclic phosphorylation of the RS domain. In contrast, SRSF2, another member of the SR family, is unable to shuttle due to the presence of a nuclear retention sequence (NRS) at the C-terminus of its RS domain. When this NRS is fused to SRSF1, it prevents nucleocytoplasmic shuttling of the SRSF1-NRS fusion protein. In addition to its nuclear roles, SRSF1 is directly associated with the translation machinery and can activate mRNA translation of target transcripts via an mTOR-dependent mechanism. The specific mRNA translational targets that SRSF1 serves to regulate encode numerous factors including RNA processing factors and cell-cycle proteins. The aim of this work is to study the physiological relevance of SRSF1 cytoplasmic functions, as previous data have relied on overexpression systems. CRISPR/Cas9 editing was used to knock-in the NRS naturally present in SRSF2 at the SRSF1 genomic locus, creating an SRSF1-NRS fusion protein. After numerous attempts, it was only possible to obtain a single viable homozygous clone in mouse embryonic stem cells (mESCs), despite being able to successfully tag the genomic SRSF1 locus. This strongly suggests that the ablation of SRSF1 shuttling ability is highly selected against in mESCs. To assess the physiological importance of SRSF1 nucleocytoplasmic shuttling during development, a mouse model for SRSF1-NRS was also developed. SRSF1-NRS homozygous mice are born at correct Mendelian ratios, but are small in size and present with severe hydrocephalus. Finally, proteomics was used to identify interactors of endogenous cytoplasmic SRSF1 and those that bind the NRS of SRSF2 to gain insights into the mechanism of nuclear retention for non-shuttling SR proteins. In summary, this work analyses the physiological relevance of cytoplasmic SRSF1 function and the consequences of the SRSF1-NRS allele in mouse development.

A mucopolissacaridose tipo I (MPS I) é causada pela deficiência de alfa-L-iduronidase (IDUA), responsável pelo catabolismo de glicosaminoglicanos (GAGs), levando ao acúmulo multissistêmico de sulfato de heparano e dermatano. Este estudo tem por objetivo avaliar o potencial de sistemas lipídicos nanoestruturados como carreadores do plasmídeo do sistema CRISPR/Cas9 e um vetor doador da sequência do gene IDUA/Idua para edição gênica em fibroblastos de pacientes e em modelo murino de MPS I. Foram produzidos lipossomas (DOTAP, DOPE e DSPE-PEG) e nanoemulsões (e TCM) por homogeneização à alta pressão e microfluidização. O DNA foi associado às formulações por adsorção, ou por encapsulamento dos complexos pré-formados DNA/DOTAP no núcleo oleoso da nanoemulsão. A eficiência de transfecção dos complexos foi avaliada em fibroblastos de pacientes MPS I e ocorreu um aumento significativo da atividade de IDUA em 2, 15 e 30 dias após os tratamentos, que promoveu uma redução na quantidade de lisossomos nos fibroblastos tratados. A caracterização físico-química de formulações produzidas por microfluidização complexadas a somente um plasmídeo ou juntamente com um oligonucleotídeo foi verificada e pode-se afirmar que a capacidade de complexação e transfecção depende diretamente do tipo celular e da relação de cargas, e não há implicações quanto ao tamanho das sequências de ácidos nucleicos. Camundongos MPS I receberam os complexos lipossomais por injeção hidrodinâmica e sua biodistribuição foi detectada principalmente no pulmão, coração e fígado. A atividade sérica de IDUA normal aumentou em cerca de 6% e foi mantida por seis meses. A atividade aumentada no pulmão, coração, fígado e rim após eutanásia promoveu redução dos GAGs na urina e nos mesmos tecidos, corroborando com as análises histológicas. Em um estudo em andamento, foi realizada uma investigação mais aprofundada do efeito do tratamento lipossomal na morfologia óssea, sistemas cardiovascular e respiratório, e funções cerebrais dos animais tratados. A análise ecocardiográfica demonstrou uma melhora na hipertrofia e contratilidade do coração, porém não houve melhora na espessura das válvulas. O diâmetro da aorta foi similar ao de animais normais, porém as quebras de elastina ficaram entre o grupo normal e o não tratado. A morfologia facial dos animais tratados foi intermediária, assim como a espessura do osso zigomático. Entretanto, o osso femoral demonstrou espessura comparável ao normal. Já a resistência pulmonar apresentou uma tendência de redução nos animais tratados em relação aos animais MPS I. O conjunto de resultados demonstra o potencial das nanoestruturas lipídicas co-complexadas com o plasmídeo CRISPR/Cas9 e um vetor doador da sequência IDUA/Idua para terapia gênica da MPS I. / Mucopolysaccharidosis type I (MPS I) is caused by the deficiency of alpha-L-iduronidase (IDUA), responsible for the catabolism of glycosaminoglycans (GAGs), leading to multisystemic accumulation of heparan and dermatan sulfate. This study aims to evaluate the potential of lipid-based nanostructures as carriers of the CRISPR/Cas9 plasmid and a vector donor of the IDUA/Idua sequence for gene editing in patients’ fibroblasts and in a murine model of MPS I. Liposomes (DOTAP, DOPE, and DSPE-PEG) and nanoemulsions (also MCT) were produced through high-pressure homogenization or microfluidization. DNA was associated with liposomes and nanoemulsions by adsorption or by encapsulation of DNA/DOTAP preformed complexes in the oil core of nanoemulsions. The transfection efficiency of complexes was evaluated in fibroblasts from MPS I patients and a significant increase in IDUA activity was demonstrated at 2, 15, and 30 days after treatments. It was also possible to observe a significant reduction in lysosomal amount in treated fibroblasts. The physicochemical characterization of liposomes and nanoemulsions produced through microfluidization complexed with a single plasmid or along with an oligonucleotide has been verified and it can be stated that the complexing and transfection capacity of the complexes depends directly on the cell type and the charge ratio, and there are no implications of the size of the nucleic acid sequences. MPS I mice received the liposomal complexes by hydrodynamic injection and their immediate biodistribution was detected mainly in the lung, heart, and liver. An increase of about 6% in normal serum IDUA activity was maintained for six months, in addition to increased lung, heart, liver, and kidney activity after euthanasia. The enhanced enzymatic activity promoted a significant GAGs reduction in urine and in the same tissues, corroborating with histological analysis. In an ongoing study, a deeper investigation was carried out on the effect of liposomal treatment on bone morphology, cardiovascular and respiratory systems, and brain function. The echocardiographic analysis showed an improvement in the parameters of hypertrophy and contractility of the heart, but there was no improvement in heart valves. Aorta diameter was similar to that of normal animals, but elastin breaks were between the normal and untreated groups. Facial morphology of treated animals was intermediate, as well as the analysis of zygomatic bone thickness. However, femoral bone showed thickness comparable to normal animals. Lung resistance, on the other hand, showed a tendency to reduction in treated animals when compared to MPS I. The set of results demonstrates the potential of the co-complexed lipid nanostructures with the CRISPR/Cas9 plasmid and a donor vector of the IDUA/Idua sequence for MPS I gene therapy.

During normal cell development, many genes are activated and repressed, usually through epigenetic mechanisms. These are modifications of the DNA and protein within the nucleus that result in changes in gene expression without alteration in DNA sequence. Key proteins for epigenetic modifications are the histone proteins bound to DNA in the nucleus. The best-characterised epigenetic complexes that modify histone proteins are the polycomb group proteins (PcG), comprising polycomb repressive complexes 1 (PRC1) and 2 (PRC2). The repressive modifications generated by these complexes can be removed, and the blocked genes reactivated, by enzymes that are the subject of this project. PRC1 repressive marks are removed by deubiquitinases USP12, USP16 and BAP1, whereas PRC2 marks are removed by demethylases KDM6A, KDM6B and potentially UTY. During the development of cancer, the regulation of many genes becomes abnormal, allowing the cells to escape normal growth restrictions. In this thesis, the expression of this set of chromatin-modifying enzymes in a leukaemia cell line was investigated. The FANTOM consortium has been helping to understand patterns of gene expression for over 10 years. The FANTOM4 dataset described changes in gene expression and promoter usage during differentiation of the THP-1 acute monocytic leukaemia cell line, using CAGE (Cap Analysis of Gene Expression) technology. This human monocyte-like cancer cell line can be stimulated with phorbol esters to halt proliferation and differentiate into macrophages. However, the FANTOM4 time course did not capture detailed mechanisms of regulatory factors in macrophage differentiation due to sparse time points and low read coverage. The main aim of this project was therefore to repeat the time course with tighter time points and deeper sequencing of the transcriptome to develop a very precise picture of sequential activation of gene expression, transcription start site (TSS) usage and the activity of enhancers during the transition from proliferating monocytes to differentiated macrophage phenotype of the THP-1 leukaemia cell line, using CAGE. The focus of this research was on the chromatin-modifying enzymes, but other key cell cycle and macrophage genes have also been examined. The differentiation time course was repeated in triplicate. RNA was extracted and CAGE libraries generated for 18 time points, including the 6 originally studied in FANTOM4. Sequencing results were analysed and normalised using bioinformatics tools. It was shown that analysing 8 samples on one Illumina HiSeq 2500 lane yielded enough read coverage to detect activity from even low expression TSSs, such as those associated with enhancer activity. Clusters of genes which were up- and downregulated at different time points during the differentiation process were identified and characterised. CAGE results for key genes encoding chromatin modifying enzymes and macrophage markers were validated by qRT-PCR. There was a rapid increase of histone demethylase KDM6B mRNA once differentiation was initiated. Histone deubiquitinase USP12 mRNA was also upregulated early in the process. Histone deubiquitinase BAP1 mRNA shows an interesting cyclic regulation pattern which was not seen in the more limited samples of FANTOM4. These interesting chromatin-modifying enzymes and their close paralogues (deubiquitinases USP12, USP16 and BAP1, together with demethylases KDM6A, KDM6B and UTY) were investigated by bioinformatics and genetic tools. USP16 knockout THP-1 cell line was successfully created using CRISPR-Cas9 and its ability to differentiate into macrophages was examined using cell cycle analysis and CAGE sequencing. The USP16 knockout cell line, along with siRNA knock downs of USP12, USP16 and BAP1, was also compared to wildtype THP-1 differentiation using CAGE. Unfortunately, creating other mutant THP-1 cell lines was unsuccessful due to low THP-1 viability after single cell sorting. Investigating KDM6A, KDM6B and UTY using bioinformatics showed that UTY and KDM6A gene expression is positively correlated and this is disrupted in cancer samples. Gene expression and sequence comparison suggested that KDM6A and UTY are coregulated and may act in a similar way in histone demethylation. In summary, the results in this thesis show the transcriptomic changes as the leukaemia cell line ceases proliferation and commences differentiation. Detailed examination suggests that histone modifications are important in the transition between proliferation and differentiation and provide better understanding of regulatory factors in macrophage differentiation and leukaemia.

Ten-Eleven-Translocation (TET) proteins form a family of enzymes responsible for active DNA demethylation by oxidation of 5-methylcytosine. TET proteins play a key role in genomic reprogramming in vitro and in vivo. Although TET proteins are expressed in embryonic stem cells (ESCs), their role in regulating pluripotency remains unclear. In addition, the mechanisms by which TET proteins are recruited to chromatin are largely unknown. To visualise TET protein dynamics during pluripotency and differentiation, the endogenous Tet1/2/3 alleles were fused to epitope tags in ESCs using CRISPR/Cas9. Characterisation of these cell lines showed that TET1 is the highest expressed TET protein in both naïve and primed pluripotent cells. In contrast, TET2 is expressed heterogeneously in ESCs and marks cells with a high self-renewal capacity. To assess the function of Tet genes in pluripotent stem cells, the endogenous Tet1/2/3 ORFs were removed using CRISPR/Cas9. Comparative analysis of single and combined Tet gene knockout ESC lines indicated that Tet1 and Tet2, but not Tet3, play redundant roles to promote loss of pluripotency. Furthermore, Tet-deficient cells retained a naïve morphology in differentiating conditions, suggestive of a LIF-independent self-renewal phenotype. To characterise physiological TET1 protein-protein interactions, TET1 protein partners were identified in ESCs by mass spectrometry and co-immuno-precipitations. This revealed that TET1 interacts with multiple epigenetic and pluripotency-related factors in ESCs. Moreover, detailed characterisation of the interaction between TET1 and NANOG identified three regions of TET1 involved in protein-protein interactions that are conserved in evolution. To investigate TET1 chromatin binding in ESCs, both at the molecular and cellular levels, TET1 was characterised by ChIP-seq analysis and live imaging experiments. Interestingly, TET1 is targeted to chromatin by two different mechanisms, involving distinct protein regions. The interaction with multiple protein partners, including NANOG, might enable TET1 to be targeted to specific chromosomal locations. Additionally, TET1 has the unusual ability to bind mitotic chromatin through its N-terminus, independently of its interaction with NANOG. Together these analyses provide a new understanding of the role of TET proteins in pluripotent cells, as well as a detailed map of TET1 residues involved in protein-protein interactions and mitotic chromatin binding.

Osteosarcoma is the most common primary malignancy of bone. 5-year survival has remained stable at around 60-70% for 40 years. However, a number of patients will suffer from recurrent and/or metastatic disease representing a large unmet clinical need. CSPG4 is a transmembrane protein which is expressed on a number of progenitor cells and tumour types. Preliminary work had found CSPG4 present in osteosarcoma tumour samples. In this study, CSPG4 mRNA and protein expression was demonstrated in clinical samples and model cell lines. CSPG4 mRNA is overexpressed in osteosarcoma samples compared to mature osteoblast cells, the putative cell of origin for osteosarcoma. In a cohort of patients, CSPG4 protein expression was found on 86% of samples. Furthermore, CSPG4 expression was demonstrated in U2OS, MG63, HOS, HOS-MNNG and 143B osteosarcoma cell lines. CSPG4 protein expression was successfully deleted in 143B cells using CRISPR/Cas9 technology. Two stable CSPG4-negative cell lines were produced. CSPG4 expression was then reintroduced into negative cell lines, as well as the parental 143B cell line. This created a panel of 6 cell lines with differing CSPG4 expression. Furthermore, siRNA treatment of U2OS, MG63, 143B and U87MG cell lines reduced CSPG4 expression. These cells provided another panel with varying CSPG4 expression for in vitro investigation. In vitro experiments failed to demonstrate a role for CSPG4 in osteosarcoma tumorigenesis. The CRISPR/Cas9 cell panel found that CSPG4 expression did not influence cell proliferation, adhesion and spreading on fibronectin or collagen-I, cell migration, chemosensitivity or anchorage-independent growth. Similarly, the siRNA cell panel found that CSPG4 expression did not influence cell proliferation or anchorage-independent growth. In vivo experimentation did not demonstrate a role for CSPG4 in mediating osteosarcoma tumour growth or metastatic spread. Treatment with a sc-Fv antibody fragment failed to demonstrate specific toxicity of CSPG4-positive cell lines. These results indicate that CSPG4 plays no role in osteosarcoma tumour cell behaviour. However, due to its wide expression pattern it represents a viable therapeutic option for drug targeting.

abstract: The research question explored in this thesis is how CRISPR mediated editing is influenced by artificially opened chromatin in cells. Closed chromatin poses a barrier to Cas9 binding and editing at target genes. Synthetic pioneer factors (PFs) are a promising new approach to artificially open condensed heterochromatin allowing greater access of target DNA to Cas9. The Haynes lab has constructed fusions of enzymatic chromatin-modifying domains designed to remodel chromatin and increase Cas9 editing efficiency. With a library of PFs available, this research focuses on analyzing the behavior of Cas9 in chromatin that has been artificially opened by PFs. The types and frequency of INDELs (insertions & deletions) were determined after non-homologous end joining (NHEJ) in PF and Cas9-treated cells using quantitative Sanger sequencing and Synthego’s ICE software. Furthermore, NOME-seq analysis was carried out to map nucleosome position in PF and Cas9 treated cells. Although this experiment was unsuccessful, the heat map generated with data obtained from Synthego ICE predicts a possible presence of nucleosome in the vicinity suggesting that perhaps a fully open chromatin state was not achieved. Linear Regression analysis with certain assumptions confirms that with the increase in distance downstream of cut-site, the editing frequency decreases exponentially. Nevertheless, further experimental work should be carried out to investigate this hypothesis. / Dissertation/Thesis / Masters Thesis Biomedical Engineering 2019

Proteoglycans (PGs) are composed of highly sulfated glycosaminoglycans chains (GAGs) attached to specific core proteins. They are present in extracellular matrices, on the cell surface and in storage granules of hematopoietic cells. Heparan sulfate (HS) and chondroitin/dermatan sulfate (CS/DS) GAGs play indispensable roles in a wide range of biological processes, where they can serve as protein carriers, be involved in growth factor or morphogen gradient formation and act as co-receptors in signaling processes. Protein binding abilities of GAGs are believed to be predominantly dependent on the arrangement of the sugar modifications, sulfation and epimerization, into specific oligosaccharide sequences. Although the process of HS and CS/DS assembly and modification is not fully understood, a set of GAG biosynthetic enzymes have been fairly well studied and several mutations in genes encoding for this Golgi machinery have been linked to human genetic disorders. This thesis focuses on the zebrafish N-deacetylase/N-sulfotransferase gene family, encoding key enzymes in HS chain modification, as well as glycosyltransferases responsible for chondroitin/dermatan sulfate elongation present in zebrafish. Our data illustrates the strict spatio-temporal expression of both the NDST enzymes (Paper I) and CS/DS glycosyltransferases (Paper II) in the developing zebrafish embryo. In Paper III we took advantage of the four preexisting zebrafish mutants with defective GAG biosynthesis. We could demonstrate a relation between HS content and the severity of the pectoral fin defects, and additionally correlate impaired HS biosynthesis with altered chondrocyte intercalation. Interestingly, altered CS biosynthesis resulted in loss of the chondrocyte extracellular matrix. One of the main findings was the demonstration of the ratio between the HS biosynthesis enzyme Extl3 and the Csgalnact1/Csgalnact2 proteins, as a main factor influencing the HS/CS ratio. In Paper IV we used the newly developed CRISPR/Cas9 technique to create a collection of zebrafish mutants with defective GAG biosynthetic machineries. Lack of phenotypes linked to null-mutations of most of the investigated genes is striking in this study.

Asthma is a major public health epidemic that presents a heavy burden on those who suffer from the disease. Little is currently understood about the genetic signature that distinguishes one type of asthma from another. Recently, the single nucleotide polymorphism (SNP) rs968567 was found to have a high degree of association in asthmatic patients (Sharma et al., 2014). This particular SNP is in the promoter region of the FADS2 gene that synthesizes the enzyme delta-6-desaturase (D6D). D6D mediates the formation of pro-inflammatory factors that lead to exacerbation of asthmatic symptoms. We engineered a novel, customized CRISPR-Cas9 construct to induce the SNP rs968567 in the HUES9 human embryonic stem cell (hESC) line. Our results show success in generating the custom CRISPR-Cas9 construct for use in stem cells, while efficiency in expressing the desired mutation in our cell line is currently being optimized. Disease modeling in the genomic era of medicine provides an opportunity for the development of personalized medical treatment. Future projects aim to differentiate stem cell lines edited with our CRISPR-Cas9 construct to lung progenitor cells to study the cellular phenotype of this mutation in context of asthma pathogenesis.

In this study, for the first time we used CRISPR/Cas9 gene editing technology in sturgeons i.e., sterlets (Acipenser ruthenus). The sequences of sgRNA and primers were designed based on published dnd1 sterlet sequence. Each pair of sgRNA oligos after ligation ready duplex DNA fragment was cloned into vector pX330-U6-Chimeric_BB-CBh-hSpCas9 backbone and thereafter the transformation to competent cells Escherichia coli DH5 was done. The plasmid carried sgRNA was extracted for downstream applications. We diluted extracted plasmid with 10% of 2 M KCl and injection into animal pole of fertilized eggs of sterlets at one to four-cell-stage, 4 hours post fertilization (hpf). At the same time, second microinjection with 2.5% FITC-biotin-dextrans was injected into vegetal pole for labelling PGCs. In the control groups, the eggs were only injected by 2.5% FITC into vegetal pole. PGCs of sterlet were visualized and photographed using a uorescent stereo microscope Leica M165 FC. To confirm the presence or deletion/insertion occurring in the target gene, we used MCE-202 MultiNA microchip electrophoresis system for DNA analysis, in which the targeted gene after amplifying by PCR was analyzed. Mutations in both treated and control embryos of sterlet were further assessed by Sanger sequencing of the PCR product. In present study, we successfully established basic protocols such as preparation of competent cells, construction of vector carrying sgRNA and its transformation into competent cells to carry out the CRISPR/Cas9 technology in sturgeons. Less number of PGCs was observed in embryos that were treated with CRISPR/Cas9; however, sequencing did not provide us a reliable evidence for mutation of the targeted gene probably due to an unspecific PCR. Therefore, more authentication of dnd1 knockout should be done in future by more specific PCR and repeated sequencing.