O carcinoma de cabeça e pescoço (HNSCC) é um dos tipos de câncer que mais acomete as pessoas ao redor do mundo, sendo responsável por 300 mil mortes anualmente. Essa alta taxa de mortalidade está diretamente associada ao diagnóstico tardio, ausência de biomarcadores e tratamento inespecífico. Diversos estudos mostraram que a expressão desregulada da classe de serino proteases do tipo II está intimamente relacionada com a etiologia de diversos tipos de carcinomas, como é o caso da via proteolítica da matriptase. Ainda, a matriptase, no contexto normal da descamação epitelial, ativa as KLKs 5 e 7, as quais são inibidas por LEKTI. A proteína LEKTI, codificada pelo gene SPINK5, possui 15 domínios diferentes, os quais são secretados de forma individual para a matriz extracelular, onde participa da inibição das KLKs 5 e 7. Resultados ainda não publicados do nosso laboratório mostraram que LEKTI é capaz de inibir o fenótipo pré-maligno causado pela superexpressão da matriptase na camada basal do epitélio de camundongos, quando superexpresso nessa mesma região. Ainda, marcações imunohistoquímicas de LEKTI, em amostras de carcinomas orais de humanos, mostraram que essa proteína está também presente nos carcinomas de cabeça e pescoço. Dessa forma, a hipótese levantada neste estudo é a de que LEKTI possui papel inibitório durante a carcinogênese de cabeça e pescoço. Sendo assim, para testar nossa hipótese, a presente dissertação buscou nocautear LEKTI, por meio da tecnologia de edição gênica CRISPR/Cas9, em linhagens de queratinócito imortalizado, através da dissecção por biologia celular, da função de LEKTI nestas células. Os resultados, obtidos por meio de Western Blot, imunofluorescência e sequenciamento das células potencialmente nocauteadas, mostram as dificuldades e desafios de nocautear células hipotetraplóides, como é o caso da linhagem celular utilizada neste estudo. / Head and neck squamous cell carcinoma (HNSCC) is one of the most common type of cancers around the world and it\'s responsible for 300.000 deaths every year. This high mortality rate is directly related with late diagnosis, lack of biomarkers and specific treatment. A fair amount of studies have shown that the deregulated expression of type II serine proteases, such as the matriptase proteolytic pathway, is intimately related with the etiology of a large number of carcinomas. Still, in a normal epithelial desquamation context, matriptase is able to activate KLKs 5 and 7, which are inhibited by LEKTI. The protein LEKTI, encoded by SPINK5 gene, has 15 different domains, which are secreted individually to the extracellular matrix, where acts inhibiting KLKs 5 and 7. Non published results from our laboratory has shown that LEKTI is able to inhibit the pre-malignant phenotype caused by the super-expression of matriptase in the epithelia\'s basal layer of mice, when super-expressed in the same region. Moreover, immunohistochemistry staining of LEKTI in human oral carcinomas showed that this protein is also present in head and neck carcinomas. In this way, the hypothesis of this study is that LEKTI has an inhibitory role during carcinogenesis of head and neck carcinomas. Therefore, to test our hypothesis, this dissertation aimed to knockout LEKTI in an immortalized keratinocyte cell line, using CRISPR/Cas9 editing technology, through cell biology dissection of LEKTI function in those cells. The results, obtained by Western Blot, immunofluorescence and sequencing of the cells, shows the difficulties and challenges to knockout cells that are hypo-tetraploids, just like HaCaT cell line.

The ground state naive pluripotency is established in the epiblast of the blastocyst and can be captured by culturing mouse embryonic stem cells (mESCs) with MEK and GSK3 inhibitors (2i). The transcription network that maintains pluripotency has been extensively studied with the indispensable core factors being Oct4, Sox2 and Nanog, together with other ancillary factors reinforcing the network. However, how this network is dissolved at the onset of differentiation is still not fully understood. To identify genes required for differentiation in an unbiased fashion, I conducted a genome-wide CRISPR-Cas9-mediated screen in Rex1GFPd2 mESCs. This cell line expresses GFP specifically in the naive state and rapidly down-regulate upon differentiation. I differentiated mutagenised mESCs for two days and sorted mutants that kept higher GFP expression. gRNA representation was subsequently analysed by sequencing. I identified 563 and 8 genes whose mutants showed delayed and accelerated differentiation, respectively, at a false discovery rate (FDR) cutoff of 10%. The majority of the previously known genes were identified in my screen, suggesting faithful representation of genes regulating differentiation. Detailed screening result analysis revealed a comprehensive picture of pathways involved in the dissolution of naive pluripotency. Amongst the genes identified are 19 mTORC1 regulators and components of the mTORC2 complex. Deficiency in the TSC and GATOR complexes resulted in mTORC1 upregulation in consistent with previous studies. However, they showed opposite phenotype during ESC differentiation: TSC complex knockout cells showed delayed differentiation, whereas GATOR1 deficiency accelerated differentiation I found that the pattern of GSK3b phosphorylation is highly correlated with differentiation phenotype. I conclude that mTORC1 is involved in pluripotency maintenance and differentiation through cross-talk with the Wnt signalling pathway. My screen has demonstrated the power of CRISPR-Cas9-mediated screen and provided further insights in biological pathways involved in regulating differentiation. It would be interesting to explore the remaining unstudied genes for better understanding of the mechanisms underlying mESC differentiation.

Approximately half of all adults diagnosed with acute leukemia (AL) relapse after standard chemotherapy, highlighting the need for alternative treatment options. We have previously shown that vaccination with irradiated autologous tumour cells infected with an oncolytic virus (OV) can elicit a durable, tumour specific, T cell- mediated response in a mouse model of AL. In the context of this AL infected cell vaccine (ICV) model, infection of autologous cells ex vivo with an OV is essential for stimulating a lasting immune response. While the murine AL line L1210 can be robustly infected with Maraba MG1, creating a potent infected cell culture, this ICV still has room for improvement as ICV-vaccinated mice with high tumour burden still die from leukemia. Therefore, we sought to utilize a genome wide CRISPR-Cas9 screen to identify genetic factors that mediate OV resistance in this model of AL. L1210 cells stably expressing Cas9 were transduced with the mouse GeCKOv2 library, which contains 130,209 gRNAs against 20,611 genes within the mouse genome. Following selection, cells were treated with Maraba MG1 and genomic DNA from resistant populations was sequenced to identify genes enriched in resistant cells relative to mock treated cells. Our screen identified several genes that mediate susceptibility to OV infection including those involved in viral entry (Ldlr), receptor-mediated endocytosis (Atp6v1g2), intracellular signaling (Cav1), the cytoskeleton (Filip1 and Tmod4), as well as autophagy and exosome production (Atg5). We aim to use the findings from this work to improve therapeutic efficacy in otherwise OV resistant tumour models as well as identify biomarkers, to determine the feasibility of administering an ICV using patient derived tumour cells.

Forward genetic screening allows for the identification of any genes important for a particular biological process or phenotype. While the power of this approach is broadly agreed on, the efficacy of currently available tools limits the strength of conclusions drawn from these experiments. This thesis describes a method to optimize molecular tools for high-throughput screening, both for shRNA and sgRNA based reagents. Using large shRNA efficacy datasets, we first designed an algorithm predicting the potency of shRNAs based on sequence determinants. Combined with a novel shRNA backbone that further improves the processing of synthetic shRNAs, we built a library of potent shRNAs to reliably and efficiently knock-down any gene in the human and mouse genomes. We then went on to apply a similar approach to identify sgRNAs with increased activity. We complemented this with conservation and repair prediction to increase the likelihood of generating functional knock-outs. With these tools in hand, we constructed, sequence-verified and validated arrayed shRNA and sgRNA libraries targeting any protein coding gene in the human genome. These resources allow large-scale screens to be performed in a multiplexed or arrayed format in a variety of biological contexts. I have also applied these tools to identify therapeutic targets to circumvent cancer resistance to treatment in two different contexts. To overcome the shortfalls of single target therapy, I have developed multiplexed multidimensional shRNA screening strategy, where two genes are knocked down simultaneously in each cell. This strategy allows the identification of gene pairs that could be targeted in tandem to maximize therapeutic benefits. As a proof of concept, I have used it with a subset of druggable genes in melanoma cell lines. Moreover, we have applied our genome wide shRNA libraries to a different resistance context, stroma-mediated resistance to gemcitabine in PDAC. In this project, we performed screens in a PDAC-CAF coculture setting to try and identify cancer vulnerabilities specifically in the presence of stroma. Overall, the tools developed in this thesis allow for the efficient knockdown or knockout of any gene, both in an individual or combinatorial setting. Apart from providing a resource that will be useful for many fields, we have performed several proof-of-concept studies where we have applied our tools to identify potential cancer drug targets.

Acute myeloid leukaemia (AML) is an aggressive cancer with a poor prognosis, for which mainstream treatments have not changed for decades. To identify novel therapeutic targets in AML, I have optimized a genome-wide clustered regularly interspaced short palindromic repeats (CRISPR) screening platform and use it to identify genetic vulnerabilities in AML cells. This work led to the identification of 492 AML-specific cell-essential genes, including several established therapeutic targets such as that represent new clinically actionable candidates. I have validated selected genes using DOT1L, BCL2, MEN1 and many other genes genetic and pharmacological inhibition, and chose candidates for downstream studies. Both the epigenetic modifier KAT2A and SRPK1 as promising KAT2A and spliceosome kinase SRPK1 inhibition demonstrated anti-AML activity by inducing myeloid differentiation and apoptosis, and suppressed the growth of primary human AMLs while sparing normal hemopoietic stem-progenitor cells. My findings propose that KAT2A and SRPK1 inhibition should be investigated as new therapeutic strategies in AML and also provide a large number of novel genetic vulnerabilities of this leukaemia that can be pursued in downstream studies. As these screens were performed in immortalised AML cell lines, I then went on to develop a method for the performance of dropout screens in genotypically-defined primary murine AMLs developed in our lab and arising in Cas9-expressing mice. Through this work, I successfully carried out the first such screen in AML cells driven by mutant Npm1 (NPM1c) and Flt3-ITD, the commonest two-mutation combination in human AML. Downstream analysis of the results revealed the excellent potential of this type of screen and enabled me to investigate the molecular effects on mutant Npm1, which are currently poorly understood. Overall, my results demonstrate that unbiased CRISPR dropout screens can identify novel therapeutic targets in cancer while, in parallel, revealing novel biological insights.

Master of Science / Department of Biomedical Sciences / Raymond R. R. Rowland / Porcine Reproductive and Respiratory Syndrome Virus (PRSSV) is one of the most economically important diseases in the global swine industry, costing producers an estimated $660 million annually. PRRSV is genetically diverse with a low replication fidelity, due to it being an RNA virus, resulting in multitudes of isolates being produced. This virus has a tropism for cells of the monocyte/macrophage lineage. Cluster of Differentiation 163 (CD163) is considered the primary PRRSV receptor located on porcine alveolar macrophages (PAMs). CRISPR/Cas9 technology was utilized to knock out CD163 via a frameshift mutation, resulting in pigs of the CD163 Null genotype. Also, a domain of porcine CD163 was deleted and replaced with the insertion of a CD163 homolog of human-like domain and neomycin cassette to serve as a genetic marker. This swap resulted in pigs that possessed a CD163L1 domain 8 mimic of porcine homolog human CD163-like (hCD163L-1) of SRCR domain 8. Previous work has demonstrated that CD163 Null pigs were resistant to one genotype 2 PRRSV isolate. An in vivo study was performed to observe whether hCD163L-1 pigs were also resistant to infection. Various diagnostic tests were performed to determine the presence or absence of PRRSV viremia levels in serum, CD163 receptor surface expression levels on PAMs, IgG antibody levels and haptoglobin (Hp) levels in serum. hCD163L-1 pigs did not support genotype 1 PRRSV replication, but were susceptible to genotype 2 PRRSV infections. In addition, in vitro infection experiments were performed on PAMs and macrophages derived from peripheral blood mononuclear cells (PBMCs) to determine resistance to multiple isolates. hCD163L-1 macrophages showed reduced infection with genotype 2 and no infection with genotype 1 PRRSV during in vitro infections. Null PAMs and PBMCs derived macrophages did not support infection towards any isolate of either PRRSV genotype.

Glioblastoma is the most common and most lethal primary brain tumor to affect adults. While current treatment options provide temporary recourse, the majority of patients experience tumor recurrence and few survive five years past their initial diagnosis. Recently, tumor microtubes (TMs) were identified in an in vivo model of glioblastoma. These membrane-bound structures formed physical connections between tumor cells, over short and long distances, and facilitated intratumoral communication, invasion, treatment resistance, and post-treatment tumor recovery. To date, this is the first instance of TMs being reported in glioblastoma. The lack of an in vitro model for these structures has delayed further characterization of how TMs form between cells, facilitate intercellular exchange, and how they can be therapeutically targeted to increase treatment susceptibility. The study presented here is the first instance of TMs characterized in an in vitro model of primary glioblastoma (PriGO) cells. These TMs recapitulated many of the structural and functional properties of those observed in vivo, making it a suitable model for further experimentation. Using this model, Rac1, a known orchestrator of cytoskeletal remodeling and motility, was shown to be integral to establishing a TM network between PriGO cells, as demonstrated by siRNA-mediated protein knockdowns. PREX1, a GEF necessary for Rac1 signaling activity, also played a role in PriGO TM formation as evidenced by CRISPR/Cas9-based knockouts. Re-introducing a PREX1 domain with Rac-GEF activity into cells lacking the protein led to a functional rescue of TM growth, thus confirming PREX1’s involvement. Characterizing a cell culture model of glioblastoma TMs is a necessary first step in the study of these structures, ultimately paving the way for future development of therapies that disrupt this network.

Viral diseases constitute a major threat to the agricultural production and food security throughout the world. Plants cope with the invading viruses by triggering immune responses and small RNA interference (RNAi) systems. In prokaryotes, CRISPR/Cas systems function as an adaptive immune system to provide bacteria with resistance against invading phages and conjugative plasmids. Interestingly, CRISPR/Cas9 system was shown to interfere with eukaryotic DNA viruses and confer resistance against plant DNA viruses. The majority of the plant viruses have RNA genomes. The aim of this study is to test the ability of the newly discovered CRISPR/Cas13a immune system, that targets and cleaves single stranded RNA (ssRNA) in prokaryotes, to provide resistance against RNA viruses in plants. Here, I employ the CRISPR/Cas13a system for molecular interference against Turnip Mosaic Virus (TuMV), a plant RNA virus. The results of this study established the CRISPR/Cas13a as a molecular interference machinery against RNA viruses in plants. Specifically, my data show that the CRISPR/Cas13a machinery is able to interfere with and degrade the TuMV (TuMV-GFP) RNA genome. In conclusion, these data indicate that the CRISPR/Cas13 systems can be employed for engineering interference and durable resistance against RNA viruses in diverse plant species.

Secondary metabolites play important roles in tree defense. Proanthocyanidins (PAs), one of the most common secondary metabolites, are widely distributed in trees and woody plants, and are abundant in poplar. In my research, molecular biology and biochemistry techniques were used to investigate the function of two important transcription factors, MYB115 and MYB134, in regulating the PA pathway in hybrid poplars. The importance of these transcription factors in regulating PA synthesis in leaves has recently emerged, but their roles in roots are not known. MYB134- and MYB115-overexpressing transgenic poplars showed a strong high-PA phenotype in leaves, but how these two regulators interact in vivo is still a mystery. This research aims to test the function of both MYBs in the regulation of PAs in poplar roots, and to explore the antimicrobial functions of root PAs. Both alleles of the MYB genes were sequenced in wild type poplars to design gRNAs for creating transgenic poplars with knocked-out (KO) MYB115 and MYB134 using the CRISPR Cas9 system. Both hairy root and whole plant transgenics with respective single- and double knock-outs were generated. Chemical and genetic characterization of both mutant types showed reduced PA content and down-regulated flavonoid genes in leaves. In poplar roots, only double-KOs showed a significant change in PA and salicinoid metabolism. These results indicated that the regulatory pathways for PA biosynthesis may differ in poplar leaves and roots. Significant PA concentrations remained in double-KO plants, suggesting other transcription factors for PA regulation are active. Because poplars accumulate large amounts of PAs in roots, potential functions of root tannins were also investigated. Antimicrobial activity of PAs was tested by disc inhibition assay in vitro and mycorrhizal co-culture sandwich assay in vivo. Pure PAs showed no inhibition towards the pathogenic fungi Armillaria ostoyae and A. sinapina but displayed slight inhibition to the mycorrhiza fungus Laccaria bicolor. These results provide preliminary insight into the functions of PAs in roots. / Graduate / 2023-04-24

Thesis advisor: Jia Niu / O-linked β-N-acetylglucosamine (O-GlcNAc) is a post-translational modification plays important roles in cellular network/diseases such as autophagy, transcriptional activity, protein stability, phosphorylation modification competition, cancers, neurodegenerative disorders, etc. However, there are still lots of unknown roles of it in gene regulation especially while there is existence of more than 1000 transcription factors and many other coactivators/repressors which might have influence in expression level with or without O-GlcNAc modification. In this project, we use CRISPR technology with catalytically inactive Cas9 from Streptococcus pyogenes fused with the only writer and eraser of O-GlcNAc to selectively target to the DNA sequence of interest by guide RNA to see what the result of additional modification or cleavage of O-GlcNAc on proximal proteins in gene regulation is. A cost and time efficient way for guide RNA construction is developed in the project with one-piece PCR and Gibson assembly for 2 different backbones of guide RNA: 11 guide RNA for each. Other different cloning methods have also been used for the future work. For the future work, second reporter will be introduced to normalize the luciferase signal. In addition, new metabolic O-GlcNAc reporter and Y289L GalT/ UDP-GalNAz could be used to find out proteins which might be modify by dCas9-OGT/OGA with click reaction. Furthermore, split OGA/Turbo ID system could also be used to reduce the background and find out proteins/modification sites which might be important to the gene regulation for DNA-protein or protein-protein interaction. / Thesis (MS) — Boston College, 2022. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.