A anemia aplástica (AA) é uma doença hematológica rara caracterizada pela hipocelularidade da medula óssea, o que provoca pancitopenia. Esta pode ser de origem genética (associada a encurtamento telomérico) ou adquirida (não-associada a desgaste excessivo dos telômeros). Na forma adquirida, a ativação anormal de linfócitos T provoca a destruição das células hematopoéticas. O mecanismo que leva a essa destruição ainda não foi elucidado. Um dos tratamentos mais eficazes para repovoar a medula óssea hipocelular é o transplante com célulastronco hematopoéticas (CTHs). Porém, uma grande porcentagem de pacientes não se beneficia de nenhum tratamento, fazendo-se necessário o desenvolvimento de novas alternativas para terapia. A geração de células-tronco pluripotentes induzidas (iPSCs) a partir de células somáticas (reprogramação) representa uma ferramenta promissora para o estudo de doenças e para o desenvolvimento de possíveis terapias paciente-especificas, como transplantes autólogos. Neste trabalho, avaliamos a capacidade de reprogramação de fibroblastos e eritroblastos de pacientes com AA adquirida. Metodologias de reprogramação utilizando lentivírus ou plasmídeos epissomais não integrativos foram testadas em células de quatro pacientes e de um controle saudável. Eritroblastos dos quatro pacientes e do controle foram reprogramados utilizando os plasmídeos não integrativos. As iPSCs geradas apresentaram-se similares a células-tronco embrionárias quanto à morfologia, expressão dos marcadores de pluripotência OCT4, SOX2, NANOG, SSEA-4, Tra-1-60 e Tra-1-81, e capacidade de diferenciação in vitro em corpos embrioides (EBs). A dinâmica telomérica das células pré- e pós-reprogramação foi avaliada em diferentes passagens utilizando a técnica de flow-FISH. O comprimento telomérico foi aumentado nas iPSCs quando comparado às células parentais o que indica que a célula foi completamente reprogramada. No presente trabalho, células de pacientes com AA adquirida foram reprogramadas a um estado de pluripotência por meio de um método não integrativo. As iPSCs geradas serão essenciais para futuros ensaios de diferenciação hematopoética, o que poderá contribuir para o entendimento dos mecanismos envolvidos no desenvolvimento dessa doença. Além disso, a diferenciação dessas células livres de transgenes poderá servir como uma alternativa terapêutica para os pacientes com AA como, por exemplo, em transplantes autólogos / Aplastic anemia (AA) is a rare hematological disease characterized by bone marrow hypocellularity that leads to pancytopenia. Its origin can be genetic (associated with telomere shortening) or acquired (non-associated with telomere shortening). The acquired form exhibit T lymphocytes abnormal activation, which leads to hematopoietic cells destruction. The mechanisms behind this phenomenon are still unclear. One of the most effective treatments for hypocelullar bone marrow repopulation is hematopoietic stem cell (HSCs) transplantation. However, a large percentage of patients do not benefit from any of the available treatments. This highlights the need to develop new therapeutic strategies. The generation of induced pluripotent stem cells (iPSCs) from somatic cells (reprogramming) represents a powerful tool for disease modeling and for the development of patient-specific therapies such as autologous transplants. In this study, we evaluate the capacity of reprogramming acquired AA patients\' fibroblasts and erythroblasts. Reprogramming methods using lentivirus or non-integrative episomal plasmids were tested in four patients\' cells and in cells from one healthy donor. Erythroblasts from these four patients and healthy donor were reprogrammed using non-integrative plasmids. The iPSCs resembled human embryonic stem cells in morphology, in the expression of pluripotent markers such as OCT4, SOX2, NANOG, SSEA-4, Tra-1-60 and Tra-1-81, and in in vitro differentiation (capacity to form embryoid bodies). The telomere dynamics of the cells before and after reprogramming was assessed along passaging using flow-FISH. The telomere length in the iPSCs was increased when compared to the parental cells. Thus, acquire AA patients\' cells could be reprogrammed to a pluripotent state by a nonintegrative method. The iPSCs will be essential for future hematopoietic differentiation assays that could contribute to the understanding of the mechanisms involved in the disease development. Furthermore, the differentiation of transgene-free cells may serve as an alternative therapy for patients with AA such as autologous transplants

Amyotrophic lateral sclerosis (ALS) is a late onset neurodegenerative disorder characterised by selective loss of upper and lower motor neurons (MNs). Recently, the GGGGCC (G4C2) hexanucleotide repeat expansion in chromosome 9 open reading frame 72 (C9orf72) has been identified as the most common genetic cause of ALS, highlighting the importance of studying the pathogenic mechanisms underlying this mutation. Accumulating evidence implicates that ALS is a multisystem and multifactor disease. Specifically, non-neuronal cells, astrocytes in particular, are also affected by toxicity mediated by ALS-related mutations, and they can contribute to neurodegeneration, suggesting astrocytes as a key player in ALS pathogenesis. Here, a human induced pluripotent stem cells (iPSCs)-based in vitro model of ALS was established to investigate the impact of the C9orf72 mutation on astrocyte behaviour—both cell- and non-cell autonomous. Work in this study shows that patient iPSC-derived astrocytes recapitulate key pathological features associated with C9orf72-mediated ALS, such as formation of G4C2 repeat RNA foci, production of dipeptide repeat (DPR) proteins and reduced viability under basal conditions compared to controls. Moreover, C9orf72 mutant astrocytes in co-culture result in reduced viability and structural defects of human MNs. Importantly, correction of the G4C2 repeat expansion in mutant astrocytes through targeted gene editing reverses these phenotypes, strongly confirming that the C9orf72 mutation is responsible for the observed findings. Altogether, this iPSC-based in vitro model provides a valuable platform to gain better understandings of ALS pathophysiology and can be used for future exploration of potential therapeutic drugs.

Chemical modification of the cytosine base via the addition of a methyl group to form 5-­‐methylcytosine (5-­‐mC) is a well-­‐studied example of an epigenetic mark, which contributes to regulation of gene expression, chromatin organisation and other such cellular processes without affecting the underlying DNA sequence. In recent years it was shown that 5-­‐mC is not the only DNA modification found within the vertebrate genome. 5-­‐hydroxymethylcytosine (5-­‐hmC) was first described in 1952 although it wasn’t until 2009 when it was rediscovered in mammalian tissues that it sparked intense interest in the field. Research has found that unlike the 5-­‐mC base from which it is derived, 5-­‐hmC displays variable levels and patterns across a multitude of tissue and cell types. As such the patterns of these DNA modifications can act as an identifier of cell state. This thesis aims to characterize the methyl and hydroxymethyl profiles of induced pluripotent stem cells (iPSCs), derived from control mouse embryonic fibroblast cell line (p53-­‐/-­‐) as well as and methylation hypomorphic (p53-­‐/-­‐, Dnmt1 -­‐/-­‐) mutant cell lines. As such both somatic cells were subject to reprogramming with Yamanaka factors (Oct4, cMyc, Klf4 and Sox2) via the piggyback transposition technique. Successful reprogramming was confirmed by a number of techniques and outcomes, including the de novo expression of a number of key pluripotency related factors (Nanog, Sall4 and Gdf3). Reprogrammed cells were then analysed for transcriptomic changes as well as alterations to their methyl and hydroxymethyl landscapes that accompany reprogramming. Through this work I have shown that the reprogramming of MEF derived cell lines results in a global increase in 5-­‐hmC for both p53-­‐/-­‐ and (p53-­‐/-­‐, Dnmt1 -­‐/-­‐) hypomorphic mutant cell lines – possibly through the reactivation of an alternative form of DNMT1. I demonstrate by both antibody based dot blot assay and genome wide sequencing that the reprogramming of the (p53-­‐/-­‐, Dnmt1 -­‐/-­‐) somatic cells towards a pluripotent state brings about an increase in methylation levels within the cells. This latter observation may indicate that the reprogramming of the cells is driving them towards a more wild type phenotypic state. My studies suggest that lack of DNMT1 function is not a barrier to reprogramming of somatic cells.

Parkinson's disease (PD) primarily manifests as loss of motor control through the degeneration of nigrostriatal dopaminergic neurons. The microtubule-associated protein tau (MAPT) locus is highly genetically associated with PD, wherein the H1 haplotype confers disease risk and the H2 haplotype is protective. As this haplotype variation does not alter the amino acid sequence, disease risk may be conferred by altered gene expression, either of total MAPT or of specific isoforms, of which there are six in adult human brain. To investigate haplotype-specific control of MAPT expression in the neurons that die in PD, induced pluripotent stem cells (iPSCs) from H1/H2 heterozygous individuals were differentiated into dopaminergic neuronal cultures that expressed all six mature isoforms of MAPT after six months' maturation. A reporter construct using the human tyrosine hydroxylase locus was also generated to identify human dopaminergic neurons in mixed cultures. Haplotype-specific differences in the inclusion of exon 3 and total MAPT were observed in iPSC-derived dopaminergic neuronal cultures and a novel variant in MAPT intron 10 increased the inclusion of exon 10 by two-fold. RNA interference tools were generated to knockdown total MAPT or specific isoforms, wherein knockdown of the 4-repeat isoform of tau protein increased the velocity of axonal transport in iPSC-derived neurons. MAPT knockdown also reduced p62 levels, suggesting an impact of tau on macroautophagy, likely through altered axonal transport. These results demonstrate how variation at a disease susceptibility locus can alter gene expression, thereby impacting on neuronal function.

Submitted by Caroline Periotto (carol@ufscar.br) on 2016-09-12T14:10:40Z No. of bitstreams: 1 DissCHM.pdf: 2847929 bytes, checksum: db6163924f9983d42120de5673f3df0a (MD5) / Approved for entry into archive by Marina Freitas (marinapf@ufscar.br) on 2016-09-13T14:25:07Z (GMT) No. of bitstreams: 1 DissCHM.pdf: 2847929 bytes, checksum: db6163924f9983d42120de5673f3df0a (MD5) / Approved for entry into archive by Marina Freitas (marinapf@ufscar.br) on 2016-09-13T14:25:18Z (GMT) No. of bitstreams: 1 DissCHM.pdf: 2847929 bytes, checksum: db6163924f9983d42120de5673f3df0a (MD5) / Made available in DSpace on 2016-09-13T14:25:25Z (GMT). No. of bitstreams: 1 DissCHM.pdf: 2847929 bytes, checksum: db6163924f9983d42120de5673f3df0a (MD5) Previous issue date: 2015-11-30 / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Mitochondrial dysfunctions caused by mutations in the mitochondrial DNA (mtDNA) represent an important group of human pathologies. However, it is not possible to predict with accuracy the risk of a woman with mutant mtDNA to transmit her pathology to her descendants. This is mainly due to out limited understanding of the molecular basis of mitochondrial inheritance. Since development of a technology that enabled derivation of induced pluripotent stem cells (iPSCs) from in vitro culture of somatic cells, iPSCs have become an interesting model to study mitochondrial inheritance. Derivation of iPSCs from patients with pathogenic mtDNA mutations has revealed that the mutant load decreases through in vitro culture of iPSCs, suggesting the existence of a specific mechanism that eliminates mutant mtDNA in the germ line. Thus, the aim of this work was to use iPSCs derived from patients with mitochondrial disorders to investigate the existence of a mechanism that eliminates mtDNA molecules with pathogenic mutations. In this way, we used heteroplasmic fibroblasts harboring a point mutation A3243G in mtDNA causing mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes (MELAS); heteroplasmic fibroblasts harboring a deletion in mtDNA causing Kearn-Sayre Syndrome (KSS) and homoplasmic fibroblasts containing only wild-type mtDNA (Control). The KSS lineage derivation resulted in iPSCs with low levels of mutant mtDNA (<0,1%), and the elimination of mutant molecules during the culture. The MELAS derivation resulted in iPSCs with high levels of mutant mtDNA (> 98%), and indication of mutant molecules elimination as well. However, unexpectedly, there was no reduction of mtDNA content in iPSCs compared to fibroblasts in all lineages. On contrary, mtDNA copy number increased in MELAS and KSS iPSCs, perhaps due to the high levels of mutations in the cells. No effect of Rapamycin (mitophagy inductor) treatment was detected on the yield of colony formation in MELAS iPSCs. Additionally, Rapamycin did not affect the mutation levels in MELAS iPSCS compared to untreated iPSCs. Finally, gene expression analysis of MELAS iPSCs provided evidences of an autophagic mechanism directed towards the mitochondrion. / Disfunções mitocondriais causadas por mutações no DNA mitocondrial (mtDNA) representam um importante grupo de patologias humanas. No entanto, não é possível predizer com acurácia o risco de uma mulher acometida por uma mutação no mtDNA transmitir a patologia para seus descendentes. Isso se deve, em parte, ao desconhecimento dos mecanismos moleculares que controlam a herança mitocondrial. Com o desenvolvimento de metodologias que possibilitam a derivação de células pluripotentes induzidas (iPSCs) a partir de células somáticas cultivadas in vitro, as iPSCs se tornaram um interessante modelo para o estudo da herança mitocondrial. A derivação de iPSCs de pacientes com mutações patogênicas no mtDNA tem revelado que a porcentagem de moléculas mutantes diminui ao longo do cultivo, sugerindo a existência na linhagem germinativa de mecanismos específicos para eliminação de mtDNAs mutantes. Portanto, o presente trabalho investigou em iPSCs derivadas de pacientes com desordens mitocondriais a existência de um mecanismo celular que elimina as moléculas de mtDNA com mutações patogênicas. Para tanto, foram utilizados fibroblastos heteroplásmicos portadores da mutação pontual A3243G no mtDNA causadora de encefalomiopatia mitocondrial, acidose lática e episódios tipo acidente vascular cerebral (MELAS); fibroblastos heteroplásmicos portadores de uma deleção de 4,9 kb no mtDNA causadora da Síndrome de Kearns-Sayre (KSS) e fibroblastos Controle, contendo apenas mtDNA selvagem. A derivação de linhagens portadoras de KSS resultou em iPSCs com baixos níveis de mtDNA mutante (< 0,1%), e na eliminação de moléculas mutantes ao longo do cultivo. A derivação de linhagens portadoras de MELAS resultou em iPSCs com alta taxa de mutação (> 98%), também com indícios de diminuição da quantidade de moléculas mutantes ao longo do cultivo. No entanto, ao contrário do esperado, não houve diminuição da quantidade de cópias de mtDNA nas iPSCs em relação aos fibroblastos em todas as linhagens (Controle, KSS e MELAS), sendo que as iPSCs de MELAS e KSS apresentaram um aumento significativo na quantidade de cópias de mtDNA, provavelmente devido a efeitos causados pela mutação no mtDNA. Ao analisar o efeito do tratamento com Rapamicina (indutor de mitofagia) durante a derivação de MELAS não observamos aumento na eficiência de formação de colônias, além de o tratamento não afetar a quantidade de mtDNA mutante, resultando em iPSCs com níveis de mutação similares aos encontrados nas iPSC MELAS não tratadas com o rapamicina. Por fim, resultados de expressão gênica das iPSCs do grupo MELAS revelaram indícios de mecanismos autofágicos direcionados a mitocôndria provavelmente devido ao efeitos causados pela a alta taxa da mutação. / 2013/13869-5

A anemia aplástica (AA) é uma doença hematológica rara caracterizada pela hipocelularidade da medula óssea, o que provoca pancitopenia. Esta pode ser de origem genética (associada a encurtamento telomérico) ou adquirida (não-associada a desgaste excessivo dos telômeros). Na forma adquirida, a ativação anormal de linfócitos T provoca a destruição das células hematopoéticas. O mecanismo que leva a essa destruição ainda não foi elucidado. Um dos tratamentos mais eficazes para repovoar a medula óssea hipocelular é o transplante com célulastronco hematopoéticas (CTHs). Porém, uma grande porcentagem de pacientes não se beneficia de nenhum tratamento, fazendo-se necessário o desenvolvimento de novas alternativas para terapia. A geração de células-tronco pluripotentes induzidas (iPSCs) a partir de células somáticas (reprogramação) representa uma ferramenta promissora para o estudo de doenças e para o desenvolvimento de possíveis terapias paciente-especificas, como transplantes autólogos. Neste trabalho, avaliamos a capacidade de reprogramação de fibroblastos e eritroblastos de pacientes com AA adquirida. Metodologias de reprogramação utilizando lentivírus ou plasmídeos epissomais não integrativos foram testadas em células de quatro pacientes e de um controle saudável. Eritroblastos dos quatro pacientes e do controle foram reprogramados utilizando os plasmídeos não integrativos. As iPSCs geradas apresentaram-se similares a células-tronco embrionárias quanto à morfologia, expressão dos marcadores de pluripotência OCT4, SOX2, NANOG, SSEA-4, Tra-1-60 e Tra-1-81, e capacidade de diferenciação in vitro em corpos embrioides (EBs). A dinâmica telomérica das células pré- e pós-reprogramação foi avaliada em diferentes passagens utilizando a técnica de flow-FISH. O comprimento telomérico foi aumentado nas iPSCs quando comparado às células parentais o que indica que a célula foi completamente reprogramada. No presente trabalho, células de pacientes com AA adquirida foram reprogramadas a um estado de pluripotência por meio de um método não integrativo. As iPSCs geradas serão essenciais para futuros ensaios de diferenciação hematopoética, o que poderá contribuir para o entendimento dos mecanismos envolvidos no desenvolvimento dessa doença. Além disso, a diferenciação dessas células livres de transgenes poderá servir como uma alternativa terapêutica para os pacientes com AA como, por exemplo, em transplantes autólogos / Aplastic anemia (AA) is a rare hematological disease characterized by bone marrow hypocellularity that leads to pancytopenia. Its origin can be genetic (associated with telomere shortening) or acquired (non-associated with telomere shortening). The acquired form exhibit T lymphocytes abnormal activation, which leads to hematopoietic cells destruction. The mechanisms behind this phenomenon are still unclear. One of the most effective treatments for hypocelullar bone marrow repopulation is hematopoietic stem cell (HSCs) transplantation. However, a large percentage of patients do not benefit from any of the available treatments. This highlights the need to develop new therapeutic strategies. The generation of induced pluripotent stem cells (iPSCs) from somatic cells (reprogramming) represents a powerful tool for disease modeling and for the development of patient-specific therapies such as autologous transplants. In this study, we evaluate the capacity of reprogramming acquired AA patients\' fibroblasts and erythroblasts. Reprogramming methods using lentivirus or non-integrative episomal plasmids were tested in four patients\' cells and in cells from one healthy donor. Erythroblasts from these four patients and healthy donor were reprogrammed using non-integrative plasmids. The iPSCs resembled human embryonic stem cells in morphology, in the expression of pluripotent markers such as OCT4, SOX2, NANOG, SSEA-4, Tra-1-60 and Tra-1-81, and in in vitro differentiation (capacity to form embryoid bodies). The telomere dynamics of the cells before and after reprogramming was assessed along passaging using flow-FISH. The telomere length in the iPSCs was increased when compared to the parental cells. Thus, acquire AA patients\' cells could be reprogrammed to a pluripotent state by a nonintegrative method. The iPSCs will be essential for future hematopoietic differentiation assays that could contribute to the understanding of the mechanisms involved in the disease development. Furthermore, the differentiation of transgene-free cells may serve as an alternative therapy for patients with AA such as autologous transplants

There is an unmet need to develop and test treatments for rare lysosomal disease (LD). Most LDs are present in childhood and do not currently have approved therapies. Rare diseases individually are uncommon but taken together account for a population prevalence of 3.5-5.9% worldwide. Due to their rarity, it often takes significant time and effort to diagnose rare diseases. New diagnostic tools, especially for early detection, will offer an advantage in avoiding this diagnostic odyssey. This dissertation is focused on investigating novel diagnostic and treatment methods in vitro for two neurodegenerative LDs: Gaucher disease (GD) and mucopolysaccharidosis IIIB (MPS IIIB). Mutations in NAGLU and GBA1, the genes that encode for lysosomal hydrolases required for degradation of heparan sulfate and glucocerebrosides, lead to the observed pathogenesis in MPS IIIB and GD, respectively. Since many LDs, including MPS IIIB and some forms of GD, are neurodegenerative, cell and gene-based therapeutic strategies are of significant interest. Therapeutics that offer some symptom mitigation in other LDs, such as enzyme replacement or substrate reduction therapies, do not offer appreciable disease mitigation in MPS IIIB or neurodegenerative GD. Here, a novel compound heterozygous mutation, NAGLUY140C/R297X, that results in approximately 50% residual NAGLU protein and 0.6% NAGLU enzyme activity is reported in NAGLU. Furthermore, a RFLP and site-directed mutagenesis strategy was developed to identify the presence of the relatively common p.R297X mutation in patient cell samples, in addition to two other novel molecular assays for the detection of the p.E153K mutation in NAGLU and p.N370S mutation in GBA1. MPS IIIB and GD human skin fibroblasts were reprogrammed to iPSCs using non-integrating Sendai viral vectors with a reprogramming efficiency of 0.2% and 0.3%, respectively. Resulting iPS cell lines were confirmed as being pluripotent through a barrage of analyses for markers of pluripotency and differentiation. Intriguingly, early passage MPS IIIB iPSCs were found to exhibit increased cell death and spontaneous differentiation to embryoid body-like structures, which was hypothesized to be caused by fibroblast growth factor 2 (FGF2) sequestration or degradation due to inherent heparan sulfate dysregulation. Supplemental FGF2 (100 ng/mL) was found to significantly increase confluency of MPS IIIB iPSCs after 48 hours (n = 5, p ≤ 0.05) and persisting to 96 hrs (n = 5, p ≤ 0.05), thus providing evidence for an important role of FGF2-heparan sulfate interactions in the maintenance of stem cell pluripotency. These findings highlight the importance of considering inherent disease pathology when developing disease models. Three genome editing strategies, CRISPR-Cas9, base and prime editing, are addressed throughout this dissertation. Genome editing outcomes in NAGLU and GBA1, as well as a control gene, HPRT1, are reported in HEK293 cells, human skin fibroblasts, and induced pluripotent stem cells (iPSCs). Although CRISPR-HDR failed to yield mutation correction, base editing of the common p.N370S (c.1226 A>G) in GD skin fibroblasts using with 42% efficiency is reported. Base editing of HPRT1 in HEK293 cells with an overall editing efficiency of 6 ± 0.5% (n = 3), but interestingly, when base editing at the centered nucleotide was analyzed, the editing efficiency increases to 27 ± 4.3% (n = 3). These findings align with other reports of a centered nucleotide preference for base editors and will help direct genome editing strategies in the future. This dissertation describes the first genome editing in NAGLU, and the first base editing in GBA1, and underscores the importance of optimizing genome editing strategies when targeting disease-causing mutations in patient-derived cells. The findings reported here will direct future genome editing strategies for developing cell and gene-based therapies for MPS IIIB and GD. / Graduate / 2021-12-15

The repetitive sequence composes nearly half of human and mouse genome, most of which are scattered repeats of transposable elements (TEs). The non-LTR retrotransposons are the most accumulated TEs in the mammalian genome and L1s are the most active and abundant autonomous retrotransposons. L1s are highly activated during the epigenetic reprogramming of early mammalian embryos and have the highest level of expression among all retrotransposons throughout the preimplantation state. Moreover, the reprogramming of somatic cells into iPSCs is associated with an increase in L1 expression. The transcription of L1 during the early embryogenesis is necessary to regulate developmental genes and prevent heterochromatin formation to maintain cellular pluripotency state, that guarantying an appropriate future differentiation. However, the role of L1 reactivation during the somatic cell reprogramming remains unclear. Therefore, aim of this work is to study the impact of L1 transcription during the reprogramming process of the iPSCs. We used CRISPR-mediated gene activation (CRISPRa) system that fuse a deactivated Cas9 (dCas9) with transactivation domains (VPR). We confirm the ability to overexpress L1 in Human Embryonic Kidney cells (HEK293) and Human Dermal Fibroblasts (HDFs) by utilizing CRISPR activation system and this will provide a good opportunity to study the role of L1 transcripts during the reprogramming of HDFs into iPSCs. Furthermore, we established stable HDFs that able to express combinations of “Yamanaka” reprogramming factors. The model system will allow to investigate the effect of overexpressing L1 with reprogramming factors to answer the question of whether L1 can trigger or facilitate the reprogramming processes and its underlying mechanism.

Klinefelter Syndrome (KS) is a multisystemic disorder associated with a plethora of phenotypic features including mental retardation, cardiac abnormalities, osteoporosis, infertility, gynecomastia, type two diabetes and increased cancer risk. KS is the most common aneuploidy in humans (with a prevalence of 1:500 to 1:1000 born males) and is characterized by one or more supernumerary X-chromosomes (47-XXY, 48-XXXY, and 49-XXXXY karyotypes). While X-chromosome inactivation (XCI) represses extra Xs, few genes called “escape genes” elude the XCI mechanism and are actively transcribed from X inactive. The overdosage of escape genes has been considered the molecular landscape that underlies KS clinical features. In this project, we exploit an integration-free reprogramming method to generate the largest described cohort of iPSCs from seven patients with KS and healthy donor fibroblasts from two relatives. The unicity of this cohort relies on the derivation of 47-XXY iPSCs and their isogenic 46-XY healthy counterparts, along with multiple rare 48-XXXY and 49-XXXXY iPSC lines. Through X chromosome inactivation (XCI) assessment, we show consistent retention of n-1 XCI in all derived KS-iPSCs. We identify the genes within the PAR1 region as the most susceptible to dosage-dependent transcriptional dysregulation and therefore putatively responsible for the progressively worsening phenotype in higher grade X aneuploidies. Moreover, we explore the transcriptional impact of X overdosage on autosomes and identify that the X-dosage-sensitive autosomal transcription factor NRF1 is a master regulator of the X-linked escape gene ZFX. Finally, we dissect the potential pathological impact of the escape gene KDM6A on low- and high-grade supernumerary X iPSCs and differentiated derivatives. We highlight a considerable proportion of KDM6A targets that could be responsible for paradigmatic clinical manifestations of KS.

Raman spectroscopy is a noninvasive technique that can provide complex information on the vibrational state of the molecules. It defines the unique fingerprint that allow the identification of the various chemical components within a given sample. The aim of the following thesis work is to analyze Raman maps related to three pairs of different cells, highlighting differences and similarities through multivariate algorithms. The first pair of analyzed cells are human embryonic stem cells (hESCs), while the other two pairs are induced pluripotent stem cells (iPSCs) derived from T lymphocytes and keratinocytes, respectively. Although two different multivariate techniques were employed, ie Principal Component Analysis and Cluster Analysis, the same results were achieved: the iPSCs derived from T-lymphocytes show a higher content of genetic material both compared with the iPSCs derived from keratinocytes and the hESCs . On the other side, equally evident, was that iPS cells derived from keratinocytes assume a molecular distribution very similar to hESCs.