Orientador: Maria Marluce dos Santos Vilela / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-24T17:02:47Z (GMT). No. of bitstreams: 1 Ramalho_VanessaDomingues_D.pdf: 1441004 bytes, checksum: a45d3f4f8825288e2959115ef25fc13a (MD5) Previous issue date: 2014 / Resumo: A agamaglobulinemia ligada ao X (XLA; OMIM#300755) é caracterizada por um bloqueio na diferenciação dos linfócitos B na medula óssea, levando à profunda hipogamaglobulinemia e reduzido número ou ausência de linfócitos B periféricos. Os pacientes são susceptíveis a infecções recorrentes por bactérias encapsuladas e enterovírus. XLA é causada por mutações no gene da tirosina quinase de Bruton (BTK). Contudo, não há estudos de relação entre expressão protéica e o tipo de mutação, nem sobre as conseqüências da retenção intracelular do excesso de proteínas mal formadas. Os objetivos deste trabalho foram avaliar a expressão de BTK e sua relação com o tipo de mutação em pacientes com XLA, assim como verificar suas conseqüências nos sensores de estresse do retículo endoplasmático. O diagnóstico de XLA foi baseado em infecções recorrentes, níveis significativamente reduzidos de IgM, IgG e IgA, linfócitos B circulantes <2% e mutação identificada no gene BTK. A expressão dos transcritos de BTK foi avaliada por PCR quantitativo em tempo real em oito pacientes XLA e oito controles. Pela mesma técnica, foi avaliada a expressão de 10 genes do estresse do retículo endoplasmático em seis pacientes e seis controles. Foram caracterizadas quatro mutações missense, uma mutação nonsense, dois frameshifts e um defeito em sítio de splicing. As mutações do tipo nonsense, frameshift e defeito em sítio de splicing levaram à formação de stop codon prematuro. Foi detectado um perfil de expressão de BTK diferenciado nos pacientes com mutações com stop codon prematuro em comparação aos pacientes com mutações missense e controles saudáveis. Especificamente, os pacientes com mutações com stop codon prematuro apresentaram redução da expressão de BTK (P = 0,004). No entanto, verificamos que as mutações missense não afetaram a expressão de BTK. Por meio de imunocitoquímica, encontramos que as mutações com stop codon prematuro levaram à deficiência da expressão da proteína BTK e as do tipo missense resultaram na localização anormal da proteína no citoplasma celular, o que evidencia a síntese de proteína não funcional. Os pacientes com XLA apresentaram expressão aumentada do marcador de estresse do retículo endoplasmático XBP1 (P = 0,002). Em conclusão, a quantificação da expressão de mRNA para BTK é uma ferramenta para diferenciar as conseqüências mutacionais em pacientes com XLA. Ela também pode contribuir para o estudo de transcritos em outras doenças genéticas com diferentes tipos de mutação. Este é o primeiro relato de estresse do retículo endoplasmático na agamaglobulinemia ligada ao X / Abstract: X-linked agammaglobulinemia (XLA, OMIM # 300755) is characterized by a block in differentiation of B lymphocytes in the bone marrow, leading to profound hypogammaglobulinemia and few or no peripheral B lymphocytes. Patients are susceptible to recurrent infections by encapsulated bacteria and enteroviruses. XLA is caused by mutations in the Bruton tyrosine kinase gene (BTK). However, there have been no studies on the relationship between protein expression and the type of mutation, nor on the consequences of the disruption of protein folding that results in intracellular retention. The objectives of this study were to evaluate BTK expression and its mutation type in patients with XLA, as well as to verify their consequences on the endoplasmic reticulum stress sensors. The XLA diagnosis was based on recurrent infections, significantly reduced levels of IgM, IgG and IgA, circulating B lymphocytes <2% and BTK gene mutation identified. The expression of BTK transcripts was assessed by quantitative real-time PCR in eight XLA patients and eight control subjects. By the same technique, the expression of 10 endoplasmic reticulum stress genes was measured in six patients and six controls. Four missense mutations, one nonsense mutation, two frameshifts and a splice site defect were characterized. Mutations of the nonsense type, frameshift and splice site defect led to a premature stop codon formation. A differential profile of expression of BTK was detected in patients with mutations that led to a premature stop codon compared to patients with missense mutations and healthy controls. Specifically, patients with mutations resulting in a premature stop codon exhibited reduced expression of BTK gene (P = 0.004). However, it was found that missense mutations did not affect BTK expression. By immunocytochemistry, we found that mutations with a premature stop codon impaired expression of BTK protein and that missense mutations led to an abnormal localization of the protein in the cell cytoplasm, showing the synthesis of a non-functional protein. Patients with XLA showed increased expression of the endoplasmic reticulum stress marker XBP1 (P = 0.002). In conclusion, the quantification of mRNA expression for BTK is a tool to differentiate mutational consequences in patients with XLA. It can also contribute to the study of transcripts in other genetic diseases with different types of mutation. This is the first report on endoplasmic reticulum stress in X-linked agammaglobulinemia / Doutorado / Saude da Criança e do Adolescente / Doutora em Ciências

Controlling influenza A virus (IAV) infections remains a challenge largely due to the high replication and mutation rates of the virus. IAV is a negative-sense RNA virus with two main surface proteins — hemagglutinin (HA) and neuraminidase (NA). HA recognizes and binds sialic acid on host cell receptors to initiate virus entry. NA also recognizes sialic acid on host cell receptors but functions by cleaving sialic acid interactions to release progeny virus. Because both HA and NA interact with sialic acid on the host cell surface with opposing effects, their balance is essential for optimal viral infectivity. However, the evolutionary constraints that maintain HA and NA function, while conserving a functional balance, are not fully understood. I adapted the comprehensive and systematic mutational scanning technology, termed EMPIRIC (Exceedingly Meticulous and Parallel Investigation of Randomized Individual Codons), to investigate the local fitness landscape of regions of HA under standard conditions and under drug pressure. We observed that synonymous substitutions had a higher mean absolute fitness effect in the signal than a neighboring HA region used as a control. Folding ∆G calculations revealed a hairpin loop that appeared to be differentially enriched between human and swine IAV variants in sequences of circulating strains. However, the molecular mechanism resulting in the observed host species-specific constraints remains undefined. Studying the fitness landscape of the receptor binding site of HA revealed the high sensitivity of this region to mutation. However, modulating the levels of NA activity by mutation and by using the NA inhibitor oseltamivir enabled the identification of HA mutations with adaptive potential under selection pressure by oseltamivir. These results highlight the importance of the HA-NA functional balance virus replication and in the development of resistance to oseltamivir inhibitors. These studies provide improved understanding of IAV biology, and can inform the development of improved antiviral agents with reduced likelihood for resistance.

(EN) Humans and cells in their bodies are exposed to various mutagens in their lifetime that cause DNA damage and mutations, which affect the biology and physiology of the target cell, and can lead to the expansion of an immortalized cell clone. Genome-wide massively parallel sequencing allows the identification of DNA mutations in the coding sequences (whole exome sequencing, WES), or even the entire genome of a tumour. Mutational signatures of individual mutagenic processes can be extracted from these data, as well as mutations in genes potentially important for cancer development ('cancer drivers', as opposed to 'passengers', which do not confer a comparative growth advantage to a cell clone). Many known mutational signatures do not yet have an attributed cause; and many known mutagens do not have an attributed signature. Similarly, it is estimated that many cancer driver genes remain to be identified. This Thesis proposes a system based on immortalization of mouse embryonic fibroblasts (MEF) upon mutagen treatment for modelling of mutational signatures and identification and testing of cancer driver genes and mutations. The signatures extracted from WES data of 25 immortalized MEF cell lines, which arose upon treatment with a variety of mutagens, showed that the assay recapitulates the...

Genetic changes likely occur in wild fish populations as a consequence of interactions with cultured fish, but to what extent do those changes threaten the maintenance of natural genetic diversity and population viability? Following a review and categorization of numerous processes suspected of being agents of post-release genetic change in recipient wild populations (Chapter 1), I focus on risks relating to the magnitude and duration of releases -- but with a twist. That is, I assume that the mean fitness of released, cultured individuals does not differ from that of the recipient natural population. Throughout, attention is devoted to potential post-release changes in inbreeding (NeI) and variance (NeV) effective population sizes -- indicators of expected rates of population-level change in inbreeding and drift variance, respectively. The reductive effect that large-scale releases exert on NeI in recipient populations can be significant. The effect is shown to be a threshold process (Chapter 2) and thus suggestive of an approach for determining risk-adverse stocking (or release) rates. This approach is utilized in Chapter 3, which describes genetic recommendations for an incipient marine stocking program. Several discordant contemporary NeI models are examined mathematically and by computer simulation (Chapter 4). I show that certain published results pertaining to the effect of multiple paternity on NeI are erroneous; a general model is described which accounts for inbreeding and relatedness in and among parents. That model is utilized in an empirical study of gene correlation in a hatchery cohort (Chapter 5). Propagation-related causes of reductions in NeI are also investigated in this cohort. Finally, extending mutational meltdown theory to accommodate fluctuating population sizes and recessive selective effects, I show that when large reductions in NeV occur (such as those that accompany admixtures of cultured and wild fish), the expected time to population inviability is significantly reduced (Chapter 6). Although a more comprehensive theoretical approach is needed, a precautionary inference may be drawn -- aquaculture-induced reductions in Ne, even though they may be transient, can lead to adverse genetic impacts. Avoidance of Ne-reductions cannot be accomplished, in a practical sense, without considering the stocking or release rates of cultured fish.

Pancreatic ductal adenocarcinoma (PDAC) is a serious oncological disease, which ranks among cancers with the worst prognosis and a three-year life expectancy of 10%. Ex-vivo organoid cultures derived from cancer tissue are popular and reliable research models, which reflect the morphology and histology of the original tissue. Genetic background leading to development PDAC confer typical alterations in genes KRAS, TP53, SMAD4 a CDKN2A. The aim of this thesis was to determine mutations present in organoid cultures derived from human PDAC. We used online genomic databases to estimate specific mutations typical for PDAC. Based on that research we designed protocols for the detection of PDAC genetic alterations and optimized those methods using cultured cells. We applied the approach on primary ex- vivo organoids derived from surgical cancer specimens and detected mutations in KRAS, TP53, SMAD4, or deletion of exons in CDKN2A. Alternatively, we proposed improvements for the analysis of genetic background in PDAC. The data obtained within this thesis will be used for the stratification of metabolomics and biochemical analyses further in the project.

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This dissertation proposes tools and analysis of mutational signatures in human cancer and their application to the stratification of patients for drug response. To provide a comprehensive workflow for preprocessing, analysis, and visualization of mutational signatures, I created the Mutational Signature Comprehensive Analysis Toolkit (musicatk) package. musicatk enables users to select different schemas for counting mutation types and easily combine count tables from different schemas. Multiple distinct methods are available to deconvolute signatures and exposures or to predict exposures in individual samples given a pre-existing set of signatures. Additional exploratory features include the ability to compare signatures to the COSMIC database, embed tumors in two dimensions with UMAP, cluster tumors into subgroups based on exposure frequencies, identify differentially active exposures between tumor subgroups, and plot exposure distributions across user-defined annotations such as tumor type. I then use musicatk to analyze the largest tumor sequencing dataset from a Chinese population to date. I identified differences in the levels of signature exposures compared to similar data from a Western cohort. Specifically, COSMIC signature SBS25 was higher in the Chinese dataset for Melanoma and Renal Cell Carcinoma patients and Melanoma patients had lower levels of SBS7a/b (Ultraviolet Light). My analysis also revealed a putative novel signature enriched in pancreatic cancers. Lastly, I assess the ability of mutational signatures to identify patients who may respond to irofulven, a drug for late-stage cancer patients who have defects in the Transcription Coupled Nucleotide Excision Repair (TC-NER) pathway. As the functional understanding of which mutations successfully disrupt this pathway is incomplete, I develop an approach that classifies patients based on evidence of this pathway being disrupted based on levels of mutational signatures. I build a model that successfully predicts patients who will respond to treatment without a known relevant mutation in the TC-NER pathway. The work from this study furthers our understanding of mutational signatures in different populations and demonstrates the feasibility of using mutational signatures to identify patients eligible for drug trials.

Thesis advisor: Abhishek Chatterjee / Thesis advisor: Jia Niu / Directed Evolution has emerged as an invaluable tool for advancing protein functions in both research and industry. Our lab has pioneered a directed evolution platform in mammalian cells, utilizing an AAV delivery vector to package a DNA library and linking the biomolecule of interest to AAV production. During my tenure in Prof. Chatterjee's lab, I focused on harnessing our lab’s directed evolution platform, known as Virus-Assisted Directed Evolution of tRNA (VADER), to develop highly efficient tRNAs for genetic code expansion. Additionally, I contributed to the development of the AAV-based selection platform, termed Virus-Assisted Mutational Profiling (VAMP), as a profiling tool. Through the utilization of VAMP, I conducted comprehensive profiling of tRNA and RNA polymerase III promoter sequences. This enabled me to gain insights into regions of flexibility and evolution, ultimately leading to the construction of improved constructs with enhanced activity relative to the starting sequence. / Thesis (PhD) — Boston College, 2024. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

As neoplasias mieloproliferativas (NMPs) BCR-ABL1 negativas compreendem a mielofibrose primária (PMF), trombocitemia essencial (TE) e a policitemia vera (PV). A patogênese e progressão dessas NMPs não estão completamente elucidadas. As metaloproteinases de matriz (MMPs) degradam a matriz extracelular, ativando citocinas e fatores de crescimento que, por sua vez, participam da tumorigênese e angiogênese. O objetivo deste estudo foi avaliar a relação da expressão gênica das MMPs, TIMPs, HIF1-&#945; e SPARC com os marcadores angiogênicos bFGF e VEGFA em pacientes com MF e TE, considerando o status mutacional; bem como avaliar a regulação desses genes em camundongos submetidos à hipóxia, e em modelos HIF1-&#945;(-/-) e VHL(-/-). Foram incluídos 21 pacientes com MF, 21 com MF pós-TE, 6 com MF pós-PV, 23 com TE e 78 indivíduos controle. As análises realizadas foram: dosagem sérica e expressão de RNAm de MMP2, MMP9, TIMP1, TIMP2 e SPARC, hemograma, determinação da proteína C reativa ultrassensível, determinação das concentrações de VEGFA e bFGF e avaliação das mutações nos genes JAK2, cMPL e CALR. A avaliação da densidade microvascular da medula óssea foi feita em 30 dos pacientes incluídos. Os pacientes com MFP, MFPTE e TE apresentaram maior expressão de MMP2, SPARC, TIMP1, TIMP2 e bFGF quando comparados aos seus controles (P<0,05), enquanto MMP9 foi mais expressa nos pacientes com MFPTE e TE (P= 0,011 e P=0,047, respectivamente). Os pacientes com TE apresentaram maior expressão de HIF1-&#945; e VEGFA em relação ao grupo controle (P<0,05). Pacientes com MF JAK2V617F positivos apresentaram maiores concentrações de MMP9, TIMP2, bFGF e VEGFA quando comparados aos pacientes portadores de mutações na CALR (P<0,05). Os pacientes com TE JAK2V617F positivos apresentaram maiores concentrações de MMP2 e TIMP2 (P=0,049 e P=0,020, respectivamente). As concentrações das proteínas estudadas não apresentaram correlação com a carga alélica de JAK2V617F e nem com a densidade microvascular da medula óssea. Células de medula óssea de camundongos submetidos à hipóxia apresentaram maior expressão de MMP2 e TIMP1 comparados aos camundongos em normóxia. Camundongos VHL(-/-) apresentaram aumento na expressão dos genes MMP2, MMP9, TIMP1, TIMP2 e VEGFA. Diferentemente, embriões HIF1-&#945;(-/-) não foram considerados um bom modelo para este estudo devido ao envolvimento das MMPs na embriogênese/organogênese. Frente aos resultados encontrados, pode-se sugerir que a maior expressão de MMP2, SPARC e de bFGF estão associadas às NMPs. A mutação JAK2V617F foi associada a maiores concentrações de MMPs, TIMP2 VEGFA e bFGF. HIF1-&#945; foi mais expresso na PV e na TE, sugerindo uma possível regulação da expressão das MMPs e TIMPs nessas doenças. / Myeloproliferative neoplasms (MPNs) BCR-ABL1-negative include primary myelofibrosis (PMF), essential thrombocythemia (ET) and polycythemia vera (PV). The mechanisms underlying the pathology and disease progression in MPN are not completely elucidated. The matrix metalloproteinases (MMPs) cleave extracellular matrix, activating cytokines and growth factors that, in turn, regulate tumorigenesis and angiogenesis. The aim of this study was to evaluate the relationship of MMPs, TIMPs, HIF1-&#945 and SPARC gene expression with angiogenic markers bFGF and VEGFA in patients with MPN considering their mutational status; as well as to assess the regulation of these genes in animal models HIF1-&#945 and VHL knockouts. Twenty-one MF, 21 MF post-ET, 6 MF post-PV, 23 ET patients and 78 controls were enrolled. The analysis performed in peripheral blood were: serum and mRNA expression of MMP2, MMP9, TIMP1, TIMP2 and SPARC, blood count, high-sensitivity C-reactive protein determination and VEGFA and bFGF measurements in plasma. We also evaluate mutations in JAK2, MPL and CALR. The assessment of microvascular density (MVD) in bone marrow was performed in 30 patients. Patients with MFP, MFPET and ET presented higher expression of MMP2, SPARC, TIMP1, TIMP2 and bFGF compared to their controls (P <0.05), while MMP9 expression was higher in patients with MFPET and ET (P=0.011 and P=0.047, respectively). Higher expression of HIF1-&#945; and VEGFA was found in ET patients compared to the controls (P <0.05). PMF JAK2V617F patients had higher concentrations of MMP9, TIMP2, bFGF and VEGFA compared to CALR mutated ones (P <0.05). ET patients JAK2V617F positive had higher levels of MMP2 and TIMP2 (P=0.049 and P=0.020, respectively). The JAK2V617F allele burden was not associated with MVD in the bone marrow. Bone marrow cells from mice in hypoxia condition showed higher MMP2 and TIMP1 expression compared to the control. VHL(-/-) mice exhibited increased expression of MMP2, MMP9, TIMP1, TIMP2 and VEGFA. In contrast, the HIF1-&#945;(-/-) embryos were not considered an applicable model for this study due to MMPs role in embryogenesis/organogenesis. In view of these findings, we can conclude that increased expression of MMP2, SPARC and bFGF are associated with MPN. The JAK2V617F mutation was associated with higher concentrations of MMPs, TIMP2 VEGFA and bFGF. HIF1-&#945; is upregulated in PV and ET and perhaps regulate the MMPs and TIMPs expression in these diseases.