Orientadora : Profª Drª Roseli Terezinha Boschilia / Dissertação (mestrado) - Universidade Federal do Paraná, Setor de Ciências Humanas, Programa de Pós-Graduação em História. Defesa: Curitiba, 04/03/2015 / Inclui referências / Resumo: Vladimir Kozák é um nome significativo para estudiosos dos indígenas brasileiros. No entanto, sua trajetória como pesquisador é praticamente desconhecida no país e pouco se sabe sobre sua vida pessoal, suas relações de amizade, seus desejos e interesses. Qualificá-lo como engenheiro, fotógrafo, cineasta, artista ou mesmo etnólogo é pouco esclarecedor, pois era uma figura multifacetada que sempre esteve envolvida simultaneamente em diversas atividades. Esta pesquisa apoia-se, no estudo do seu arquivo pessoal, com ênfase na documentação epistolar. Analisamos a prática epistolar de Vladimir Kozák, observando o meio em que viveu, buscando interpretar as redes de amizade e os sentimentos que uniram essas pessoas. A amizade é observada como uma relação humana capaz de romper distâncias e ampliar horizontes, consolidando um espaço de conversação franca e de troca cultural e pessoal. Relações que podem desvendar aspectos da trajetória deste homem que viveu entre o espaço da ciência e o espaço das artes, mas não se sentia partícipe de nenhum desses ambientes. Destacamos alguns aspectos da biografia e da autobiografia, bem como os limites da escrita biográfica, para chegar aos dados esclarecedores que registraram a trajetória pessoal e institucional de Vladimir Kozák. Observamos as cartas como forma íntima de comunicação que possibilitam a exposição de ideias e trocas de experiência entre os correspondentes e amigos, que permite pensar as relações de amizade como formas de sentir e de agir que aproximam homens e mulheres. E, fundamentada nessa noção de amizade, que buscamos os correspondentes mais assíduos e permanentes, que propiciaram a compreensão do personagem e dos sentimentos que envolveram suas relações pessoais e profissionais: o casal de antropólogos americanos, Robert Carneiro e Gertrude Dole, e a canadense Marjory Baillon. Os dois primeiros referem-se a uma comunicação direcionada a questões mais profissionais, enquanto a segunda trata-se da amiga escolhida que mantém um diálogo mais individualizado e pessoal. Palavras Chave: Vladimir Kozák. Arquivos. Biografia. Amizade. Índios. / Abstract: Vladimir Kozák is a meaningful name for all brazilian indians researchers. However, his career as a researcher is virtually unknown and little is known about his personal life, his friendships and interests. Name him as an engineer, photographer, filmmaker, artist or even ethnologist is rather vague as he was a multifaceted figure who has always been involved in several activities simultaneously. This research is based on the study of his personal archive, with emphasis on epistolary documentation. Analyzed the epistolary practice of Vladimir Kozák, observing the environment in which he lived, seeking to interpret the friendship networks and the feelings that united these people. Friendship is seen as a human relationship capable of breaking distances and expand horizons, consolidating one free conversational space and cultural and personal exchange. Relationships that can unravel aspects of the trajectory of this man who lived between space science and space of the arts, but he was not a participant in any of these environments. We highlight some aspects of biography and autobiography, as the limits of biographical writing, to reach the illuminating data that recorded the personal and institutional trajectory of Vladimir Kozák. We observed the cards as intimate form of communication that allow exposure of ideas and experience exchange between the correspondents and friends, which suggests friendly relations as ways of feeling and acting that bring men and women. Based on this notion of friendship, we seek the corresponding most assiduous and permanent, which led to the understanding of the character and feelings surrounding their personal and professional relationships: the couple of american anthropologists, Robert Carneiro and Gertrude Dole, and the Canadian Marjory Baillon. The first two refer to a more directed communication professional issues, while the second is selected from the friend who maintains a more individualized and personal dialogue. Keywords: Vladimir Kozák. Files. Biography. Friendship. Indians.

More than 300 human conditions, ranging from cancer predisposition to developmental and neurological mendelian disorders, are caused by haploinsufficiency (HI), a genetic condition by which mutational inactivation of a single allele leads to reduced protein levels and is enough to produce the disease phenotype. Therefore, translational enhancement of the spare allele could exert a therapeutic effect. Here we propose a novel approach for the potential rescue of haploinsufficiency disease loci based on the insertion of specific single nucleotide changes in the Kozak sequence. Since this sequence controls translation by regulating start codon recognition, we aimed at identifying and introducing specific nucleotide variations to enhance translation and rescue haploinsufficiency. To do so, we used CRISPR-Cas base editors, able to generate single nucleotide changes in genomic DNA without the need of a donor DNA and without creating double-strand breaks. We performed a high-throughput screening to evaluate the strength of the Kozak sequences of 231 haploinsufficient genes. We compared the translational efficiency of each wild-type sequence to that of several variants using FACS-seq, which combines fluorescence-activated cell sorting and high-throughput DNA sequencing. We thus selected 5 candidate genes (PPARGC1B, FKBP6, GALR1, NRXN1, and NCF1) and several nucleotide variations able to up-regulate translation. Finally, we used CRISPR-Cas base editors to reproduce the most efficient variants of NCF1 in a cell model relevant for the associated haploinsufficient disease and verified the increase of protein levels. This study proposes a novel therapeutic strategy to rescue haploinsufficiency and sheds new insights into the regulatory mechanisms underlying the translational process. On a broader level, the possibility of modulating gene expression by acting exclusively on translation expands the CRISPR-Cas genome editing applications.

O mecanismo responsável pela exposição da fosfatidilserina (PS) nas membranas celulares não está bem definido. Uma atividade dependente de ATP está envolvida, provavelmente uma ATPase tipo-P. ATPases tipo P são uma família de proteínas transmembranares envolvidas no transporte de metais, íons e fosfolipídios através da membrana plasmática. As P4 ATPases translocam aminofosfolipidios (APTLs) como a PS durante a apoptose. No entanto, o sentido do transporte de PS pela APLT não está claramente definido. Os macrófagos reconhecem a PS exposta na superfície das células apoptóticas, o que inibe sua capacidade microbicida. Formas promastigotas e amastigotas de Leishmania ssp. sofrem apoptose, porém a exposição de PS na superfície dos promastigotas sempre leva à morte, enquanto que nos amastigotas não está necessariamente associada à morte e permite a internalização desses protozoários e sua sobrevivência no macrófago. Esse trabalho teve como objetivo a caracterização molecular da APLT de L. (L.) amazonensis e a avaliação de seu papel na exposição de PS nesse parasita. / The mechanism responsible for phosphatidylserine (PS) exposure in biological membranes is still an open subject. An ATP-dependent activity is involved, probably a Type P- ATPase. Type P ATPases are a family of transmembrane proteins involved in the transport of metals, ions and phospholipids across plasma membrane. P4 ATPases mediate phospholipid transport (APLT) as PS during the process of cell death by apoptosis. However, the direction (inwards or outwards) of this translocation has not been defined. Macrophages recognize exposed PS on the surface of apoptotic cells, what inhibits their microbicidal capacity. Promastigotes and amastigotes of Leishmania ssp. die by apoptosis, but PS exposure on promastigotes always leads to apoptosis, whereas PS exposure by amastigotes is not necessarily associated to death and allows their internalization and survival in the macrophage. This work aimed to characterize APLT from L. (L.) amazonensis and to evaluate its role in PS exposure in this parasite.

O mecanismo responsável pela exposição da fosfatidilserina (PS) nas membranas celulares não está bem definido. Uma atividade dependente de ATP está envolvida, provavelmente uma ATPase tipo-P. ATPases tipo P são uma família de proteínas transmembranares envolvidas no transporte de metais, íons e fosfolipídios através da membrana plasmática. As P4 ATPases translocam aminofosfolipidios (APTLs) como a PS durante a apoptose. No entanto, o sentido do transporte de PS pela APLT não está claramente definido. Os macrófagos reconhecem a PS exposta na superfície das células apoptóticas, o que inibe sua capacidade microbicida. Formas promastigotas e amastigotas de Leishmania ssp. sofrem apoptose, porém a exposição de PS na superfície dos promastigotas sempre leva à morte, enquanto que nos amastigotas não está necessariamente associada à morte e permite a internalização desses protozoários e sua sobrevivência no macrófago. Esse trabalho teve como objetivo a caracterização molecular da APLT de L. (L.) amazonensis e a avaliação de seu papel na exposição de PS nesse parasita. / The mechanism responsible for phosphatidylserine (PS) exposure in biological membranes is still an open subject. An ATP-dependent activity is involved, probably a Type P- ATPase. Type P ATPases are a family of transmembrane proteins involved in the transport of metals, ions and phospholipids across plasma membrane. P4 ATPases mediate phospholipid transport (APLT) as PS during the process of cell death by apoptosis. However, the direction (inwards or outwards) of this translocation has not been defined. Macrophages recognize exposed PS on the surface of apoptotic cells, what inhibits their microbicidal capacity. Promastigotes and amastigotes of Leishmania ssp. die by apoptosis, but PS exposure on promastigotes always leads to apoptosis, whereas PS exposure by amastigotes is not necessarily associated to death and allows their internalization and survival in the macrophage. This work aimed to characterize APLT from L. (L.) amazonensis and to evaluate its role in PS exposure in this parasite.

Gene expression regulation encompasses a wide range of mechanisms that govern cellular processes. Among these, post-transcriptional regulation, including translation control, plays a pivotal role in ensuring precise protein synthesis, timing, and quantity. Perturbations of mechanisms such as RNA modifications, and interactions between RNA-binding proteins (RBPs) and specific RNA motifs, can lead to dysregulation of essential cellular processes. These alterations contribute to the development of various disorders, including cancer, neurodegenerative diseases, and metabolic disorders. Many publicly available datasets and studies offer opportunities to investigate the link between alterations in these mechanisms and disease manifestations. However, the limited availability of datasets for certain conditions or notable inconsistencies among reported associations prevent complete understanding of the underlying processes. Therefore, extending the investigations to encompass a diverse range of genes and/or diseases will enhance our comprehension of these intricate regulatory and disease mechanisms, aiding in the identification of potential therapeutic targets and innovative interventions to mitigate pathological conditions. In particular, we focused on three separate aspects involved in gene expression regulation: RNA modifications, RBPs interactions with RNA secondary structures, and the Kozak consensus sequence as a translational modulator. Each part uncovers essential mechanisms that govern post-transcriptional control of gene expression, shedding light on their roles in cellular processes and disease contexts. At first, we performed a comprehensive exploration of 15 RBPs involved in the regulation of the N6-methyladenosine (m6A) methylation. Leveraging data from The Cancer Genome Atlas (TCGA), we conducted a pan-cancer analysis across 31 tumor types to uncover the distribution of alterations of these factors, and we developed a user-friendly web application to enable users to conduct similar analyses. Additionally, we performed a parallel analysis focused on neuroblastoma, using data from publicly available and in-house datasets. These investigations unveil the potential impact of a subset of m6A factors on cancer development and progression. While in the first case, VIRMA and YTHDF reader proteins, emerged as the most frequently altered genes with significant pan-cancer prognostic implications, in the context of neuroblastoma, the writer METTL14 and the reader ALKBH5, showed the most prominent roles. Subsequently, our focus shifted to a distinct subset of RBPs capable of interacting with RNA secondary structures, particularly with RNA G-quadruplexes (RG4s). We established a comprehensive database cataloging RBPs with potential RG4-binding capabilities. This resource represents a valuable tool for researchers aiming to explore the intricate interplays between RBPs and RG4s, and their putative implications in diverse biological processes and diseases. Finally, attention was directed to the Kozak sequence, a pivotal determinant of the regulation of translation initiation. Exploiting the power of base editors, we developed a method to optimize translation initiation by modifying the Kozak sequence. This strategy offers promise in addressing haploinsufficiency-related disorders, where enhancing the functional protein is essential. Overall, these findings present opportunities for the identification of potential therapeutic targets and precision medicine strategies to alleviate a spectrum of pathological conditions, thus fostering advancements in the field of molecular biology and disease management.

The subject of my thesis was to genetically modify a potato for increased resistance against its pathogens and pests. In developing a resistant plant, it is quite common to use the same type of molecules that plants use themselves in their defense reactions. In this work I used the gene SPI-2 originating from a honeycomb moth (Galleria mellonella). The protein SPI-2 is a member of serine protease inhibitors. Since the previous attempts of the team to detect the protein in transformed plants haven't been successful, the basic form of the gene was modified by adding a Kozak sequence near the start codon, which should have increased the translation initiation and hence increase the level of the protein. Two constructs were prepared for the transformation: SPI-2-T a SPI-2-Y. They differ by one amino acid, which slightly changes their inhibitory activity. First, the construct SPI-2-T was used for a transient transformation of tobacco Nicotiana benthamiana by agroinfiltration of its leaves. Then both constructs were used for a stable transformation of Solanum tuberosum cv. Desireé. The detection of the protein has not been successful, although the inserted gene was transcribed and his sequence was verified by sequencing. It is therefore most likely that the protein has a low stability in the cytoplasm....