After r After refering oxygen and the law of energy conservation as typical efering oxygen and the law of energy conservation astypicalexamples of “silmultaneous discoveries”, the author reflects upon some of the XXth century discoveries in medicine. Recovering of Mendel’s laws, insulin and DNA are illustrative examples how scientists can fi nd similar solutions for the some problems at the some time.

The A. writes about his personal vision of 53years of genetics history (1900-1953), whichfinishes with the Watson and Crick’s double helix model.Starting with the re-discovery of Mendel’s lawsin 1900, he then describes the different contributions of Morgan, Avery, Pauling and MaxDelbrück, leader of the “phage group”, to modern genetics.The relationships between quantum physicsand the birth of molecular biology are particularly highlighted

Nucleic acids store and control genetic information and, beyond the canonical B-form DNA double helix, can adopt a variety of non-canonical architectures, including G-quadruplex (G4s), i-motifs (iMs), triplexes, R-loops, and Z-DNA/Z-RNA. These structures are widespread across genomes and transcriptomes and play important biological roles (transcription, replication, translation, and genome stability). They are recognized as regulatory hotspots enriched at promoters, enhancers, telomeres, untranslated regions, and viral genomes and their involvement in cancer, viral infection, neurodegeneration, and inflammatory disorders. Their in vivo functions have also opened therapeutic avenues both as drug targets and therapeutic tools. As drug targets, these non-canonical structures can be modulated to alter cellular behavior, while as therapeutic agents, nucleic acids can be engineered to adopt non-canonical folds that bind key cellular partners and exert biological effects. This dual role is unusual for other approaches and positions non-canonical structures at the forefront of modern drug discovery. These structures are emerging as important tools in oncological research and therapeutic development. Proto-oncogenes encode proteins essential to normal physiology; however, when mutated or amplified become oncogenes, they drive unchecked growth and survival. The transcription factors (TFs) are a particularly important class of proto-oncogenes that bind to DNA and recruit co-regulators, orchestrating broad gene-expression programs. Their sweeping control of transcription makes them powerful cancer drivers but also challenging drug targets. Unlike enzymes with well-defined catalytic pockets, TFs often rely on flexible, intrinsically disordered regions for interactions, contributing to their reputation as “undruggable.” Numerous TFs have been reported as dysregulated in cancer and are frequently correlated with poor prognosis and resistance to chemotherapy. Other TF are gaining relevance, such as B-MYB (MYBL2), a transcription factor from the MYB family required for normal cell-cycle progression, which acts as an oncogene when overexpressed or deregulated. Functionally, the B-MYB protein serves as a master cell-cycle regulator by integrating into multiprotein assemblies, most notably the DREAM and MMB complexes, that coordinate cell-cycle gene expression. Beyond these physiological functions, B-MYB is upregulated in multiple cancers, such as breast and lung, and its overexpression is correlated with aggressive disease, treatment resistance, and unfavorable prognosis. The molecular mechanisms linking B-MYB to tumorigenesis include gene amplification, cell cycle deregulation, genomic instability, apoptosis suppression, post-transcriptional and post-translational modifications, or can contribute to epithelial-to-mesenchymal transition. Thus, B-MYB TF can be seen as a relevant clinical biomarker and one of the main orchestrators of carcinogenesis; however, until now, no pharmacological therapy against B-MYB has been developed, also related to its “undruggable” profile. Also, it’s known, by bioinformatic analysis of the human genome, that oncogene promoter regions are G/C rich around transcription start sites (TSS), which allows the formation of structures called G4 or iM. The G4s are formed by the self-association of four guanine bases in a quasi-planar arrangement via Hoogsteen bonds and are described as having a gene regulatory function, namely at the transcriptional level, while the iM is formed by Hoogsteen bonds between protonated and deprotonated cytosines. Motivated by these observations, the central objective of this thesis was to explore alternative routes to target the B-MYB oncogene using non-canonical nucleic-acid structures: first as drug targets and later as therapeutics. The thesis started by the identification of G-rich sequences at the B-MYB promoter capable of forming G4 structures. The identification of G-rich sequences was performed using the G4Hunter algorithm, and their conservation across mammalian species was also verified. The experimental validation of their formation was performed by combining 10 biophysical and biochemical methods. Later, the in-cell relevance of G4 structures was evaluated employing the G4access method, which reveals that from the predicted sequences, only the most stable one (B-MYB 43R) was shown to be significantly formed in cells, evidencing a potential impact on the transcription of this gene to its location closest to the TSS. Next, the ability of C-rich sequences to form iM structure was evaluated. Again, the iM-forming sequences were predicted using the G4Hunter algorithm and the experimental validation started by circular dichroism (CD) and nuclear magnetic resonance (NMR) to determine if the sequences fold into an iM structure. Then, stability parameters such as pHT (pH transitional midpoint) were determined by acquiring spectra at different pH values (between pH=5 and pH=8; 0.25-unit increments). The thermal stability and thermodynamic parameters were also calculated using the denaturation and renaturation melting curves. Then, the in-cell formation was assessed using iM-CUT&Tag experiments in HEK293T, which revealed the formation of the iB-MYB 43 that was characterized to have the highest Tm and the highest pHT among the studied sequences. After this characterization, the interaction of small molecules with iB-MYB structures was assessed. As a general tendency, the ligands did not affect the CD spectral shape; however, some of them evidenced changes in secondary structure or thermal destabilization. After evaluating the capacity to form G4 or iM, we continued to validate the G4 formation within a cellular environment using a G4-triggered fluorogenic hybridization probe. This strategy circumvents the limitations of the antibodies and small-molecule probes, which show a lack of structural selectivity labeling diverse DNA and RNA G4s indiscriminately, and cannot specifically target the desired G4. The molecular probe was composed of G4-recognizing light-up ligand (acridine derivative) with an antisense oligonucleotide that hybridizes adjacent region of B-MYB G4. Before probe synthesis using a click chemistry approach, the ligands were photophysically characterized, biophysically evaluated against G4, and further validated spectroscopically and in cells. Cellular studies confirmed the co-localization between the molecular probe and B-MYB G4 in-cell, offering promise for future applications in cancer research in terms of targeted therapies and monitoring of G4. Although G4s are highly dynamic and topologically diverse, promoter G4s are commonly parallel, whereas antiparallel forms are underreported and less characterized. In earlier work, we identified a B-MYB promoter sequence (B-MYB 26RA), that forms an antiparallel G4. Thus, a biophysical pipeline, starting in solution NMR spectroscopy alongside in-cell NMR studies, was made to predict a three-dimensional model (antiparallel quadruplex-duplex junction) and to assess the formation in a live complex environment of B-MYB 26RA. Interestingly, B-MYB 26RA evidenced an ionic sensitivity to K+ and Na+, increasing their topological dynamics, and demonstrated that, when associated with PhenDC3 ligand, a conformational shift from hybrid to antiparallel topology happens. Then, we moved to B-MYB 43R G4 to discuss the therapeutic relevance and the capacity to be targeted by small molecules. Thus, using spectroscopic methods, the G4 formation and its interaction with a panel of G4-stabilizing ligands were confirmed. From the tested ligands, PhenDC3 and TMPyP4 demonstrated the highest binding affinity and stabilization and revealed that both ligands inhibited proliferation and migration in two lung cancer cell lines (A549 and H1299), with PhenDC3 showing potent cytotoxic and cytostatic effects. Furthermore, PhenDC3 upregulated B-MYB expression despite its strong phenotypic effects, highlighting the complexity of G4-targeting mechanisms and supporting the growing evidence that ligands do not always downregulate their target genes. Finally, we explored the use of Polypurine Reverse-Hoogsteen (PPRH) hairpins as an alternative therapeutic approach to target the B-MYB promoter. PPRH are DNA oligonucleotides that fold into intramolecular hairpins and bind complementary polypyrimidine, forming stable DNA triplexes, impeding transcription, and displacing the G-rich strand, inducing G4 formation, enabling dual-level regulation of oncogene expression. Thus, using a bioinformatic tool (TFO Searching Tool), a PPRH was designed against the G4-forming region at the B-MYB promoter (B-MYB 43R), and then experimentally assessed the triplex formation using biophysical methods. The biological effects of designed PPRH, evaluated in lung cellular models (A549, H1299 and MRC-5), demonstrated a low cell viability and clonogenic capacity accompanied by mRNA expression reduction and proteomic profile alterations. Thus, the combination of triplex-based approaches with G4 biology could be a future venue for therapy. Overall, this thesis establishes non-canonical nucleic-acid structures as both actionable targets and therapeutic agents. As a target, the formation of G4 and iM structures at the promoter region of the B-MYB oncogene; was provided new insights about structure and topological dynamics according to the surrounding environment; was validated their formation in-cell, as well as explored the interaction with small molecules. On the other side, the therapeutic potential was explored using PPRH, which revealed a selective and powerful tool to target B-MYB. Together, these findings provide fundamental insights and open new avenues for translational research on the B-MYB oncogene.

Minicircle DNA (mcDNA) technology is in the vanguard of vectors designed for gene therapy, since the absence of prokaryotic sequences confers to mcDNA higher biosafety in comparison to other DNA vectors. However, the presence of other isoforms and non-recombined parental molecules hampers the isolation of supercoiled (sc) mcDNA with the chromatographic methods already established for plasmid purification. In this work, two monolithic supports were modified with lysine and its decarboxylated derivative, cadaverine, to explore their performance in the sc mcDNA purification. Increasing NaCl gradients and different pH values (from 6 to 9) were tested in both modified monoliths. In general, cadaverine modified support established stronger interactions with mcDNA than lysine modified monolith, at acidic pH. For instance, at pH 6.0 the retention time for RNA and DNA molecules in lysine modified monolith was 11.58 and 14.59, respectively, while for cadaverine modified monolith was 20.32 and 27.12, respectively. The lysine modified monolith was able to successfully isolate sc mcDNA from the lysate sample. However, recovery yield was significantly sacrificed to guarantee high purity levels of sc mcDNA. The cadaverine modified monolith showed better selectivity than the previous monolith, achieving the successful sc mcDNA isolation from the lysate sample. The final sc mcDNA sample, obtained by the column that showed the best performance, was characterized by real-time PCR, presenting 98.4% purity and 78.6% recovery yield. The impurities content, namely genomic DNA, proteins and endotoxins, was found within the criteria established by regulatory agencies. Overall, a simple and practical chromatographic strategy to purify sc mcDNA was for the first time implemented by exploring a modified monolithic column, with no significant reduction on the purity and recovery and without resorting to backbone modification or specific enzymatic digestion. Such features will surely be crucial in the industrial scale-up of this chromatographic strategy since it will not be associated with significant cost-increase.

The vast majority of human genes express multiple mRNAs by alternative splicing of their pre-mRNAs. This mechanism allows the expression of many different mRNAs from the same gene, and greatly increases the complexity of “cell-specific” protein function. It can alter the function of proteins by removing or adding specific domains (nuclear localization signals, transcription activation domains, DNA or RNA binding domains, trans-membrane domains), post-translation modification sites, or by causing substantial changes in protein structure. The existence of several variant RNA transcripts has been described for many steroid receptors. However, the information on alternative spliced mRNAs of androgen receptor (AR) gene is scarce. A previous work in our research group (Laurentino et al. 2006, results not published) allowed the identification for the first time of AR alternative transcripts in human testis. Two transcripts result from exon 2 (ARΔ2) and exon 3 (ARΔ3) skipping. Transcripts lacking exon 2 but retaining part of intron 2 of the AR gene (AR55), and lacking part of exon 4 (AR94) were also detected. The aims of the present study are: 1) characterize the distribution of AR transcripts in different human tissues (heart, kidney, liver, and lung); 2) perform an evolutionary analysis by characterizing the expression of AR transcripts in testis of chicken (Gallus gallus), dog (Cannis lupus), rat (Rattus norvegicus) and sea bream (Sparus aurata). For this purpose the following experimental approach was established: 1) exon spanning primers suitable for the detection of alternative spliced ARs were designed; 2) Total RNA was extracted and used for cDNA synthesis; 3) RTPCR was carried out and the amplified products were cloned and sequenced; 4) the obtained sequences were analysed using bioinformatics tools. In human heart, kidney, liver and lung was observed the presence of ARΔ2 and ARΔ3 transcripts previously identified in testis. In seabream, rat and dog testis were detected several AR transcripts some of which are homologous to those identified in human testis.

Well-defined oligo(ethylene glycol) methyl ether methacrylate (OEOMA) based block copolymers with cationic segments composed by N,N-(dimethylamino) ethyl methacrylate (DMAEMA) and/or 2-(diisopropylamino) ethyl methacrylate (DPA) were developed under biorelevant reaction conditions. These brush-type copolymers were synthesized through supplemental activator and reducing agent (SARA) atom transfer radical polymerization (ATRP) using sodium dithionite as SARA agent. The synthesis was carried out using an eco-friendly solvent mixture, very low copper catalyst concentration, and mild reaction conditions. The structure of the block copolymers was characterized by size exclusion chromatography (SEC) analysis and 1H nuclear magnetic resonance (NMR) spectroscopy. The pH-dependent protonation of these copolymers enables the efficient complexation with plasmid DNA (pDNA), yielding polyplexes with sizes ranging from 200 up to 700 nm, depending on the molecular weight of the copolymers, composition and concentration used. Agarose gel electrophoresis confirmed the successful pDNA encapsulation. No cytotoxicity effect was observed, even for N/P ratios higher than 50, for human fibroblasts and cervical cancer cell lines cells. The in vitro cellular uptake experiments demonstrated that the pDNA-loaded block copolymers were efficiently delivered into nucleus of cervical cancer cells. The polymerization approach, the unique structure of the block copolymers and the efficient DNA encapsulation presented can open new avenues for development of efficient tailor made gene delivery systems under biorelevant conditions.

Schiff bases (SB) obtained from S-methyl dithiocarbazate and aromatic aldehydes: salicylaldehyde (H2L1), o-vanillin (H2L2), pyridoxal (H2L3) and 2,6-diformyl-4-methylphenol (H3L4), and their corresponding Zn(II)-complexes (1-4), are synthesized. All compounds are characterized by elemental analyses, infrared, UV-Vis, nuclear magnetic resonance spectroscopy and mass spectrometry. The structures of H2L2 and [Zn2(L1)2(H2O)(DMF)] (1a) (DMF = dimethylformamide) are solved by single crystal X-ray diffraction. The SB coordinates the metal center through the Ophenolate, Nimine and Sthiolate atoms. The radical scavenging activity is tested using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay, with all ligand precursors showing IC50 values ~40 μM. Cytotoxicity studies with several tumor cell lines (PC-3, MCF-7 and Caco-2) as well as a non-tumoral cell line (NHDF) are reported. Interestingly, 1 has relevant and selective antiproliferative effect against Caco-2 cells (IC50 = 9.1 μM). Their antimicrobial activity is evaluated in five bacterial strains (Klebsiella pneumoniae, Acinetobacter baumannii, Listeria monocytogenes, Pseudomonas aeruginosa and Staphylococcus aureus) and two yeast strains (Candida albicans and Candida tropicalis) with some compounds showing bacteriostatic and fungicidal activity. The minimal inhibitory concentration (MIC90) of HnL against Mycobacterium tuberculosis is also reported, with H2L2 and H3L4 showing very high activity (MIC90 < 0.6 μg/mL). The ability of the compounds to bind bovine serum albumin (BSA) and DNA is evaluated for H3L4 and [Zn2(L4)(CH3COO)] (4), both showing high binding constants to BSA (ca. 106 M-1) and ability to bind DNA. Overall, the reported compounds show relevant antitumor and antimicrobial properties, our data indicating they may be promising compounds in several fields of medicinal chemistry.

Os marcadores mitocondriais (mtDNA) e cromossomo Y têm sido utilizados para avaliar o grau de miscigenação. No caso da América Latina, três estoques principais originaram a população atual: europeus, ameríndios e africanos. No Rio Grande do Sul além de portugueses e espanhóis, foi marcante a imigração de outros europeus, especialmente alemães e italianos. O presente trabalho busca avaliar a contribuição europeia, ameríndia e africana, para a formação da população gaúcha. Para isso foram utilizados os dois sistemas uniparentais e sobrenomes dos indivíduos. Foram analisados 190 voluntários, nascidos nas sete mesorregiões que compõem o Rio Grande do Sul, dos quais se coletou uma amostra de sangue. Observou-se uma elevada frequência de contribuição europeia (87% no cromossomo Y e 76% no mtDNA), condizente com a vinda de casais de imigrantes portugueses, alemães e italianos. Os dados de sobrenomes demonstram também serem estes os sobrenomes mais encontrados na população gaúcha.

The characterization of the plasmid pLK39.was the aim of work.pLK39 was isolated from a endophytic Gram-negative, bactéria isolated from leaves form Solanum lycocarpum (Lobeira). In order to obtain a selection marker which would characterization in Escherichia coli the kanamycin, resistence gene from pUC4K. was subcloned into the PstIsite of pLK39. The characterization of PLK39 was basedon studies of stability, incompatibility, determination of the copy number and through DNA sequencing. The stability was carried out in Escherichia coli XL10 cells. Cells transformed with pLK39 were inoculated in Lúria broth containing Kanamycin (50&#956;g/ml) during24 hours. After 24 hours of incubation one sample of the culture was in medim with and without selective pressure, and inoculated in Lúria broth without pressure médium, for 240 generations. After 240 generations 81,5% of cells maintained the plasmid, showing that pLK39is highly stable in Escherichia coli to make the incompatibility test, cells of Escherichia coli XL10 were co-transformed with pLK39/pUC18; pBR322 and pLK39/pACYC184. After 240 generations, the plasmid pLK39 iof detected in all systems used, showing the compatibility of pLK39 with the plasmids tested. The copý number pLK39 was estimated by the intensity of plasmidial bands in agarose gel, electrophoresis using a photodocumentation and analysis system (KODAK EDAS). The copý number of pLK39 was estimated as 25 copies per cell. PLK39 was digested with Sau3AI restriction and subcloned into the BamHI site of plasmid pUC18. The recombinant clones were sequenced and 1637 pb reading fragment was obtained. This sequence showed high homology with pSW200, pSW100, pEC3, pUCD5000 and pBERT, which were isolated from Gram-negative bacteria. This sequence possesses two possible genes. The ORF I showed high homology with mobB gene from plasmid pSW200 (96%), pSW100 (96%), pEC3 (94%), pUCD5000 (94%) and pBERT (87%). The ORF II showed homology with mobD gene from plasmids pSW200 (92%), pSW100 (90%), pEC3 (90%) and PUCD5000 (89%).

A identificação de restos mortais a partir de tecido ósseo pode ser realizada com o uso de ferramentas de diferentes campos da Ciência Forense, sendo a Genética a mais avançada. O uso do DNA em casos forense possui uma grande importância em processos criminais e estudos voltados para o aprimoramento das técnicas existentes ainda são necessários no caso de ossos. Restos esqueletizados são frequentemente encontrados nos cenários de crimes, das guerras e das grandes catástrofes, envolvendo civis e militares, inclusive em ambientes aquáticos ou similares, sendo alvo de emprego de metodologias moleculares. Entretanto, estudos demonstram dificuldades em gerar perfis genéticos dessas amostras, visando sua identificação. O objetivo foi identificar alterações em amostras de tecido ósseo, sob em água do rio, em nível estrutural e genético, que possam corroborar para o melhor conhecimento da análise forense a partir dessas amostras. Para isto, foi utilizado amostras de fragmentos ósseos do acervo do Laboratório de Diagnóstico por DNA (UERJ). As amostras foram expostas à água de rio durante 30, 60 e 90 dias. Análise por microscopia eletrônica de varredura (MEV-EDS) e espectroscopia de Raman foram realizadas para avaliar a condição estrutural da amostra. Analise quantitativa da molécula de Ácido Deoxinucleíco (DNA) preparados das amostras foram realizadas por amplificação quantitativa por Reação em Cadeia da Polimerase em Tempo Real (qPCR). Foi identificado alterações morfológicas presentes, principalmente, nos ossos corticais e trabeculares. Modificações iônicas foram observadas, primordialmente na superfície externa e na porção trabecular dos ossos, com presença da diminuição da porcentagem dos componentes principais e aumento de elementos, como Manganês, Ferro, Alumínio e Silício. Os resultados da quantificação por qPCR demonstraram não haver diferença significativa entre as quantidades de DNA em função da exposição ambiental nem do tempo estudado. De modo semelhante, a análise dos géis de agarose permitiu inferir que não houve redução significativa da qualidade do DNA mitocondrial (DNAmt) em função da exposição ambiental e do tempo estudado. Apenas íons positivos foram encontrados. Esses íons podem alterar a conformação do DNA e interferir no processo de anelamento de primers na PCR, o que pode diminuir a eficiência do processo de amplificação e interferir na qualidade do perfil genético gerado. Entretanto, são necessários estudos voltados diretamente para a estrutura do DNA para confirmação da nossa hipótese.