Pesquisas de genômica e transcriptômica para o estudo de ncRNAs com foco em parasitas tropicais

Submitted by Maina Lourenco ([email protected]) on 2020-02-14T06:04:58Z No. of bitstreams: 1 TeseCompleta.pdf: 4922139 bytes, checksum: 70ec429bdab637dba0d10c4513d14d2a (MD5)Rejected by Eliane Araujo ([email protected]), reason: Prezada Mainá, AUTOARQUIVAMENTO REJEITADO! Conforme as “Diretrizes para Normalização de Trabalhos Acadêmicos da UFMG” detectamos que no Arquivo do seu trabalho Acadêmico não consta a Ficha Catalográfica e a Ata de Defesa ou Folha de Aprovação devidamente assinada. Considerando a visibilidade nacional e internacional do RI/UFMG, os trabalhos disponibilizados devem seguir padrões e protocolos de integração qualificados e normalizados, a fim de valorizar e facilitar a disseminação do conteúdo. Diante disso, gentileza executar as correções e inserções devidas acessando o link abaixo. 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Atenciosamente, Eliane Araujo Repositório Institucional/UFMG Comunidade - TRABALHOS ACADÊMICOS Biblioteca Universitária - SB/UFMG 3409.4625 - 4620 - 5513 on 2020-02-21T17:37:52Z (GMT)Submitted by Maina Lourenco ([email protected]) on 2020-02-23T03:11:47Z No. of bitstreams: 2 TeseComFicha.pdf: 5238729 bytes, checksum: a780c5c5a72bc433822a626e2126d400 (MD5) ATA MAINA801.pdf: 776036 bytes, checksum: 7449a5f648be36b22cecf53a58d75eee (MD5)Rejected by Camila Silva ([email protected]), reason: Prezada Maina, Gentileza incorporar a ata de defesa posteriormente à ficha catalográfica em arquivo único. Após adequações no arquivo, pedimos que exclua os dois anexos em seu atual depósito e faça upload do arquivo correto. Aguardamos alterações para emissão do seu atestado. Atenciosamente, Equipe Repositório Institucional on 2020-02-27T16:52:57Z (GMT)Submitted by Maina Lourenco ([email protected]) on 2020-02-27T20:33:55Z No. of bitstreams: 1 TeseParaBiblioteca.pdf: 6029038 bytes, checksum: 17e1eb2dbb9ea05ca8ebe18a12945641 (MD5)Approved for entry into archive by Eliane Araujo ([email protected]) on 2020-03-02T18:32:25Z (GMT) No. of bitstreams: 1 TeseParaBiblioteca.pdf: 6029038 bytes, checksum: 17e1eb2dbb9ea05ca8ebe18a12945641 (MD5)Made available in DSpace on 2020-03-04T18:24:23Z (GMT). No. of bitstreams: 1 TeseParaBiblioteca.pdf: 6029038 bytes, checksum: 17e1eb2dbb9ea05ca8ebe18a12945641 (MD5) Previous issue date: 2015-02-20CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível SuperiorNon-coding RNAs (ncRNAs) have been studied in great detail in the last decade, culminating in the current view of widespread molecules playing important roles in virtually all cell processes in all kingdoms of life. The tropical parasites Schistosoma mansoni (the causative agent of schistosomiasis) and Trypanosoma cruzi (the causative agent of Chagas disease) have complex life-cycles involving different hosts and environments and thus requiring a refined regulation of gene expression. In this sense, ncRNAs are known to be broad regulators of gene expression at multiple levels and more detailed surveys to identify such RNAs and the mechanisms in which they are involved are of great importance in the context of these parasites. To assess the roles and features of ncRNAs in tropical parasites, I have studied the mechanism of spliced-leader trans-splicing (SLTS) i n S. mansoni and further expanded the study to involve all species in which this mechanism is currently known and also performed a thorough survey to identify new ncRNAs in the genome of T. cruzi. The SLTS mechanism is known to be present in species of seven different phyla. The molecular characteristics of the spliced-leader (SL) RNA and the set of transcripts processed by the SLTS RNA were studied both in S. mansoni and further in ~150 species. Main findings include the sequence of the SL RNA exon being conserved among different species of a given phylum and more divergent when species from different phyla are compared, however the overall structure of the SL RNA is more conserved in all phyla than its sequence. Additionally, when analyzing the transcripts that receive the SL sequence, I have observed that these include genes from unrelated classes and have no clear bias to any specific function. The genome of T. cruzi was scanned in search of non-annotated ncRNAs, using both sequence-based and structure-based search algorithms and different databases. As a result, I report 1,595 unique ncRNA candidates, more than 700 of these representing new findings. Interestingly, the most abundant classes of new ncRNAs include snoRNAs, RNase P RNAs and miRNAs. The first are known to be widespread in the genome of Trypanosoma brucei (the causative agent of the sleeping sickness) and play important roles in the modification and putative stabilization of rRNAs. RNase P RNAs were not previously reported in trypanosomatids and to date only a protein-only RNase P complex was believed to be present in such organisms. The identification of miRNAs candidates in the genome of T. cruzi was surprising given that the parasite does not harbour a classic RNAi machinery and therefore further analyses should be conducted to prove this finding. Lastly, RNA candidates involved with regulation of gene expression that are believed to be exclusive of prokaryotes were also found, raising questions about how their function could be performed in this eukaryotic parasite.RNAs não-codificadores têm sido estudados em detalhes na última década, culminando na visão recente de que estes são moléculas ubíquas que desempenham papel importante em virtualmente todos os processos biológicos em todos os reinos da vida. Os parasitos tropicais Schistosoma mansoni (agente causador da esquistosomose) e Trypanosoma cruzi (agente causador da Doença de Chagas) têm ciclos de vida complexos envolvendo diferentes hospedeiros e ambientes, necessitando portanto de uma refinada regulação da expressão gênica. Nesse sentido, os ncRNAs são reconhecidamente reguladores da expressão gênica em vários níveis e inspeções mais detalhadas para identificar tais RNAs e os mecanismos nos quais estes estão envolvidos são de grande importância no contexto destes parasitos. Para avaliar as funções e as características dos ncRNAs em parasitos tropicais, o mecanismo de spliced-leader transsplicing (SLTS) foi estudado em S. mansoni e, posteriormente, o estudo foi expandido para incluir todas as espécies nas quais o mecanismo é atualmente conhecido. Além disso, uma inspeção minuciosa foi realizada para identificar novos ncRNAs no genoma de T. cruzi. O mecanismo de SLTS já foi descrito em espécies de sete diferentes filos. As características moleculares do RNA spliced-leader (SL) e o conjunto de transcritos processados pelo mecanismo de SLTS foram estudados em ambos em S. mansoni e posteriormente em ~150 espécies. Os principais achados incluem a sequência do éxon do SL RNA, a qual é conservada dentre diferentes espécies de um mesmo filo e mais divergentes quando espécies de diferentes filos são comparadas. No entanto, a estrutura tridimensional geral do SL RNA é mais conservada em todos os filos do que sua sequência. Adicionalmente, quando os transcritos que recebem a sequência SL foram analisados, observou-se que estes incluem genes de classes não relacionadas e não parecem estar enviesados para nenhuma função especifica. O genoma do T. cruzi foi escaneado em busca de ncRNAs não anotados, utilizando tanto algoritmos de busca baseados em estrutura quanto em sequência e diferentes bases de dados. Como resultado foram encontrados 1.595 candidatos a ncRNA únicos, mais de 700 destes representando novos achados. Interessantemente, as classes mais abundantes de novos ncRNAs incluem snoRNAs, RNase P RNAs e miRNAs. Os primeiros são conhecidos por serem ubíquos no genoma de Trypanosoma brucei (o agente causador da tripanosomíase africana) e desempenham papéis importantes na modificação e putativa estabilização dos rRNAs. RNAs integrantes do complexo RNase P não haviam sido anteriormente descritos em tripanosomatídeos e atualmente apenas um complexo RNase constituído somente de proteínas era conhecido nestes organismos. A identificação de miRNAs candidatos no genoma de T. cruzi foi surpreendente, dada a ausência de uma maquinaria clássica de RNAi neste parasito e portanto análises subsequentes devem ser conduzidas para provar este resultado. Finalmente, candidatos a RNA envolvidos na regulação da expressão gênica que acredita-se ser exclusivos de procariotos também foram encontrados, levantando questionamentos sobre como sua função pode ser desempenhada neste parasito eucarioto

Using Human iPSC-Derived Neurons to Uncover Activity-Dependent Non-Coding RNAs

Humans are arguably the most complex organisms present on Earth with their ability to imagine, create, and problem solve. As underlying mechanisms enabling these capacities reside in the brain, it is not surprising that the brain has undergone an extraordinary increase in size and complexity within the last few million years. Human induced pluripotent stem cells (hiPSCs) can be differentiated into many cell types that were virtually inaccessible historically, such as neurons. Here, we used hiPSC-derived neurons to investigate the cellular response to activation at the transcript level. Neuronal activation was performed with potassium chloride (KCl) and its effects were assessed by RNA sequencing. Our results revealed the involvement of long non-coding RNAs and human-specific genetic variants in response to neuronal activation and help validate hiPSCs as a valuable resource for the study of human neuronal networks. In summary, we find that genes affected by KCl-triggered activation are implicated in pathways that drive cell proliferation, differentiation, and the emergence of specialized morphological features. Interestingly, non-coding RNAs of various classes are amongst the most highly expressed genes in activated hiPSC-derived neurons, thus suggesting these play crucial roles in neural pathways and may significantly contribute to the unique functioning of the human brain

Genes with human-specific features are primarily involved with brain, immune and metabolic evolution

Background: Humans have adapted to widespread changes during the past 2 million years in both environmental and lifestyle factors. This is evident in overall body alterations such as average height and brain size. Although we can appreciate the uniqueness of our species in many aspects, molecular variations that drive such changes are far from being fully known and explained. Comparative genomics is able to determine variations in genomic sequence that may provide functional information to better understand species-specific adaptations. A large number of human-specific genomic variations have been reported but no currently available dataset comprises all of these, a problem which contributes to hinder progress in the field. Results: Here we critically update high confidence human-specific genomic variants that mostly associate with protein-coding regions and find 856 related genes. Events that create such human-specificity are mainly gene duplications, the emergence of novel gene regions and sequence and structural alterations. Functional analysis of these human-specific genes identifies adaptations to brain, immune and metabolic systems to be highly involved. We further show that many of these genes may be functionally associated with neural activity and generating the expanded human cortex in dynamic spatial and temporal contexts. Conclusions: This comprehensive study contributes to the current knowledge by considerably updating the number of human-specific genes following a critical bibliographic survey. Human-specific genes were functionally assessed for the first time to such extent, thus providing unique information. Our results are consistent with environmental changes, such as immune challenges and alterations in diet, as well as neural sophistication, as significant contributors to recent human evolution

Viability of TcNTH1 transfected epimastigotes submitted to oxidative stress.

<p>TcNTH1 overexpressing epimastigotes and its control (parasites transfected with empty vector) were treated for 30 min with different H₂O₂ initial concentrations (A) or with a glucose-glucose oxidase system producing sustained H₂O₂ concentrations for 24 hours (B). Viability was determined by AlamarBlue assays. *p value: 0,01</p

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Expression of TcNTH1 DNA glycosylase in <i>T</i>. <i>cruzi</i> cellular forms.

<p><b>A:</b> TcNTH1 polyclonal antibodies prepared in mice specifically identifies a purified recombinant TcNTH1 (lane 1) and a TcNTH1 expressed in recombinant bacterial homogenates (lane 2). This protein was not recognized in non-expressing bacteria (lane 3). <b>B:</b> Western blot detection of TcNTH1 in total protein homogenates from epimastigotes (lane 1), trypomastigotes (lane 2) and amastigotes (lane 3). Lanes 4 and 5 correspond to TcNTH1 purified from transformed <i>E</i>. <i>coli</i> and to the same protein from recombinant over-expressing <i>T</i>. <i>cruzi</i> epimastigote homogenate, respectively. <b>C:</b> Same as B but using an anti-HIS antibody. <b>D:</b> Loading control for epimastigotes (lane 1), trypomastigotes (lane 2) and amastigotes (lane 3) using an alpha-tubulin antibody. All electrophoretic separations were performed in 12%SDS-PAGE.</p

The catalytic residues disposition and its effects on lesion recognition by TcNTH1.

<p><b>A:</b> Protein-DNA complexes of <i>G</i>. <i>stearothermophilus</i> Endonuclease III (PDB 1P59, cyan) as determined by X-ray crystallography and TcNTH1 from <i>T</i>. <i>cruzi</i> (magenta) as determined by molecular docking. Only protein backbones are shown. <b>B:</b> The same protein-DNA complexes in a different view and depicting the protein surface. Structurally divergent residues are labeled to suggest regions of interest for further analyses. The DNA-interacting region is circulated and the lesion site is shown inside the rectangle. The DNA molecule represented is from 1P59, which is in a similar position when compared to the TcNTH1-DNA complex, as shown in A.</p

Expression and the Peculiar Enzymatic Behavior of the <i>Trypanosoma cruzi</i> NTH1 DNA Glycosylase - Fig 1

<p><b>Multiple amino acid sequences alignment (A) and deduced cladogram (B) of <i>T</i>. <i>cruzi</i> NTH1 with NTH1 from <i>Escherichia coli</i>, <i>Geobacillus stearothermophilus</i>, <i>Homo sapiens</i>, <i>Schizosaccharomyces cerevisiae</i> and <i>Leishmania infantum</i>.</b> (*) are identical residues match and (“:” and “.”) are chemically similar residues. Black highlighted are the residues involved in DNA recognition and binding. Gray highlighted are the residues that generates the lesion DNA base recognition pocket. Boxes are critical residues for catalysis. Arrow-heads (▼) are [4Fe-4S]²⁺ clusters union residues. Overline indicates the helix-hairpin-helix (HhH) motif.</p

TcNTH1 does not present mono nor bifunctional DNA glycosylase activities but an AP endonuclease activity.

<p><b>A, B and C:</b> A [γ-32P]ATP labeled 32 mer oligonucleotide containing a thymine glycol residue at position 18 incubated without enzyme (negative control, lane 1) or with <i>E</i>. <i>coli</i> Endo III (bacterial NTH1, positive control, lane 2). <b>A:</b> Lanes 3 and 4, same oligo incubated with native TcNTH1 purified from transformed bacteria or purified from transfected epimastigotes, respectively. <b>B:</b> Lane 3 same oligo co-incubated with native TcNTH1 purified from transformed bacteria and with native TcAP1 endonuclease. Lanes 4 and 5 same oligo incubated with native TcNTH1 purified from transformed bacteria or incubated with native TcAP1, respectively. <b>C:</b> Lanes 3 and 4 same oligo incubated with epimastigote or trypomastigote homogenates, respectively. <b>D:</b> A [γ-³²P]ATP labeled 25-mer oligonucleotide with an AP site at position 8, was incubated with <i>E</i>. <i>coli</i> Endo III (AP lyase, positive control, lane 3), with native TcNTH1 purified from transformed bacteria (lane 4) and with native TcNTH1 purified from transfected epimastigotes (lane 5). Lane 1 same oligo incubated without enzyme (negative control). Lanes 2 and 6 same oligo incubated with <i>E</i>. <i>coli</i> Exo III (canonic AP endonuclease, positive control) or with TcAP1 AP endonuclease, respectively. A densitometric analysis of bands was performed using the Quantity One version 4.6.3 program (Bio Rad). S: substrate, P: product.</p