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Objective:To analyze the epidemic characteristics and virus gene sequence of hemorrhagic fever with renal syndrome (HFRS) in an industrial park in Daishan County, Zhejiang Province, and to provide clues and basis for local HFRS prevention and control.Methods:According to the case questionnaire in the "National Surveillance Program for Hemorrhagic Fever with Renal Syndrome", general and epidemiological investigation of HFRS cases was carried out in the epidemic-related industrial park. Serum samples of the cases, people and host animals in the same living environment were collected for hantavirus antibody or nucleic acid detection, the M, S gene amplification and sequence determination. MEGAX 10.1.8 software was used to construct the phylogenetic tree of M and S genes for virus genotyping and evolutionary analysis.Results:A total of 3 confirmed cases of HFRS were reported. They were all workers in the epidemic-related industrial park, male, who lived in the park for more than half a year and had no history of HFRS vaccination. There were no rodent-proof facilities in the industrial park's dormitories and canteens, and the living items were placed in a disorderly manner, the rodents and its excrement could be seen; a total of 38 host animals were captured in the same living environment with cases, all of which were Rattus norvegicus. The 3 reported cases of HFRS were all mild, with atypical clinical manifestations in the early stage of onset, mainly fever and fatigue. The serum specific antibodies of hantavirus IgG and IgM were positive (3/3), and the antibodies of people in the same living environment were negative (100.0%, 100/100). The serum samples of 2 reported cases of HFRS and 4 Rattus norvegicus were positive for nucleic acid, all of which were SEOV type hantavirus. The M gene segment homology of 6 positive serum samples was 100.0%, which was closely related to Rod/2012/QHD/4/Gc isolated from Hebei and RuianRn180 isolated from Ruian Zhejiang Province; the homology of S gene segment was 99.6% to 99.8%, which was closely related to JiangxiXinjianRn-09-2011, a strain isolated from Jiangxi Province. Conclusions:The HFRS epidemic in the industrial park is caused by the transmission of SEOV type hantavirus to humans via Rattus norvegicus; poor living environment, poor hygiene habits of personnel and lack of vaccination are all related to the incidence of HFRS; the main epidemic strains shows high homology and geographical aggregation.
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Triterpenoids are one of the most diverse compounds in plant metabolites, and they have a wide variety of physiological activities and are of important economic value. Oxidosqualene cyclases catalyze the cyclization of 2, 3-oxidosqualene to generate different types of sterols and plant triterpenoids, which is of great significance to the structural diversity of natural products. However, the mechanism of the diversified cyclization of 2, 3-oxidosqualene catalyzed by oxidosqualene cyclases remains unclear. This review summarized the research progress of oxidosqualene cyclases from the aspects of catalytic function, molecular evolutionary relationship between genes and proteins, protein structure, molecular simulation and molecular calculations, which may provide a reference for protein engineering and metabolic engineering of triterpene cyclase.
Subject(s)
Intramolecular Transferases/metabolism , Metabolic Engineering , Plants/genetics , Squalene/chemistry , TriterpenesABSTRACT
Genes encoding structurally independent phosphotriesterases (PTEs) are identified in soil bacteria. These pte genes, often identified on mobilizable and self-transmissible plasmids are organized as mobile genetic elements. Their dissemination through lateral gene transfer is evident due to the detection of identical organophosphate degradation genes among soil bacteria with little orno taxonomic relationship. Convergent evolution of PTEs provided selective advantages to the bacterial strain as they convert toxic phosphotriesters (PTs) into a source of phosphate. The residues of organophosphate (OP) compounds that accumulate in a soil are proposed to contribute to the evolution of PTEs through substrate-assisted gain-of-function. This review provides comprehensiveinformation on lateral transfer of pte genes and critically examines proposed hypotheses on their evolution in the light of the short half-life of OPs in the environment. The review also proposes alternate factors that have possibly contributed to the evolution and lateral mobility of PTEs by taking into account their biology and analyses of pte genes in genomic and metagenomic databases.
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Objective To amplify and sequence Coxl and Nadl genes in Echinococcus multilocularis isolates from Qinghai Province, and to create phylogenetic trees and molecular clocks, so as to provide evidence for estimating the evolutionary relationships and origins of E. multilocularis in Qinghai Province. Methods Twenty-two post-surgical specimens of patients with hepatic alveolar echinococcosis were sampled from Qinghai Provincial People’s Hospital in 2017. The Coxl and Nadl genes were amplified from E. multilocularis samples and sequenced. Then, the gene sequences were aligned to the Coxl and Nadl genes of Echinococcus species in GenBank database. The intra-species variation was observed, and the phylogenetic tree and molecular clock were created. Results All E. multilocularis samples shared more than 99% genetic homology with the sequences of Coxl and Nadl genes from the E. multilocularis Asian strain in the GenBank database. A total of 6 genotypes were identified, including 2 isolates that had no 100% homology with the sequences of known genes in the GenBank database. Phylogenetic tree analysis revealed remarkable clustering of the E. multilocularis samples with the E. multilocularis Asian strain, and the E. multilocularis isolates from Qinghai Province were estimated to date back to 94 000 years ago by the molecular clock. Conclusions The present study characterizes 6 E. multilocularis genotypes in Qinghai Province, including 2 novel genotypes. Asian strain is the predominant strain of E. multilocularis in Qinghai Province, and the E. multilocularis isolates from Qinghai Province date back to 94 000 years ago.
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Objective To amplify and sequence Coxl and Nadl genes in Echinococcus multilocularis isolates from Qinghai Province, and to create phylogenetic trees and molecular clocks, so as to provide evidence for estimating the evolutionary relationships and origins of E. multilocularis in Qinghai Province. Methods Twenty-two post-surgical specimens of patients with hepatic alveolar echinococcosis were sampled from Qinghai Provincial People’s Hospital in 2017. The Coxl and Nadl genes were amplified from E. multilocularis samples and sequenced. Then, the gene sequences were aligned to the Coxl and Nadl genes of Echinococcus species in GenBank database. The intra-species variation was observed, and the phylogenetic tree and molecular clock were created. Results All E. multilocularis samples shared more than 99% genetic homology with the sequences of Coxl and Nadl genes from the E. multilocularis Asian strain in the GenBank database. A total of 6 genotypes were identified, including 2 isolates that had no 100% homology with the sequences of known genes in the GenBank database. Phylogenetic tree analysis revealed remarkable clustering of the E. multilocularis samples with the E. multilocularis Asian strain, and the E. multilocularis isolates from Qinghai Province were estimated to date back to 94 000 years ago by the molecular clock. Conclusions The present study characterizes 6 E. multilocularis genotypes in Qinghai Province, including 2 novel genotypes. Asian strain is the predominant strain of E. multilocularis in Qinghai Province, and the E. multilocularis isolates from Qinghai Province date back to 94 000 years ago.
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Objective To investigate the molecular evolution of the Hemagglutinin (HA) and Neuraminidase (NA) genes of influenza A/H3N2 viruses in Shenzhen in the first half of 2017, so as to provide scientific basis for predicating influenza epidemic and variation. Methods A total of 40 influenza A/H3N2 viruses strains were selected and the molecular phylogenetic trees were constructed by bioinformatics software DNAStar, MEGA 7.0, etc. Then, the genetic characteristics and variation of HA and NA genes along with corresponding amino acids were analyzed. Results The homology of Shenzhen isolates reached 97.8%-100.0%, which located in the human-derived branch of Asia and North America. Compared with the vaccine strains A/Switzerland/9715293/2013(H3N2) and A/Hong Kong 14801/2014(H3N2) recommended by world Health Oraganication (WHO), there was a higher sequence similarity. Compared with the vaccine strain, HA and NA proteins had a number of amino acid sites replaced, of which HA 6 antigen sites and 2 receptor binding sites change; NA had a mutation of D151N/G located in enzyme activity sites. Potential N-glycosylation sites for HA and NA also changed. Conclusions The influenza A/H3N2 viruses in Shenzhen in the first half of 2017 has not yet formed a new subtype in the epidemic. Currently, the recommended vaccine strains still have some protective effects on the population. The replacement mutation of multiple amino acids sites of HA and NA suggests that the dynamic monitoring of molecular level of influenza A/H3N2 viruses need to be strengthened.
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The 4a and 4b proteins of the Middle East respiratory syndrome coronavirus (MERS-CoV) have been described for their antagonism on host innate immunity. However, unlike clustering patterns of the complete gene sequences of human and camel MERS-CoVs, the 4a and 4b protein coding regions did not constitute species-specific phylogenetic groups. Moreover, given the estimated evolutionary rates of the complete, 4a, and 4b gene sequences, the 4a and 4b proteins might be less affected by species-specific innate immune pressures. These results suggest that the 4a and 4b proteins of MERS-CoV may function against host innate immunity in a manner independent of host species and/or evolutionary clustering patterns.
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Humans , Camelus , Clinical Coding , Coronavirus Infections , Evolution, Molecular , Immunity, Innate , Middle East Respiratory Syndrome Coronavirus , Middle East , Open Reading Frames , Phylogeny , ZoonosesABSTRACT
Abstract Domestication is of unquestionable importance to the technological revolution that has given rise to modern human societies. In this study, we analyzed the DNA and protein sequences of six genes of the oxytocin and arginine vasopressin systems (OXT-OXTR; AVP-AVPR1a, AVPR1b and AVPR2) in 40 placental mammals. These systems play an important role in the control of physiology and behavior. According to our analyses, neutrality does not explain the pattern of molecular evolution found in some of these genes. We observed specific sites under positive selection in AVPR1b (ω = 1.429, p = 0.001) and AVPR2 (ω= 1.49, p = 0.001), suggesting that they could be involved in behavior and physiological changes, including those related to the domestication process. Furthermore, AVPR1a, which plays a role in social behavior, is under relaxed selective constraint in domesticated species. These results provide new insights into the nature of the domestication process and its impact on the OXT-AVP system.
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Abstract Alcohol dehydrogenases belong to the large superfamily of medium-chain dehydrogenases/reductases, which occur throughout the biological world and are involved with many important metabolic routes. We considered the phylogeny of 190 ADH sequences of animals, fungi, and plants. Non-class III Caenorhabditis elegans ADHs were seen closely related to tetrameric fungal ADHs. ADH3 forms a sister group to amphibian, reptilian, avian and mammalian non-class III ADHs. In fishes, two main forms are identified: ADH1 and ADH3, whereas in amphibians there is a new ADH form (ADH8). ADH2 is found in Mammalia and Aves, and they formed a monophyletic group. Additionally, mammalian ADH4 seems to result from an ADH1 duplication, while in Fungi, ADH formed clusters based on types and genera. The plant ADH isoforms constitute a basal clade in relation to ADHs from animals. We identified amino acid residues responsible for functional divergence between ADH types in fungi, mammals, and fishes. In mammals, these differences occur mainly between ADH1/ADH4 and ADH3/ADH5, whereas functional divergence occurred in fungi between ADH1/ADH5, ADH5/ADH4, and ADH5/ADH3. In fishes, the forms also seem to be functionally divergent. The ADH family expansion exemplifies a neofunctionalization process where reiterative duplication events are related to new activities.
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Polyketides are a large class of natural products with notable structural diversity and different biological activities. They have essential pharmacological value for human health. In plants, the enzymes responsible for the formation of phenolic metabolites backbone structures are collectively known as type Ⅲ polyketide synthases (PKSs), which are the key enzymes for the polyketides biosynthesis. The PKSs catalyze a series of condensation reactions of two-carbon acetate units with an acyl starter. A brief overview of this group of enzymes, including their reaction mechanisms, function modification, expression regulation, molecular evolution, and recent interesting findings are presented here.
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We used in vitro molecular evolution technology by error-prone PCR and high-throughput screening to improve thermostability of Bacillus flexus CCTCC 2015368 β-amylase. Mutant D476N with significant thermostability increase was selected by LB agar starch plate colorimetric assay and 96-well plate enzyme activity assay. The optimum pH was 6.5 for the mutant D476N, compared to 7.0 of the wild type. The optimal temperature was 55 ℃ for both mutant D476N and the wild type. The T₅₀ value of the mutant D476N was 4 ℃ higher than that of the wild type. The half-life of mutant D476N at 55 ℃ was 35 min, 95% higher than that of the wild type. The Km of the mutant D476N was 97.98 μmol/L, 1.14 times of that of the wild type (85.86 μmol/L). The thermostability of the mutant D476N was slightly lower than that of the wild type. The three-dimensional structure of wild type and mutant D476N was simulated by SWISS-MODEL and analyzed by PyMol software. The mutated amino acid residue Asn476 was located on the loop of protein surface. The molecular free energy(ΔG) of D476N was calculated by MOE software was 106.0 kcal/mol, reduced by 10.3% compared to the wild enzyme. These results were consistent with the theory that the protein molecular free energy and thermostability were negatively correlated.
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Abstract The FOXP subfamily is probably the most extensively characterized subfamily of the forkhead superfamily, playing important roles in development and homeostasis in vertebrates. Intrinsically disorder protein regions (IDRs) are protein segments that exhibit multiple physical interactions and play critical roles in various biological processes, including regulation and signaling. IDRs in proteins may play an important role in the evolvability of genetic systems. In this study, we analyzed 77 orthologous FOXP genes/proteins from Tetrapoda, regarding protein disorder content and evolutionary rate. We also predicted the number and type of short linear motifs (SLIMs) in the IDRs. Similar levels of protein disorder (approximately 70%) were found for FOXP1, FOXP2, and FOXP4. However, for FOXP3, which is shorter in length and has a more specific function, the disordered content was lower (30%). Mammals showed higher protein disorders for FOXP1 and FOXP4 than non-mammals. Specific analyses related to linear motifs in the four genes showed also a clear differentiation between FOXPs in mammals and non-mammals. We predicted for the first time the role of IDRs and SLIMs in the FOXP gene family associated with possible adaptive novelties within Tetrapoda. For instance, we found gain and loss of important phosphorylation sites in the Homo sapiens FOXP2 IDR regions, with possible implication for the evolution of human speech.
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In order to investigate the molecular evolution and spatio-temporal migration of Getah viruses (GETV) isolated around the world,the nucleotide and deduced amino acid sequence of GETVs were analyzed and phylogenetic trees were constructed by using informatics software including ClustalX1.83,MegaAlign,GeneDOC and Mega6.0.The Bayesian Stochastic Search Variable Selection (BSSVS) program in the BEAST v 1.8.1 software package was used to analyze the spatial dynamics of the Getah virus.Results showed that the full-length of Getah virus E2 gene consists of 1 266 nueleotides,encoding 422 amino acids.And the homology of nucleotide and amino acid were 94.5％ 100％ and 96.4％ 100％ respectively.The molecular evolution analysis revealed that there were no species and geographical distribution difference existing among GETV host animals (e.g.horses and pigs) and vectors (e.g.mosquitoes).Bioinformatics analysis showed that GETV originated in Malaysia,then it was spread to Japan,China,South Korea,Mongolia,Russia,etc.GETV E2 gene was relatively stable since GETV was first isolated in 1955.The differences of species and geographical distribution did not exist among GETV host animals and vectors,and the virus has spread from tropical regions to Eurasian continent.Thus,strengthening the detection and monitoring of GETV and its infections in humans and livestock is critical.
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Objective:The aim is to establish L-glutamate specific aminotransferase-L-glutamate dehydrogenase coupling 96-well high throughput screening method,which is applied to molecular evolution of aminotransferase WecE from E.coli.Methods:An optical assay for aminotransferase catalytic activity based on aminotransferase-glutamate dehydrogenase coupling system is established by optimization of coupling enzyme loading,signal molecule NADH concentration and coupling time.Mutants library of WecE is obtained by sitedirected saturation mutagenesis.Positive mutants can be screened out through 96-well preliminary screening and flask second screening.Results:The target transamination reaction is coupled with L-glutamate dehydrogenase indicative reaction system which consists of 0.5 U/ml enzyme loading and 0.4 mmol/L NADH.A positive mutant Y321F whose catalytic activity increases 3.4 fold compared to that of wild type is screened out in Tyr 321 saturation mutagenesis library of WecE.Conclusion:An accurate high throughput screening method with weak background interference is established.It offers feasible solution for molecular evolution of L-glutamate specific aminotransferase.
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Objective@#To investigate the molecular evolution characteristics of human coronavirus (HCoV) subtypes in patients with fever and respiratory tract infection in Guangzhou from 2010 to 2012.@*Methods@#Partial fragments of NP, RdRp and S genes of HCoV-OC43, HCoV-229E and HCoV-NL63 positive samples were amplified by RT-PCR and sequencing. Bioinformatics software, including Bio-edit, Mega4.0 and Clustal1.83 were used for comparison and analysis of NP, RDRp and S gene sequences. Molecular evolutionary tree of different gene regions of HCoV-OC43, HCoV-229E and HCoV-NL63 were built.@*Results@#No remarkable variation or recombinant strain of HCoV-OC43, HCoV-229E and HCoV-NL63 was found in Guangzhou during 2010—2012. The HCoV-OC43 substrains were genetically closest to the strains found in Belgium and Hong Kong (GenBank accession number JN129834 and AY903460). HCoV-229E substrains were genetically closest to those found in Amsterdam (GenBank accession number JX503060) and HCoV-NL63 most genetically close to those in Amsterdam and Beijing (GenBank accession number JX104161 and DQ445911). The NP and RDRp genes of all subtypes were highly conserved, while S gene was more variable.@*Conclusions@#There were at least 3 substrains of HCoV-OC43, HCoV-229E and HCoV-NL63 epidemic in Guangzhou during 2010—2012, and no remarkable variation or recombinant viral strain was found. The NP and RDRp genes of all subtypes were highly conserved and can be used in virus detection, while S gene was more variable and suitable for phylogenetic and variation study.
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Objective To investigate the molecular characteristics of dengue virus serotype 2 (DENV2) in Guangzhou during 2001-2015,and analyze the E gene of the strains isolated,the phylogenetic tree and molecular clock were constructed to know about the evolution of the strains.Methods The serum samples of the patients were detected by real time PCR,and positive samples were used to isolate dengue virus by using C6/36 cells.The E gene of the isolated strains were sequenced.The phylogenetic tree was constructed by using software Mega 4.0,and the molecular clock was drawn by using software BEASTv1.8.2.Results Twenty-six dengue virus strains were isolated between 2001 and 2015.They were all clustered into 2 genotypes,i.e.cosmopolitan genotype and Asian genotype Ⅰ.The strains isolated in Guangzhou shared high homology with Southeast Asian strains.The cosmopolitan genotype was divided into 2 sub-genotype at about 46 and 35 years ago.The substitution rate of dengue virus serotype 2 in Guangzhou was 7.1 × 10-4 per year per site.Conclusions There were close relationship between the Guangzhou strains and Southeast Asian strains.Guangzhou was at high risk of imported dengue fever,outbreak of dengue hemorrhagic fever and dengue shock syndrome.There might be two ways of introduction of cosmopolitan genotype.The substitution rate of the strains in Guangzhou was similar to that in the neighbor countries.
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Objective To investigate the molecular characteristics of dengue virus serotype 2 (DENV2) in Guangzhou during 2001-2015,and analyze the E gene of the strains isolated,the phylogenetic tree and molecular clock were constructed to know about the evolution of the strains.Methods The serum samples of the patients were detected by real time PCR,and positive samples were used to isolate dengue virus by using C6/36 cells.The E gene of the isolated strains were sequenced.The phylogenetic tree was constructed by using software Mega 4.0,and the molecular clock was drawn by using software BEASTv1.8.2.Results Twenty-six dengue virus strains were isolated between 2001 and 2015.They were all clustered into 2 genotypes,i.e.cosmopolitan genotype and Asian genotype Ⅰ.The strains isolated in Guangzhou shared high homology with Southeast Asian strains.The cosmopolitan genotype was divided into 2 sub-genotype at about 46 and 35 years ago.The substitution rate of dengue virus serotype 2 in Guangzhou was 7.1 × 10-4 per year per site.Conclusions There were close relationship between the Guangzhou strains and Southeast Asian strains.Guangzhou was at high risk of imported dengue fever,outbreak of dengue hemorrhagic fever and dengue shock syndrome.There might be two ways of introduction of cosmopolitan genotype.The substitution rate of the strains in Guangzhou was similar to that in the neighbor countries.
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Zika virus (ZIKV) is rapidly spreading across the America and its devastating outcomes for pregnant women and infants have driven this previously ignored pathogen into the limelight. Clinical manifestations are fever, joint pain or rash and conjunctivitis. Emergence of ZIKV started with a first outbreak in the Pacific area in 2007, a second large outbreak occurred in the Pacific in 2013/2014 and subsequently the virus spread in other Pacific islands. Threat of explosive global pandemic and severe clinical complications linked with the more immediate and recurrent epidemics necessitate the development of an effective vaccine. Several vaccine platforms such as DNA vaccine, recombinant subunit vaccine, ZIKV purified inactivated vaccine, and chimeric vaccines have shown potent efficacy in vitro and in vivo trials. Moreover, number of drugs such as Sofosbuvir, BCX4450, NITD008 and 7-DMA are ready to enter phase I clinical trial because of proven anti-ZIKV activity. Monoclonal based antibodies offer promise as an intervention effective for use in pregnant women. In this review, we describe the advances in research on ZIKV such as research strategies for the development of antiviral drugs & vaccines, molecular evolution, epidemiology emergence, neurological complications and other teratogenic outcomes as well as pathogenesis.
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Zika virus (ZIKV) is rapidly spreading across the America and its devastating outcomes for pregnant women and infants have driven this previously ignored pathogen into the limelight. Clinical manifestations are fever, joint pain or rash and conjunctivitis. Emergence of ZIKV started with a first outbreak in the Pacific area in 2007, a second large outbreak occurred in the Pacific in 2013/2014 and subsequently the virus spread in other Pacific islands. Threat of explosive global pandemic and severe clinical complications linked with the more immediate and recurrent epidemics necessitate the development of an effective vaccine. Several vaccine platforms such as DNA vaccine, recombinant subunit vaccine, ZIKV purified inactivated vaccine, and chimeric vaccines have shown potent efficacy in vitro and in vivo trials. Moreover, number of drugs such as Sofosbuvir, BCX4450, NITD008 and 7-DMA are ready to enter phase I clinical trial because of proven anti-ZIKV activity. Monoclonal based antibodies offer promise as an intervention effective for use in pregnant women. In this review, we describe the advances in research on ZIKV such as research strategies for the development of antiviral drugs & vaccines, molecular evolution, epidemiology emergence, neurological complications and other teratogenic outcomes as well as pathogenesis.
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Os microRNAs (miRNAs) são pequenos RNAs não codificadores de proteínas presentes na maioria dos eucariotos. Esses RNAs regulam a expressão gênica em nível pós-transcricional através do silenciamento de mRNAs-alvo que possuem sítios complementares às suas sequências, atuando em praticamente todos os processos celulares. Embora a estrutura e função dos miRNAs estejam bem caracterizadas, aspectos relacionados à sua organização genômica, evolução e atuação em doenças são tópicos que apresentam enormes lacunas. Nesta tese, utilizamos abordagens computacionais para investigar estes temas em três trabalhos. No primeiro, processamos e integramos um vasto volume de dados publicamente disponíveis referentes aos miRNAs e genes codificadores de proteínas para cinco espécies de vertebrados. Com isso, construimos uma ferramenta web que permite a fácil inspeção da organização genômica dos miRNAs em regiões inter e intragênicas, o acesso a dados de expressão de miRNAs e de genes codificadores de proteínas (classificados em genes hospedeiros e não hospedeiros de miRNAs), além de outras informações pertinentes. Verificamos que a ferramenta tem sido amplamente utilizada pela comunidade científica e acreditamos que ela possa facilitar a geração de hipóteses associadas à regulação dos miRNAs, principalmente quando estão inseridos em genes hospedeiros. No segundo estudo, buscamos compreender como o contexto genômico e a origem evolutiva dos genes hospedeiros influenciam a expressão e evolução dos miRNAs humanos. Nossos achados mostraram que os miRNAs intragênicos surgem preferencialmente em genes antigos (origem anterior à divergência de vertebrados). Observamos que os miRNAs inseridos em genes antigos têm maior abrangência de expressão do que os inseridos em genes novos. Surpreendentemente, miRNAs jovens localizados em genes antigos são expressos em um maior número de tecidos do que os intergênicos de mesma idade, sugerindo uma vantagem adaptativa inicial que pode estar relacionada com o controle da expressão dos genes hospedeiros, e como consequência, expondo-os a contextos celulares e conjuntos de alvos diversos. Na evolução a longo prazo, vimos que genes antigos conferem maior restrição nos padrões de expressão (menor divergência de expressão) para miRNAs intragênicos, quando comparados aos intergênicos. Também mostramos possíveis associações funcionais relacionadas ao contexto genômico, tais como o enriquecimento da expressão de miRNAs intergênicos em testículo e dos intragênicos em tecidos neurais. Propomos que o contexto genômico e a idade dos genes hospedeiros são fatores-chave para a evolução e expressão dos miRNAs. Por fim, buscamos estabelecer associações entre a expressão diferencial de miRNAs e a quimioresistência em câncer colorretal utilizando linhagens celulares sensíveis e resistentes às drogas 5-Fluoruracil e Oxaliplatina. Dentre os miRNAs identificados, o miR-342 apresentou níveis elevados de expressão nas linhagens sensíveis à Oxaliplatina. Com base na análise dos alvos preditos, detectamos uma significativa associação de miR-342 com a apoptose. A superexpressão de miR-342 na linhagem resistente SW620 evidenciou alterações na expressão de genes da via apoptótica, notavelmente a diminuição da expressão do fator de crescimento PDGFB, um alvo predito possivelmente sujeito à regulação direta pelo miR-342
MicroRNAs (miRNAs) are short non-coding RNAs found in most eukaryotic species. These RNAs regulate gene expression at post-transcriptional level by silencing target mRNAs through base-pairing of complementary sequences, thus acting on virtually all cellular processes. Although the structure and function of miRNAs are well understood, several aspects related to their genomic organization, evolution and involvement with diseases are largely underexplored. In this thesis, we employed computational methods to investigate such issues in three different studies. In the first one, we have processed and integrated a large amount of public data related to miRNAs and coding genes for five vertebrate species. Then, we developed a webtool to allow the analysis of the miRNA genomic context in inter and intragenic regions, the access of miRNA and gene expression data (classified as host and non-host genes), as well as other relevant information. We noticed that the webtool has been largely used by the scientific community, and we believe that it can facilitate hypothesis generation related to miRNA regulation, especially when they are within host genes. In the following study, we sought to understand how the genomic context and the evolutionary origin of host genes can affect the expression and evolution of human miRNAs. Our findings showed that intragenic miRNAs are preferentially embedded within old host genes (originated before the split of vertebrates). We observed that miRNAs within old genes are more broadly expressed than those within young genes. Surprisingly, young miRNAs within old genes were expressed in more tissues than their intergenic counterparts, suggesting an initial adaptive advantage which might be related to their hosts expression control, and as a consequence, exposing them to a more diverse cellular contexts and target genes. In the long run, we found that old host genes lead to expression constraints (lower expression divergence) between species for intragenic miRNAs, in respect to intergenic ones. We also showed possible functional associations related to miRNA genomic context, such as the enrichment of young intergenic miRNAs in testis, while young intragenic miRNAs were enriched in neural tissues. Thus, we propose that the genomic context and the age of the host genes are key factors in shaping the expression and evolution of miRNAs. Finally, we sought to establish associations between differential expression of miRNAs and chemoresistance in colorectal cancer using resistant and sensitive cell lines to 5-Fluoruracil and Oxaliplatin. Among differentially expressed miRNAs, miR-342 was highly expressed in sensitive cell lines to Oxaliplatin. Based on target prediction analysis, miR-342 is likely associated with apoptosis. The induced overexpression of miR-342 in SW620, a cell line resistant to Oxaliplatin, changed the expression levels of genes linked to the apoptosis pathway, notably the downregulation of PDGFB growth factor, which is a predicted target possibly subjected to direct regulation by miR-342