Efeito da depleção de MAP-Quinases em Trypanosoma brucei

Orientador : Prof. Dr. Marco Aurélio KriegerCo-orientador : Prof. Dr. Fabrício Klerynton MarchiniTese (doutorado) - Universidade Federal do Paraná, Setor de Ciências Biológicas, Programa de Pós-Graduação em Biologia Celular e Molecular. Defesa: Curitiba, 29/08/2013Inclui referênciasÀrea de concentração: Biologia celular e molecularResumo: Os Tripanossomatídeos possuem um ciclo de vida complexo, alternando entre os ambientes distintos encontrados nos seus hospedeiros. Os mecanismos envolvidos na resposta do parasita às mudanças do ambiente ainda são pouco conhecidos, apesar de muito estudados. Dentre estes mecanismos, a fosforilação de proteínas possui um papel fundamental nas adaptações do parasita ao meio. Alguns dados, como a ausência de tirosino-quinases no genoma, a super representação de famílias de quinases, a existência de proteíno-quinases únicas, entre outros, evidenciam o processo de fosforilação como um tópico de grande interesse em Tripanossomatídeos. O protozoário Trypanosoma brucei possui 176 proteínoquinases, divididas em diversas famílias, sendo que uma das mais bem estudadas, devido à sua posição central em processos biológicos essenciais, é o das quinases ativadas por mitógeno (MAPK). Tem sido descrita a participação destas proteínas em processos como divisão e diferenciação celular, resposta ao estresse celular, entre outros. No entanto, pouco é conhecido sobre a influência destas quinases na sinalização celular dos Tripanossomatídeos. Diante disso, o objetivo deste trabalho foi avaliar a influência da depleção de MAPKs sobre T. brucei. Dentre os resultados obtidos está a realização de curvas de crescimento, onde se verificou diminuição na proliferação celular em quase metade dos knockdowns analisados. O proteoma, fosfoproteoma e transcriptoma de um dos knockdowns (Tb927.10.10870) foi avaliado quantitativamente por proteômica baseada em espectrometria de massas combinada com marcação metabólica por SILAC e por RNA-Seq. No total, 1.511 proteínas, 1.510 sítios de fosforilação e cerca de 12 milhões de reads foram obtidos. Apesar da forte diminuição na proliferação celular deste knockdown, foi evidenciada pouca modulação no proteoma, fosfoproteoma e transcriptoma, indicando uma robustez na via de sinalização em estudo, corroborando com estudos publicados. Dentre as proteínas que sofreram modulação, aproximadamente um terço está relacionado à funções mitocondriais. Os sítios de fosforilação modulados após depleção da MAPK em estudo pertencem a proteínas com diferentes funções, no entanto com uma predominância de proteínas associadas à estabilização / degradação de mRNAs. A fim de se avaliar a interação entre os membros da via das MAPKs de T. brucei, fez-se a análise das interações através de duplo híbrido. Das 1.024 interações testadas, 22 foram positivas. A comparação com as interações entre os ortólogos destas proteínas, descritas em organismos modelo, não demonstrou sobreposição de nenhuma das interações positivas. Os resultados deste trabalho podem conduzir novos experimentos a fim de melhor compreender a via de sinalização das MAPKs no Tripanossomatídeos.Abstract: Trypanosomatids have a complex life cycle switching between different hosts. In this way, they find distinct environments where quick adaptation is needed. Among the cell signaling mechanisms, protein phosphorylation, driven by protein kinases and protein phosphatases, is an important process with potential to supply this necessity. Some finds on Trypanosomatids' genomes, such as, the absence of dedicated tyrosine or tyrosine kinase-like receptors, the over-representation of some kinase groups, the presence of unique kinases, and other, highlight the phosphorylation in Trypanosomatids as a hot spot topic. The flagellate parasite Trypanosoma brucei possesses 176 protein kinases classified in many families, among them, the mitogen activated protein kinase (MAPKs) family. Several publications have been documented the participation of this family in processes like cellular division and differentiation, in response to stress conditions, and other. Nevertheless little is known about its function in Trypanosomatids. In this sense, this work aimed to evaluate the effects of MAPK depletion on T. brucei. As a start point, the participation of MAPKs in the cellular proliferation was addressed. The results showed that almost a half of MAPK knockdowns had the cell proliferation rate affected. One of these knockdowns (Tb927.10.10870) was also quantitatively analyzed at the level of proteins, phosphoproteins, and mRNA transcripts by mass spectrometry based proteomics combined with metabolic labeling (SILAC) and by RNA-Seq. In total, 1,511 proteins, 1,510 phosphorylation sites, and nearly of 12 million of reads were identified. Despite the cell proliferation rate was strongly affected in this knockdown, few modulations in the proteome, phosphoproteome or transcriptome was observed. In consonance with other works, this could represent the robustness of MAPK signaling pathway in Trypanosomatids. Between the few modulations is worthy of note some mitochondrial proteins and phosphorylation sites on proteins related to mRNA stability control. As the MAPKs act in phosphorylation cascades, we also evaluate the interaction between the members of this signaling pathway by two hybrid assay. Among the 1,024 tested interactions, 22 were positive, showing no overlap with the interactions described for their ortologues in model organisms. Take together, the finds of this work have the potential to drive future projects in order to well understand the MAPK signaling pathway in Trypanosomatids

Agronomic and physiological indices for reproductive stage heat stress tolerance in green super rice

Optimum growing temperature is necessary for maximum yield-potential in any crop. The global atmospheric temperature is changing more rapidly and irregularly every year. High temperature at the flowering/reproductive stage in rice causes partial to complete pollen sterility, resulting in significant reduction in grain yield. Green Super Rice (GSR) is an effort to develop an elite rice type that can withstand multiple environmental stresses and maintain yield in different agro-ecological zones. The current study was performed to assess the effect of heat stress on agronomic and physiological attributes of GSR at flowering stage. Twenty-two GSR lines and four local checks were evaluated under normal and heat-stress conditions for different agro-physiological parameters, including plant height (PH), tillers per plant (TPP), grain yield per plant (GY), straw yield per plant (SY), harvest index (HI), 1000-grain weight (GW), grain length (GL), cell membrane stability (CMS), normalized difference vegetative index (NDVI), and pollen fertility percentage (PFP). Genotypes showed high significant variations for all the studied parameters except NDVI. Association and principal component analysis (PCA) explained the genetic diversity of the genotypes, and relationship between the particular parameters and grain yield. We found that GY, along with other agronomic traits, such as TPP, SY, HI, and CMS, were greatly affected by heat stress in most of the genotypes, while PH, GW, GL, PFP, and NDVI were affected only in a few genotypes. Outperforming NGSR-16 and NGSR-18 in heat stress could be utilized as a parent for the development of heat-tolerant rice. Moreover, these findings will be helpful in the prevention and management of heat stress in rice

ARPN Journal of Agricultural and Biological Science GENETIC VARIBAILITY AND CORRELATION ANALYSIS FOR YIELD COMPONENTS IN MUNGBEAN (Vigna radiata L. Wilezek)

ABSTRACT 20 Mungbean (Vigna radiata L. Wilezek) genotypes were tested for genetic variability and correlation among different yield contributing traits i.e., plant height, pods plant -1 , pod length, seed pod -1 , biological yield, and grain yield. Maximum plant height was observed for genotype NFM5-63-19 cm; maximum number of pods plant -1 was recorded for genotype NFM5-63-19, while; genotype NFM-12-8 and NFM-6-5 found with a maximum pod length. Similarly the maximum number of seed pod -1 , biological yield and grain yield was observed in genotype NFM-6-5, NFM-12-6 and NM-98 respectively. The high heritability was recorded for pod length (99%) and plant height (70%), while pods plant -1 (29%) and seed pod -1 (17%) had low heritability. Based on genotypic correlation analysis characters like plant height, pods plant -1 , pod length and on phenotypic basis grain yield and seed pod -1 could be the best criteria in any breeding program for increasing yield in mungbean genotypes under agro-climatic conditions of Peshawar

Association analysis uncovers the genetic basis of general combining ability of 11 yield-related traits in parents of hybrid rice

Association analysis between constructed single nucleotide polymorphism linkage disequilibrium blocks (SNPLDBs) and general combining ability (GCA) effects is a novel approach to uncover the genetic basis of GCA within the sequence genomes of parents of hybrid rice. Here, we calculated the GCA effect values of 33 parents of hybrid rice and sequenced them to identify genome-wide single nucleotide polymorphisms (SNPs). In total, 64.6 % of the uniquely mapped paired-end short reads revealed a final total of 291 959 SNPs between the 33 parental genomes and the Nipponbare reference genome. The identified SNPs were non-randomly distributed among all chromosomes of rice, whereas one-fourth of the SNPs were situated in the exonic regions with 16 % being non-synonymous. Further, the identified SNPs were merged and optimized for construction of 2612 SNPLDB markers, using linkage disequilibrium information. The single-factor analysis of variance-based association method between the constructed SNPLDB markers and GCA effects values detected 99 significant SNPLDBs for GCA of 11 yield-related traits. The associated SNPLDB markers explained 26.4 % of phenotypic variations with traits, on average. We mined 50 favourable GCA alleles at the associated SNPLDBs regions, distributed across the 33 parental genomes. The parental genomes possessed a small number of favourable GCA alleles for studied traits, with the exception of days to heading and plant height. Our results suggest that the identified GCA alleles could be used to improve the GCA performance of parents of hybrid rice through optimal crossing design. Moreover, favourable GCA alleles should be incorporated in the parental genomes through marker-assisted selection experiments, and the parental lines carrying more alleles could be utilized in breeding as superior parents for developing rice hybrids of desirable characteristics

Favorable alleles mining for gelatinization temperature, gel consistency and amylose content in Oryza sativa by association mapping

Abstract Background Improving the gelatinization temperature (GT), gel consistency (GC) and amylose content (AC) for parental grain eating and cooking qualities (ECQs) are key factors for enhancing average grain ECQs for hybrid japonica rice. Results In this study, a genome-wide association mapping (GWAS) for ECQs was performed on a selected sample of 462 rice accessions in 5 environments using 262 simple sequence repeat markers. We identified 10 loci and 27 favorable alleles for GT, GC and AC, and some of these loci were overlapped with starch synthesis-related genes. Four SSR loci for the GT trait were distributed on chromosomes 3, 5, 8, and 9, of which two SSR loci were novel. Two SSR loci associated with the GC trait were distributed on chromosomes 3 and 6, although only one SSR locus was novel. Four SSR loci associated with the AC trait were distributed on chromosomes 3, 6, 10, and 11, of which three SSR loci were novel. The novel loci RM6712 and RM6327 were simultaneously identified in more than 2 environments and were potentially reliable QTLs for ECQs, with 15 parental combinations being predicted. These QTLs and parental combinations should be used in molecular breeding to improve japonica rice average ECQs. Conclusions Among the 10 SSR loci associated with GT, GC and AC for grain ECQs detected in 27 favorable alleles, the favorable allele RM3600-90bp on chromosome 9 could significantly reduce GT, RM5753-115bp on chromosome 6 could significantly increase GC, and RM6327-230bp on chromosome 11 could significantly reduce AC in hybrid japonica rice mixed rice samples

Estimation of Genetic Variances and Stability Components of Yield-Related Traits of Green Super Rice at Multi-Environmental Conditions in Pakistan

Identifying adopted Green Super Rice (GSR) under different agro-ecological locations in Pakistan is crucial to sustaining the high productivity of rice. For this purpose, the multi-location trials of GSR were conducted to evaluate the magnitude of genetic variability, heritability, and stability in eight different locations in Pakistan. The experimental trial was laid out in a randomized complete block (RCB) design with three replications at each location. The combined analysis of variance (ANOVA) manifested significant variations for tested genotypes (g), locations (L), years (Y), genotype × year (GY), and genotype × location (GL) interactions revealing the influence of environmental factors (L and Y) on yield traits. High broad-sense heritability estimates were observed for all the studied traits representing low environmental influence over the expression of traits. Noticeably, GSR 48 showed maximum stability than all other lines in the univariate model across the two years for grain yield and related traits data. Multivariate stability analysis characterized GSR 305 and GSR 252 as the highest yielding with optimum stability across the eight tested locations. Overall, Narowal, Muzaffargarh, and Swat were the most stable locations for GSR cultivation in Pakistan. In conclusion, this study revealed that G×E interactions were an important source of rice yield variation, and its AMMI and biplots analysis are efficient tools for visualizing the response of genotypes to different locations

Inter Simple Sequence Repeat-Based Genetic Divergence and Varietal Identification of Banana in Pakistan

Banana is one of the major cash and fruit crops of Pakistan. The lack of information concerning genetic diversity and purity within locally cultivated banana varieties is a major bottleneck in improving its genetics. Due to the existence of a narrow genetic background, it’s quite important to find genomic variations in banana varieties. DNA marker-based techniques have been used to effectively characterize banana varieties. In the current study, Inter Simple Sequence Repeat (ISSR) markers were used to characterize banana cultivars and to assess the genetic diversity of 14 local banana varieties grown in Pakistan. Out of the 45 primers used, 40 primers revealed reproducible results and produced 121 polymorphic bands, which contributed a ratio of 47.87 polymorphism. The ISSR UBC-835 and UBC-834 possessed the highest PIC ranged between (86–88%) in banana varieties, while the lowest PIC (46%) was detected in the case of UBC−857 marker with (100–1500 bp) PCR product size. Pairwise Jaccard’s similarity coefficient values were also calculated, and these were ranged from 0.56–0.88. Multivariate analysis divided 14 banana varieties into two distinct groups—A and B respectively—and furthermore into subgroups, clusters, and sub−clusters. Our results indicated that at the molecular level, the banana varieties in group—A were found to be 66% similar whereas in group B were 88% similar. Nei’s genetic diversity, PCA analysis, and a minimum spanning tree depicted Fenjiao, Dajiao, and NIGAB-2 as the most diverse members as compared to all other varieties of the three populations. Out of 14 varieties used, 11 varieties were uniquely identified by 54 polymorphic ISSR bands of different sizes. Some varieties like NIGAB-2 and NIGAB-3 were uniquely identified only with one band while others were tagged by multiple unique bands. In future, this study will be utilized to establish a molecular-based protocol for the identification of banana varieties

Effects of pre-operative isolation on postoperative pulmonary complications after elective surgery: an international prospective cohort study an international prospective cohort study

We aimed to determine the impact of pre-operative isolation on postoperative pulmonary complications after elective surgery during the global SARS-CoV-2 pandemic. We performed an international prospective cohort study including patients undergoing elective surgery in October 2020. Isolation was defined as the period before surgery during which patients did not leave their house or receive visitors from outside their household. The primary outcome was postoperative pulmonary complications, adjusted in multivariable models for measured confounders. Pre-defined sub-group analyses were performed for the primary outcome. A total of 96,454 patients from 114 countries were included and overall, 26,948 (27.9%) patients isolated before surgery. Postoperative pulmonary complications were recorded in 1947 (2.0%) patients of which 227 (11.7%) were associated with SARS-CoV-2 infection. Patients who isolated pre-operatively were older, had more respiratory comorbidities and were more commonly from areas of high SARS-CoV-2 incidence and high-income countries. Although the overall rates of postoperative pulmonary complications were similar in those that isolated and those that did not (2.1% vs 2.0%, respectively), isolation was associated with higher rates of postoperative pulmonary complications after adjustment (adjusted OR 1.20, 95%CI 1.05–1.36, p = 0.005). Sensitivity analyses revealed no further differences when patients were categorised by: pre-operative testing; use of COVID-19-free pathways; or community SARS-CoV-2 prevalence. The rate of postoperative pulmonary complications increased with periods of isolation longer than 3 days, with an OR (95%CI) at 4–7 days or ≥ 8 days of 1.25 (1.04–1.48), p = 0.015 and 1.31 (1.11–1.55), p = 0.001, respectively. Isolation before elective surgery might be associated with a small but clinically important increased risk of postoperative pulmonary complications. Longer periods of isolation showed no reduction in the risk of postoperative pulmonary complications. These findings have significant implications for global provision of elective surgical care. We aimed to determine the impact of pre-operative isolation on postoperative pulmonary complications after elective surgery during the global SARS-CoV-2 pandemic. We performed an international prospective cohort study including patients undergoing elective surgery in October 2020. Isolation was defined as the period before surgery during which patients did not leave their house or receive visitors from outside their household. The primary outcome was postoperative pulmonary complications, adjusted in multivariable models for measured confounders. Pre-defined sub-group analyses were performed for the primary outcome. A total of 96,454 patients from 114 countries were included and overall, 26,948 (27.9%) patients isolated before surgery. Postoperative pulmonary complications were recorded in 1947 (2.0%) patients of which 227 (11.7%) were associated with SARS-CoV-2 infection. Patients who isolated pre-operatively were older, had more respiratory comorbidities and were more commonly from areas of high SARS-CoV-2 incidence and high-income countries. Although the overall rates of postoperative pulmonary complications were similar in those that isolated and those that did not (2.1% vs 2.0%, respectively), isolation was associated with higher rates of postoperative pulmonary complications after adjustment (adjusted OR 1.20, 95%CI 1.05–1.36, p = 0.005). Sensitivity analyses revealed no further differences when patients were categorised by: pre-operative testing; use of COVID-19-free pathways; or community SARS-CoV-2 prevalence. The rate of postoperative pulmonary complications increased with periods of isolation longer than 3 days, with an OR (95%CI) at 4–7 days or ≥ 8 days of 1.25 (1.04–1.48), p = 0.015 and 1.31 (1.11–1.55), p = 0.001, respectively. Isolation before elective surgery might be associated with a small but clinically important increased risk of postoperative pulmonary complications. Longer periods of isolation showed no reduction in the risk of postoperative pulmonary complications. These findings have significant implications for global provision of elective surgical care