Engenharia genética de Saccharomyces cerevisiae para cofermentação de xilose e glicose e produção de etanol de segunda geração

Orientador: Leandro Vieira dos SantosDissertação (mestrado) - Universidade Estadual de Campinas, Instituto de BiologiaResumo: A matriz energética mundial tem como base o uso de compostos fósseis, como o petróleo, gás e carvão. Entretanto, nas últimas décadas, a comunidade mundial manifestou uma intensa preocupação relacionada às consequências causadas pelo uso desses compostos, como o aumento dos gases causadores do efeito estufa. O etanol de segunda-geração (2G), produzido a partir biomassa lignocelulósica, surge com um grande potencial na produção de energia limpa a partir de uma matriz renovável. Entretanto, diversos desafios tecnológicos precisam ser superados visando uma produção economicamente viável de etanol 2G. O desenvolvimento de linhagens modificadas da levedura Saccharomyces cerevisiae, capazes de converter o açúcar de cinco carbonos xilose (C5) em etanol, é um passo essencial na viabilização da tecnologia 2G, tendo em vista sua significativa parcela na composição da biomassa. Um grande gargalo na implementação dessa tecnologia é a baixa eficiência de assimilação de xilose por transportadores de membrana de S. cerevisiae e a repressão catabólica, levando a inibição por glicose (C6). Proteínas de membrana capazes de transportar xilose são inibidas em altas concentrações desse açúcar, além de apresentarem baixa afinidade a xilose em relação a glicose, levando a uma competição entre os substratos e estendendo o tempo da fermentação. A xilose somente é assimilada pelas células de S. cerevisiae quando as concentrações de glicose estão baixas no ambiente extracelular. Dessa forma, este projeto teve como objetivo a caracterização de novos transportadores heterólogos de xilose eficientes e o desenvolvimento de uma linhagem capaz de co-fermentar xilose e glicose. Neste trabalho, identificamos e caracterizamos quatro novos transportadores de xilose Cs186, Cs2608, Cs3894 e Cs4130, avaliados na linhagem EBY.VW4000 modificada com a via de consumo de xilose integrada no genoma. Curiosamente, o transportador Cs4130 demonstrou superior capacidade de assimilação de xilose em altas concentrações (50 g/L) em comparação com o controle GXF1, considerado o melhor transportador heterólogo previamente descrito na literatura. Na condição descrita, GXF1 foi severamente inibido, demonstrando um comportamento oposto ao observado por Cs4130. O modelo estrutural de Cs4130 em comparação com o transportador procariótico de alta afinidade XylE e GXF1 aponta resíduos em sua arquitetura molecular que possam explicar as diferenças de comportamento observadas entre os transportadores. Dessa forma, o novo transportador Cs4130 é apresentado como um excelente candidato para engenharia genética de linhagens de S. cerevisiae para a produção de biocompostos 2G. Entretanto, os transportadores de xilose Cs4130 e GXF1 ainda apresentam uma forte inibição pela glicose na mistura dos dois açúcares. Dessa maneira, a segunda parte do projeto visa a modificação dessas proteínas de membrana e o desenvolvimento de um processo para o aumento da afinidade a xilose em relação à glicose. Para isso, desenvolvemos duas novas plataformas de evolução adaptativa, EBY_Xyl1_hxk0 derivada da linhagem EBY_Xyl1, e a linhagem JBY_hxk0 construída a partir de uma cepa industrial fermentadora de xilose. Estas novas plataformas não são capazes de metabolizar moléculas de glicose devido as deleções dos genes das hexoquinases HXK1, HXK2 e GLK1, responsáveis pela primeira etapa da via da glicolítica. As células modificadas de S. cerevisiae irão reconhecer as moléculas de glicose apenas como inibidores ao metabolismo da xilose. Por fim, a identificação de novas mutações presentes nas células evoluídas derivadas das plataformas construídas, capazes de co-fermentar xilose e glicose, visam identificar novos alvos moleculares relacionados a repressão catabólica. As informações desenvolvidas nesse projeto irão auxiliar no desenvolvimento de linhagens de S. cerevisiae capazes de fermentar simultaneamente xilose e glicose, reduzindo o tempo final de fermentação na indústria 2G.Abstract: The global energy matrix is based on fossil fuels, such as oil, gas and coal. In recent decades, the world community has expressed an intense concern related to the consequences caused by the use of these compounds, such as the increase in greenhouse gas emissions. Second-generation (2G) ethanol, produced from lignocellulosic biomass, emerges as promise sustainable advanced biofuel. However, several biotechnological challenges need to be overcome in order to produce economically viable 2G ethanol. The development of engineered Saccharomyces cerevisiae yeast strains, capable of converting xylose, the second most abundant sugar on lignocellulosic biomass, into ethanol, is an essential step to a feasible 2G process. A major bottleneck in the conversion of bioproducts from these biomass-derived sugars is the low xylose uptake by S. cerevisiae membrane transporters and catabolic repression, leading to glucose inhibition (C6). Membrane proteins capable to transport xylose are strongly inhibited on high concentrations of this sugar, in addition to presenting low affinity to xylose compared to glucose, leading to competition between substrates and extending the fermentation time. Xylose is only assimilated by S. cerevisiae cells when glucose concentrations are low in the extracellular environment. Therefore, this project aimed to identify novel and efficient heterologous xylose transporters and to develop a strain capable of co-fermenting xylose and glucose. In this work, we identified and characterized four new xylose transporters Cs186, Cs2608, Cs3894 and Cs4130, evaluated in EBY.VW4000 strain modified with the xylose consumption pathway integrated into the genome. Curiously, the Cs4130 transporter demonstrated a superior capacity of xylose assimilation at high xylose concentrations (50 g/L) compared to the GXF1 control, considered the best heterologous transporter previously described in the literature. In the condition described, GXF1 was severely inhibited, demonstrating an opposite behavior compared to that observed by Cs4130. The structural model of Cs4130 compared to the high affinity prokaryotic transporter XylE and GXF1 points out residues in its molecular architecture that may explain the differences in behavior observed between the transporters. The novel transporter Cs4130 is presented as an excellent candidate for genetic engineering of S. cerevisiae strains for 2G biocompounds production. However, the xylose transporters Cs4130 and GXF1 show strong glucose inhibition on mixtures of both sugars. Therefore, the second part of the project aims to engineering these membrane proteins and to develop a process to increase the affinity of xylose compared to glucose. We have developed two new microbial platforms for adaptive evolution, EBY_Xyl1_hxk0, derived from EBY_Xyl1 strain, and the JBY_hxk0 constructed from an industrial high-yield pentose fermenting strain. These new platforms are unable to metabolize glucose molecules due to the disruption of glycolysis pathway by knockout of hexokinase genes, HXK1, HXK2 and GLK1. The S. cerevisiae modified strains recognized glucose molecules only as an inhibitor to xylose metabolism. Therefore, new genetic mutations present in these evolved cells, capable to co-consume xylose and glucose, will provide new data to understand the genetic basis of glucose repression. The information produced in this project will help the development of S. cerevisiae strains suitable of simultaneously fermenting xylose and glucose, reducing the total fermentation time in the 2G industry.MestradoGenética de MicroorganismosMestre em Genética e Biologia Molecular0012018/00888-5CAPESFAPES

O benefício do envio da mensagem de texto no aumento de adesão à terapia antirretroviral em pacientes portadores do vírus da imunodeficiência adquirida

RESUMO: A infecção por HIV se manifesta como uma pandemia silenciosa e violenta, ainda sem cura, que depende da adesão ao tratamento para garantir melhor qualidade de vida aos pacientes. O objetivo da revisão foi identificar e analisar as produções acerca da utilização de aparelhos celulares por meio de SMS para o envio de lembretes, com o objetivo de aumentar a adesão aos portadores de HIV em tratamento. Para a concretização da presente revisão, foram utilizadas plataformas online de pesquisa como LILACS, Medline/PubMed e SciELO, com descritores que remetiam ao tema proposto, a fim de encontrar artigos para realizar a presente revisão, dos quais foram selecionados 6 artigos de 2014 a 2017. Apesar de limitações socioeconômicas e de confidencialidade, devido ao estigma sobre a doença, observou-se que a utilização de SMS é benéfica na adesão à TARV, além de contribuir como possível apoio nas diárias dificuldades vivenciadas pelos enfermos e na perspectiva favorável de qualidade de vida. Entretanto, reconhecemos que são necessários maiores estudos sobre a intervenção a longo prazo, a influência do meio (rural ou urbano), horário e dia de envio das mensagens na adesão ao tratamento, abrindo-se portas para novas pesquisas que sejam capazes de aumentar a efetividade da tecnologia e ultrapassar empecilhos como a difícil aquisição de celulares por famílias de baixa renda e, sobretudo, evitar-se constrangimentos ou até mesmo o isolamento social dos portadores do HIV &nbsp

Uso de telas pela população pediátrica e seus impactos oftalmológicos a curto e a longo prazo: uma revisão sistemática : Use of screens by the pediatric population and its short and long-term ophthalmological impacts: a systematic review

Ao se discorrer sobre a formação e formatação da nossa sociedade, pode-se notar que cada vez mais esta está permeada pela tecnologia e pelos sistemas integrativos, processo esse benéfico para comunicação, gestão e tomada de decisões para além de ser uma ferramenta de trabalho e entretenimento, porém, com a progressiva quantidade de horas que ficamos expostos a este tipo de tecnologia também podem acarretar prejuízos a acuidade visual dos usuários. Juntamente a isso, nota-se que as crianças estão tendo cada vez mais precocemente acesso os equipamentos eletrônicos e deixando de lado as brincadeiras, desta forma a partir de uma pesquisa qualitativa, feita a partir de um levantamento bibliográfico, pode-se dispor que esta busca compreender quais são os sintomas e efeitos do uso excessivo deste tipo de ferramenta e algumas formas de tratamento e profilaxia que podem ser dispostas sobre o tema, com enfoque na população pediátrica

Pervasive gaps in Amazonian ecological research

Biodiversity loss is one of the main challenges of our time,1,2 and attempts to address it require a clear un derstanding of how ecological communities respond to environmental change across time and space.3,4 While the increasing availability of global databases on ecological communities has advanced our knowledge of biodiversity sensitivity to environmental changes,5–7 vast areas of the tropics remain understudied.8–11 In the American tropics, Amazonia stands out as the world’s most diverse rainforest and the primary source of Neotropical biodiversity,12 but it remains among the least known forests in America and is often underrepre sented in biodiversity databases.13–15 To worsen this situation, human-induced modifications16,17 may elim inate pieces of the Amazon’s biodiversity puzzle before we can use them to understand how ecological com munities are responding. To increase generalization and applicability of biodiversity knowledge,18,19 it is thus crucial to reduce biases in ecological research, particularly in regions projected to face the most pronounced environmental changes. We integrate ecological community metadata of 7,694 sampling sites for multiple or ganism groups in a machine learning model framework to map the research probability across the Brazilian Amazonia, while identifying the region’s vulnerability to environmental change. 15%–18% of the most ne glected areas in ecological research are expected to experience severe climate or land use changes by 2050. This means that unless we take immediate action, we will not be able to establish their current status, much less monitor how it is changing and what is being lostinfo:eu-repo/semantics/publishedVersio

Pervasive gaps in Amazonian ecological research

Biodiversity loss is one of the main challenges of our time,1,2 and attempts to address it require a clear understanding of how ecological communities respond to environmental change across time and space.3,4 While the increasing availability of global databases on ecological communities has advanced our knowledge of biodiversity sensitivity to environmental changes,5,6,7 vast areas of the tropics remain understudied.8,9,10,11 In the American tropics, Amazonia stands out as the world's most diverse rainforest and the primary source of Neotropical biodiversity,12 but it remains among the least known forests in America and is often underrepresented in biodiversity databases.13,14,15 To worsen this situation, human-induced modifications16,17 may eliminate pieces of the Amazon's biodiversity puzzle before we can use them to understand how ecological communities are responding. To increase generalization and applicability of biodiversity knowledge,18,19 it is thus crucial to reduce biases in ecological research, particularly in regions projected to face the most pronounced environmental changes. We integrate ecological community metadata of 7,694 sampling sites for multiple organism groups in a machine learning model framework to map the research probability across the Brazilian Amazonia, while identifying the region's vulnerability to environmental change. 15%–18% of the most neglected areas in ecological research are expected to experience severe climate or land use changes by 2050. This means that unless we take immediate action, we will not be able to establish their current status, much less monitor how it is changing and what is being lost

Global burden of 288 causes of death and life expectancy decomposition in 204 countries and territories and 811 subnational locations, 1990–2021: a systematic analysis for the Global Burden of Disease Study 2021

BACKGROUND Regular, detailed reporting on population health by underlying cause of death is fundamental for public health decision making. Cause-specific estimates of mortality and the subsequent effects on life expectancy worldwide are valuable metrics to gauge progress in reducing mortality rates. These estimates are particularly important following large-scale mortality spikes, such as the COVID-19 pandemic. When systematically analysed, mortality rates and life expectancy allow comparisons of the consequences of causes of death globally and over time, providing a nuanced understanding of the effect of these causes on global populations. METHODS The Global Burden of Diseases, Injuries, and Risk Factors Study (GBD) 2021 cause-of-death analysis estimated mortality and years of life lost (YLLs) from 288 causes of death by age-sex-location-year in 204 countries and territories and 811 subnational locations for each year from 1990 until 2021. The analysis used 56 604 data sources, including data from vital registration and verbal autopsy as well as surveys, censuses, surveillance systems, and cancer registries, among others. As with previous GBD rounds, cause-specific death rates for most causes were estimated using the Cause of Death Ensemble model-a modelling tool developed for GBD to assess the out-of-sample predictive validity of different statistical models and covariate permutations and combine those results to produce cause-specific mortality estimates-with alternative strategies adapted to model causes with insufficient data, substantial changes in reporting over the study period, or unusual epidemiology. YLLs were computed as the product of the number of deaths for each cause-age-sex-location-year and the standard life expectancy at each age. As part of the modelling process, uncertainty intervals (UIs) were generated using the 2·5th and 97·5th percentiles from a 1000-draw distribution for each metric. We decomposed life expectancy by cause of death, location, and year to show cause-specific effects on life expectancy from 1990 to 2021. We also used the coefficient of variation and the fraction of population affected by 90% of deaths to highlight concentrations of mortality. Findings are reported in counts and age-standardised rates. Methodological improvements for cause-of-death estimates in GBD 2021 include the expansion of under-5-years age group to include four new age groups, enhanced methods to account for stochastic variation of sparse data, and the inclusion of COVID-19 and other pandemic-related mortality-which includes excess mortality associated with the pandemic, excluding COVID-19, lower respiratory infections, measles, malaria, and pertussis. For this analysis, 199 new country-years of vital registration cause-of-death data, 5 country-years of surveillance data, 21 country-years of verbal autopsy data, and 94 country-years of other data types were added to those used in previous GBD rounds. FINDINGS The leading causes of age-standardised deaths globally were the same in 2019 as they were in 1990; in descending order, these were, ischaemic heart disease, stroke, chronic obstructive pulmonary disease, and lower respiratory infections. In 2021, however, COVID-19 replaced stroke as the second-leading age-standardised cause of death, with 94·0 deaths (95% UI 89·2-100·0) per 100 000 population. The COVID-19 pandemic shifted the rankings of the leading five causes, lowering stroke to the third-leading and chronic obstructive pulmonary disease to the fourth-leading position. In 2021, the highest age-standardised death rates from COVID-19 occurred in sub-Saharan Africa (271·0 deaths [250·1-290·7] per 100 000 population) and Latin America and the Caribbean (195·4 deaths [182·1-211·4] per 100 000 population). The lowest age-standardised death rates from COVID-19 were in the high-income super-region (48·1 deaths [47·4-48·8] per 100 000 population) and southeast Asia, east Asia, and Oceania (23·2 deaths [16·3-37·2] per 100 000 population). Globally, life expectancy steadily improved between 1990 and 2019 for 18 of the 22 investigated causes. Decomposition of global and regional life expectancy showed the positive effect that reductions in deaths from enteric infections, lower respiratory infections, stroke, and neonatal deaths, among others have contributed to improved survival over the study period. However, a net reduction of 1·6 years occurred in global life expectancy between 2019 and 2021, primarily due to increased death rates from COVID-19 and other pandemic-related mortality. Life expectancy was highly variable between super-regions over the study period, with southeast Asia, east Asia, and Oceania gaining 8·3 years (6·7-9·9) overall, while having the smallest reduction in life expectancy due to COVID-19 (0·4 years). The largest reduction in life expectancy due to COVID-19 occurred in Latin America and the Caribbean (3·6 years). Additionally, 53 of the 288 causes of death were highly concentrated in locations with less than 50% of the global population as of 2021, and these causes of death became progressively more concentrated since 1990, when only 44 causes showed this pattern. The concentration phenomenon is discussed heuristically with respect to enteric and lower respiratory infections, malaria, HIV/AIDS, neonatal disorders, tuberculosis, and measles. INTERPRETATION Long-standing gains in life expectancy and reductions in many of the leading causes of death have been disrupted by the COVID-19 pandemic, the adverse effects of which were spread unevenly among populations. Despite the pandemic, there has been continued progress in combatting several notable causes of death, leading to improved global life expectancy over the study period. Each of the seven GBD super-regions showed an overall improvement from 1990 and 2021, obscuring the negative effect in the years of the pandemic. Additionally, our findings regarding regional variation in causes of death driving increases in life expectancy hold clear policy utility. Analyses of shifting mortality trends reveal that several causes, once widespread globally, are now increasingly concentrated geographically. These changes in mortality concentration, alongside further investigation of changing risks, interventions, and relevant policy, present an important opportunity to deepen our understanding of mortality-reduction strategies. Examining patterns in mortality concentration might reveal areas where successful public health interventions have been implemented. Translating these successes to locations where certain causes of death remain entrenched can inform policies that work to improve life expectancy for people everywhere. FUNDING Bill & Melinda Gates Foundation

14-Week exercise training modifies the DNA methylation levels at gene sites in non-Alzheimer's disease women aged 50 to 70 years

Exercise training emerges as a key strategy in lifestyle modification, capable of reducing the risk of developing Alzheimer's disease (AD) due to risk factors such as age, family history, genetics and low level of education associated with AD. We aim to analyze the effect of a 14-week combined exercise training (CT) on the methylation of genes associated with AD in non-alzheimer's disease women. CT sessions lasted 60 min, occurring three times a week for 14 weeks. Forty non-Alzheimer's disease women aged 50 to 70 years (60.7 ± 4.1 years) with a mean height of 1.6 ± 0.1 m, mean weight of 73.12 ± 9.0 kg and a mean body mass index of 29.69 ± 3.5 kg/m2, underwent two physical assessments: pre and post the 14 weeks. DNA methylation assays utilized the EPIC Infinium Methylation BeadChip from Illumina. We observed that 14 weeks of CT led to reductions in systolic (p = 0.001) and diastolic (p = 0.017) blood pressure and improved motor skills post-intervention. Among 25 genes linked to AD, CT induced differentially methylated sites in 12 genes, predominantly showing hypomethylated sites (negative β values). Interestingly, despite hypomethylated sites, some genes exhibited hypermethylated sites (positive β values), such as ABCA7, BDNF, and WWOX. A 14-week CT regimen was adequate to induce differential methylation in 12 CE-related genes in healthy older women, alongside improvements in motor skills and blood pressure. In conclusion, this study suggest that combined training can be a strategy to improve physical fitness in older individuals, especially able to induce methylation alterations in genes sites related to development of AD. It is important to highlight that training should act as protective factor in older adults.</p

Effect of age and cyclical heat stress on the serum biochemical profile of broiler chickens

Objetivou-se avaliar a influência da idade e do estresse cíclico por calor durante uma hora por dia, nas concentrações das proteínas, metabólitos e enzimas séricas em frangos de corte de 21 a 42 dias de idade. Foram utilizados 420 pintos de corte machos, da linhagem Cobb Avian48TM, em um delineamento inteiramente casualizado, composto de dois tratamentos e seis repetições. Os tratamentos foram constituídos de dois ambientes térmicos: um para frangos de corte criados em condições naturais de temperatura e umidade do primeiro ao 42° dia de idade (controle); e outro para estressados por calor à 36 °C do 16° ao 42° dia, durante uma hora do dia (12h00m às 13h00m). No 21º, 28º, 35º e 42º dias de idade, foram coletados em duas aves por repetição aproximadamente 5mL de sangue por punção cardíaca. Foram determinadas em cada amostra de soro as concentrações de proteína total, albumina, globulinas, relação A/G, aspartato aminotransferase, alanina aminotransferase, gama glutamiltransferase, ácido úrico, creatinina, colesterol total, triacilgliceróis, lipoproteínas de alta densidade, lipoproteínas de muito baixa densidade, lipoproteínas de baixa densidade e glicose. Para as concentrações de glicose, ácido úrico, alanina aminotransferase, triacilgliceróis e lipoproteínas de muito baixa densidade, não houve diferenças significativas para ambiente e idade. Observou-se interação significativa entre ambiente e idade para os valores de colesterol e lipoproteínas de baixa densidade. Aos 21 dias de idade o colesterol e as LDL-C foram maiores nas aves em estresse comparados àquelas do tratamento controle, o que não ocorreu nas demais idades. A aspartato aminotransferase nos frangos sob estresse cíclico de calor foi maior em comparação ao tratamento controle e apresentou efeito cúbico para ambiente. A idade das aves influenciou os valores de proteínas totais, albumina, globulina, albumina: globulina, creatinina, gama glutamiltransferase e das lipoproteínas de alta densidade. Conclui-se que o estresse cíclico de calor por uma hora, a 36ºC, do 16º ao 42º dia de idade aumenta aspartato aminotransferase, enquanto colesterol e LDL-C são aumentados somente aos 21 dias, indicando que as aves possam ter se adaptado. A idade influencia os valores de AST, GGT, creatinina, proteínas totais, albumina, globulinas, albumina:globulinas, colesterol, HDL-C e LDL-C.This study aimed to evaluate the influence of age and cyclical heat stress for 1 hour per day on the levels of serum proteins, metabolites, and enzymes in broiler chickens of 21-42 days of age. We used 420 male broiler chickens, Cobb Avian48TM breed, in a completely randomized experimental design, composed of two treatments and six replicates. The treatments consisted of two thermal environments: one in which broiler chickens were raised under natural conditions of temperature and humidity for the first 42 days of age (control) and another in which chickens were subjected to heat stress at 36°C, from days 16 to 42, for 1 hour per day (1200-1300 hours). At 21, 28, 35, and 42 days of age, approximately 5 mL of blood was collected by cardiac puncture in two birds per replicate. In each serum sample, levels of total protein, albumin, globulin, albumin:globulin ratio, aspartate aminotransferase (AST), alanine aminotransferase (ALT), and gamma-glutamyltransferase (GGT) activity and uric acid, creatinine, total cholesterol, triglycerides, high-density lipoproteins (HDL), very low density lipoproteins (VLDL), low-density lipoproteins (LDL), and glucose levels were determined. Concerning the environment and the age, no significant differences in the levels of glucose, uric acid, ALT, triglycerides, and very low density lipoproteins were observed. There was a significant interaction between the environment and the age for cholesterol and LDL values. At 21 days of age, cholesterol and LDL-cholesterol (LDL-C) were higher in birds under stress than in the control treatment birds, which did not occur in other age groups. Aspartate aminotransferase activity was higher in chickens under cyclical heat stress than in chickens subjected to control treatment and displayed a cubic response regarding the environment. The age of the birds influenced the values of total protein, albumin, globulin, albumin:globulin (A:G) ratio, creatinine, GGT, and HDL. In conclusion, cyclical heat stress, at 36ºC, for one hour, from days 16 to 42 of age increases serum AST, whereas cholesterol and LDL-C levels increase only at day 21, indicating that birds may have adapted to heat stress. Age influenced the activity of AST and GGT and levels of creatinine, total protein, albumin, globulin, A:G ratio, cholesterol, HDL-C, and LDL-C

A critical appraisal of multicomponent training protocols for older adults: a scoping review

Our study aimed to review the literature regarding the multicomponent training protocols prescribed for the older adult