Identification of SNPs in genes potentially affected by domestication to efficiently detect hybridization between wild and domestic cats

Tese de mestrado em Biologia Evolutiva e do Desenvolvimento, apresentada à Universidade de Lisboa, através da Faculdade de Ciências, em 2018A crescente desflorestação e expansão das áreas afectadas pela presença e actividade humana no planeta têm tido inúmeras consequências directas e indirectas na abundância e sobrevivência de espécies sensíveis aos impactos antropogénicos. Entre estas espécies em Portugal destaca-se o gato-bravo europeu (Felis silvestris silvestris). A fragmentação e a degradação do habitat e a perseguição e a caça pelo Homem obrigaram este carnívoro de médio porte a refugiar-se em áreas geográficas restritas, isoladas e dispersas da Europa. Uma ameaça adicional, críptica e ainda pouco estudada, e potencialmente uma grande ameaça à conservação do gato-bravo europeu, é o seu contacto e interacção com o gato doméstico (Felis catus ou Felis silvestris catus). O gato doméstico é um animal de estimação popular, com uma população mundial estimada em 600 milhões de indivíduos. Estudos anteriores concluíram que o gato doméstico é o resultado de gerações de seleção artificial iniciada em gatos-bravos na pré-história humana, existindo evidências arqueológicas de relações entre gatos e homens desde há 9500 anos. Análises genéticas sugeriram que a área de origem do gato doméstico terá sido o Cresceste Fértil no Médio Oriente, nos primórdios da agricultura e da pecuária, onde a função dos gatos seria principalmente de controlo de roedores. Desde essa altura até tempos recentes a seleção artificial no gato doméstico terá tido uma intensidade relativamente moderada, mas a partir do século XIX esta selecção intensificou-se e, por razões de preferências estéticas, gerou uma abundância de raças com diferentes cores e padrões de pelagem, ainda que mantendo em geral a morfologia anatómica típica da espécie ancestral. Além de outros potenciais impactos, incluindo ecológicos, comportamentais, parasitológicos e epidemiológicos, um maior contacto entre gatos domésticos e gatos-bravos, tendo em conta a raridade da segunda espécie, aumenta o risco de hibridação entre as duas espécies. Observações de campo e capturas de indivíduos têm demonstrado a existência de híbridos. Sendo estes férteis e capazes de hibridar com indivíduos parentais, a hibridação entre as duas espécies, especialmente se for comum ou frequente, pode assim constituir uma grave ameaça para o gato-bravo, uma vez que os retrocruzamentos entre híbridos e gatos-bravos puros podem levar à introgressão de genes de gato doméstico no genoma do gato-bravo. Este processo pode conduzir à quebra da integridade genética do gato-bravo e, em última análise, à extinção da espécie. Como o gato-bravo é um predador de topo em muitos ecossistemas, a sua extinção pode ter consequências graves nas cadeias tróficas e redes ecológicas. Por conseguinte, é importante implementar medidas de conservação para o gato-bravo. A fim de monitorizar, controlar e reduzir a hibridação, são necessários métodos que sejam capazes de distinguir gatos-bravos puros de híbridos. Os primeiros métodos propostos com este objectivo eram baseados em caracteres anatómicos ou morfológicos, especialmente medidas cranianas e padrões da pelagem. Contudo, vários estudos demonstraram a dificuldade em utilizar estes métodos, bem como a sua ineficiência em certos casos, devido à grande semelhança morfológica frequentemente observada entre gatos-bravos puros e híbridos. Consequentemente, tem crescido o interesse no desenvolvimento e aplicação de abordagens baseadas em marcadores moleculares. Um dos marcadores genéticos com resultados promissores neste contexto são os microssatélites, que são unidades de pequenas repetições da mesma sequência de nucleótidos (por exemplo, AGAGAGAG) e que têm elevadas taxas de mutação em comparação com outras regiões do genoma. Através do uso de microssatélites, foi possível avaliar o grau de hibridação em várias regiões europeias, desde países como a Itália e Alemanha em que as populações de gatos-bravos e gatos domésticos ainda se encontram bastante diferenciadas, até aos casos da Escócia e Hungria, onde os níveis de introgressão são elevados. No entanto, devido à sua elevada taxa mutacional, os microssatélites tendem a sofrer de homoplasia, o que pode levar à falsa detecção de híbridos e a dificuldades em identificar e distinguir híbridos de gerações diferentes e retrocruzamentos. Assim, as atenções têm-se voltado para outro tipo de marcador molecular potencialmente útil em estudos de hibridação e que, devido à sua baixa taxa de mutação têm menos problemas de homoplasia do que os microssatélites: os “single nucleotide polymorphisms” (SNPs). Os SNPs consistem em variações na sequência de ADN num só nucleótido, o que faz com que sejam os polimorfismos mais abundantes no genoma. Tendo os SNPs frequentemente apenas duas variações alélicas, se estas tiverem frequências muito diferentes entre duas espécies ou populações, e considerando a geralmente limitada homoplasia nos SNPs, nesses casos os SNPs podem revelar-se altamente diagnósticos. Estudos anteriores têm apresentado painéis de SNPs com capacidade de identificar e distinguir gatos-bravos puros, gatos domésticos puros, e híbridos até à segunda geração. Porém, esses painéis contêm um elevado número de marcadores, não sendo portanto testes prácticos e eficientes. O facto de o poder estatístico desses painéis depender aparentemente do uso de um número elevado de SNPs é possivelmente consequência da maioria deles não estar fixada para alelos diferentes nas duas espécies. É expectável que esses SNPs fixados e altamente diagnósticos estejam em regiões do genoma onde exista elevada diferenciação entre gatos-bravos e gatos domésticos. Recentemente, um estudo procurou identificar as regiões que terão sido mais afectadas pela selecção e influentes na domesticação do gato doméstico. Esse estudo baseou-se na sequenciação do genoma de seis gatos-bravos e 22 gatos domésticos pertencentes a raças com origens geográficas distantes. Regiões do genoma exibindo elevados níveis de diferenciação genética entre gatos-bravos e gatos domésticos, identificados através de valores elevados do índice de fixação (Fst), e simultaneamente baixos níveis de variação intraespecífica, indicados por valores reduzidos de heterozigosidade (Hp), foram consideradas como potencialmente impactadas por selecção positiva. Assim, identificaram-se cinco regiões possivelmente influenciadas por seleção positiva. Estas regiões contêm vários genes envolvidos em diversos processos neurais, sendo muitos desses genes em particular determinantes do comportamento. Notavelmente, vários desses genes codificam proteínas que controlam a sobrevivência e migração das células da crista neural. Esta observação está de acordo e dá suporte à hipótese da síndrome da domesticação, uma teoria que postula que os caracteres morfológicos e comportamentais modificados em mamíferos domesticados têm origem em alterações na migração das células da crista neural durante o desenvolvimento embrionário. Com base nas descobertas desse estudo e de outros semelhantes em outros mamíferos domésticos, é possível focar em genes candidatos a pesquisa de marcadores potencialmente discriminantes entre mamíferos domésticos e os seus ancestrais selvagens. O presente estudo teve como objetivo descobrir, através da análise de genes candidatos, e testar um painel de SNPs com elevado poder diagnóstico para distinguir com fiabilidade diferentes classes de gatos puros e híbridos. Os genes candidatos estudados pertencem a três categorias funcionais diferentes: i) desenvolvimento neuronal, ii) cor e estrutura da pelagem, e iii) sistema sensorial. Para cada gene selecionado, um ou dois pares de “primers” foram desenhados para amplificar fragmentos de 700-900 pares de bases em amostras de gatos-bravos de Portugal, França e Roménia, e gatos domésticos portugueses. Um conjunto de 13 genes revelou SNPs com valores elevados de diferenciação (Fst > 0.8) entre gatos-bravos e gatos domésticos. Posteriormente, genótipos de híbridos de primeira geração (F1), segunda geração (F2), e retrocruzamentos com gatos-bravos (B x FSI) e com gatos domésticos (B x FCA) foram computacionalmente simulados com base nos genótipos das classes parentais amostrados. Usando os 13 SNPs, cerca de 90% dos indivíduos analisados foram corretamente identificados e atribuídos à sua categoria. Com base nestes resultados, constata-se preliminarmente que os SNPs identificados neste estudo possuem um valor diagnóstico potencialmente comparável ao de painéis com mais SNPs reportados em estudos anteriores. O painel aqui desenvolvido, carecendo obviamente de análises adicionais que confirmem os resultados obtidos, pode futuramente constituir um método eficaz e fiável para caracterizar padrões de hibridação e introgressão em populações de gato-bravo, facilitando o trabalho de indivíduos e instituições envolvidas na conservação da espécie na Europa.The European wildcat (F. s. silvestris) is a medium-sized carnivorous mammal that has suffered significant population decline and range contraction and fragmentation during the last century. The species is also currently threatened by human-mediated hybridization with domestic cats. Their hybrid offspring is fertile and can backcross with pure wildcats, leading to the introgression of domestic cat genes into the wildcat gene pool. This poses a danger to the genetic integrity of the European wildcat, and may contribute to its extinction. Hence, methods are urgently needed that are able to distinguish between pure wildcats and hybrids. However, the accurate detection of hybridization based on available morphological characters is particularly difficult, and even the panels of molecular markers that have been reported either are not highly reliable in detecting hybrids or are too cumbersome to be of practical value for routine use. Here, I aimed to identify single nucleotide polymorphisms (SNPs) located in genes that may have been affected during the domestication process of the domestic cat (F. s. catus). A total of 51 candidate genes were selected for sequencing of exonic and flanking intronic regions in samples of wildcats and domestic cats. Analysis of the resulting sequences revealed 13 SNPs with elevated genetic differentiation (Fst > 0.8) between the two species. Subsequently, genotypes of first-generation hybrids (F1), second-generation hybrids (F2), and backcrosses with wildcats (B x FSI) and with domestic cats (B x FCA), were simulated in order to evaluate the diagnostic power of the 13 SNP set. Admixture analysis of the simulated genotypes showed that the marker panel could identify and assign each genotype to the correct category, with an accuracy of about 90%. Given the current need for efficient and reliable tools to detect and discriminate samples from wildcats, domestic cats, and their hybrids, this small panel of 13 SNPs with apparently high diagnostic power offers the promise of a convenient and effective assay for surveying and monitoring the distribution and hybridization status of European wildcats

SUBJECT-SPECIFIC MUSCULOSKELETAL MODEL TO IDENTIFY MUSCLE CONTRIBUTION TO THE ACCELERATION PHASE IN ELITE SPRINTING

The purpose of this study was to identify which lower limb joint moments of force and lower limb muscles had the highest contribution to the forward and upward acceleration of the Body Centre of Gravity (BCG) in an elite sprinter during the first step after starting from blocks in 100 meters dash. An induced acceleration analysis approach was used to mechanically ascertain this contribution. Two models were used, one based in a simple multilink rigid body’s model and a second using a musculoskeletal model developed using Opensim. Our results showed a possible synergist action between hip and ankle extensors that allow an optimal combination that resulted in a forward propulsion of the BCG, this results seem to be corroborated using an Opensim musculoskeletal model

Arquitetura muscular e treino de força : influência do tipo de ação muscular e amplitude de movimento

Doutoramento em Motricidade Humana na especialidade de BiomecânicaIntrodução: O presente estudo teve como principal objetivo a análise da adaptabilidade da arquitetura muscular e das propriedades morfológicas e mecânicas do complexo músculo-tendinoso a estímulos de treino concêntricos e excêntricos, com e sem limitação da amplitude de movimento. Métodos: Foram desenhadas duas abordagens: uma análise transversal onde se avaliaram os efeitos crónicos de diferentes estímulos na adaptabilidade das propriedades arquiteturais e mecânicas do vasto externo, e uma investigação longitudinal, com o objetivo de verificar os efeitos do regime de contração muscular (concêntrico e excêntrico) e amplitude do movimento (total e parcial) nas variáveis mencionadas. O protocolo experimental envolveu a realização de contrações máximas dos extensores do joelho, em simultâneo com a aplicação das técnicas de electroestimulação e ultrassonografia. Foi ainda quantificado, por ressonância magnética, o volume muscular e o braço de momento do tendão patelar. Resultados: Os principais resultados mostraram que o regime de contração excêntrico constitui o melhor estímulo para o aumento do comprimento do fascículo, com implicação direta nas propriedades mecânicas do complexo músculo-tendinoso, e no desempenho de ações musculares de velocidade e potência. O regime de contração concêntrico privilegiou o aumento do ângulo de penação concorrendo para um aumento da área de secção transversal fisiológica. A utilização da amplitude total de movimento potenciou o ganho de massa muscular e de força geral, enquanto a amplitude parcial desenvolveu, sobretudo, a força nos ângulos treinados. Contrações parciais concêntricas privilegiaram o aumento do ângulo de penação, enquanto as excêntricas induziram um discreto aumento do comprimento do fascículo. Desde que equalizado o volume, ambas as condições promoveram idêntica melhoria na capacidade de produção de força. Conclusões: O estímulo excêntrico é essencial para o aumento do comprimento dos fascículos, com implicações diretas nas propriedades do complexo músculo-tendinoso. A redução da amplitude de movimento tem impacto na geometria fascicular, e desde que equalizado o volume, parece não condicionar os ganhos de força.ABSTRACT: The purpose of this study was to investigate the muscle architecture plasticity and the muscle-tendon mechanical properties adaptations to concentric and eccentric muscle contraction modes, with either a full or partial range of motion (ROM). Methods: To accomplish this goal, two approaches were designed: a cross-analysis aimed to evaluate the chronic effects of different mechanical stimulus on the vastus lateralis (VL) architectural and mechanical plasticity, and a longitudinal study to investigate the effects of the muscle contraction mode (concentric vs. eccentric) and the range of motion (full vs. partial) on the mentioned variables. The experimental protocol involved maximal voluntary isometric knee extension contractions, together with superimposed stimuli, and B-mode ultrasonography analysis. VL volume and patellar moment arm were quantified by MRI. Results: The main results showed that the eccentric contraction mode is a major factor for fascicle length adaptations with direct implication on muscle-tendon properties. The eccentric stimulus was also related to speed and power performance. Instead, the concentric training induced a higher increase in pennation angle and a lower change in fascicle length than eccentric training. These adaptations lead to a greater increase in physiological cross-sectional area after concentric training. The use of the full ROM improved muscle mass and overall strength, while partial ROM mainly improved the force at the trained angles. Concentric partial ROM muscle contractions increased pennation angle while eccentric partial ROM muscle contractions were related with discrete fascicle length increases. Since the volume was equalized, both full and partial ROM training conditions showed similar strength increases. Conclusion: The eccentric stimulus is a critical factor for fascicle length adaptations with direct implications on the muscle-tendon properties. The partial ROM influences fascicle geometry and, the strength gains seem to be unaffected as long as the training volume is similar.FCT - Fundação para a Ciência e a Tecnologi

THE RELATIONSHIPS BETWEEN SPRINT RUN AND STRENGTH PARAMETERS IN YOUNG ATHLETES AND NON-ATHLETES

The aim of this study was to examine the relationships between the sprint run and strength tests, which measure the capability to produce force in isometric, pure concentric and stretch shortening cycle muscular actions. The seventy two subjects were assessed for six tests 60m sprint run, to evaluate the speed, and isometric leg-press, squat jump, countermovement jump, drop jump, and 5 horizontal jumps, to evaluate strength parameters. The 5 jumps, was the most important predictor of the 60m sprint time, maybe because of is cyclic and horizontal characteristics (more similar to the specific movement of sprint run). We suppose that the reason for results do not explain different relationship between tests and sprint performance, in young athletes can be find in multifactorial characteristics of sprinting performance and the characteristics of the subjects, youngest have reduced training adaptation

A New Approach to Express Regional Adiposity and Its Association with Blood Lipids, Inflammation and Insulin Resistance Markers

Regional adiposity is classically related with inflammation, insulin resistance (IR) and an altered blood lipid profile. Traditionally, central adiposity expressed as a ratio (%FM) = fat mass/total mass of the region is the variable most used in research. However, this ratio does not account for total bodily fat mass (TFM). So, a great accumulation in a region, where lean mass is more important than fat, could be more associated with risk factors than other with less absolute fat (kg) but higher percentage (in instance abdomen). PURPOSE: To analyse the associations between the ratio (RW) = regional fat mass (kg) / TFM (kg) and blood lilid profile, IR and inflammation. METHODS: Sixty-two pre-menopausal and healthy women were recruited for this study (age, 39.6 ±7.2 years; BMI, 29.2 ±4.2 kg.m-2; total %TFM, 42.0 ±5.9%). Weight and height were measured to the nearest 0.1 kg and 0.1 cm respectively, and body composition was estimated with dual energy x-ray aborptiometry. RWF were calculated for trunk (RWT), lower limbs (RWLL) and upper limbs (RWUL). Insulin, glycaemia and insulin sensitivity (HOMA) were the IR markers. C-reactive protein (CRP) and Tumour Necrosis Factor (TNF-α) were the inflammation variables. Total cholesterol (TC), low-density lipoprotein (LDL), high-density lipoprotein (HDL) and triglycerides (TG) were also measured. Partial correlations adjusted to TFM between RW and blood markers were carried out. RESULTS: Significant correlations were found between RWUL and HOMA (r=0.276, P<0.05), TG (r=0.292, P<0.05) and insulin (r=0.271, P<0.05). RWT was also correlated with blood lipids: TG (r=0.278, P<0.05) and LDL (r=-0.318, P<0.05). Finally, RWLL was the ratio with more number of associations: HOMA (r=-0.274, P<0.05), TG (r=-0.342, P<0.01), LDL (r=0.317, P<0.05) and insulin (r=-0.253, P<0.05). Abdominal and trunk %FM were only correlated with HOMA (r=0.380, P<0.01) and LDL (r=0.264, P<0.05) respectively. CONCLUSION: In our sample, regional fat mass contribution (RW) for whole body FM showed more associations with blood markers than classical ratios (regional %FM). These findings highlighted RW ratios were well correlated with inflammatory and IR risk factors, which in turn could be useful to express positive or negative results in future studies. This issue requires further investigation.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tec

A Novel Plant-Based Protein Has Similar Effects Compared to Whey Protein on Body Composition, Strength, Power, and Aerobic Performance in Professional and Semi-Professional Futsal Players

IntroductionThe effects of dietary protein on body composition and physical performance seemingly depend on the essential amino acid profile of the given protein source, although controversy exists about whether animal protein sources may possess additional anabolic properties to plant-based protein sources. PurposeTo compare the effects of a novel plant-based protein matrix and whey protein supplementation on body composition, strength, power, and endurance performance of trained futsal players. MethodsFifty male futsal players were followed during 8 weeks of supplementation, with 40 completing the study either with plant-based protein (N = 20) or whey protein (N = 20). The following measures were assessed: bone mineral content, lean body mass, and fat mass; muscle thickness of the rectus femoris; total body water; blood glucose, hematocrit, C-reactive protein, aspartate aminotransferase, alanine aminotransferase, creatine kinase, creatinine, and estimated glomerular filtration rate; salivary cortisol; maximal strength and 1-RM testing of the back squat and bench press exercises; muscle power and countermovement jump; VO2max and maximal aerobic speed. Subjects were asked to maintain regular dietary habits and record dietary intake every 4 weeks through 3-day food records. ResultsNo differences in any variable were observed between groups at baseline or pre- to post-intervention. Moreover, no time*group interaction was observed in any of the studied variables, and a time effect was only observed regarding fat mass reduction. ConclusionsSupplementing with either a novel plant-based protein matrix or whey protein did not affect any of the variables assessed in high-level futsal players over 8 wks. These results suggest that whey protein does not possess any unique anabolic properties over and above those of plant-based proteins when equated to an essential amino acid profile in the population studied. Furthermore, when consuming a daily protein intake >1.6 g/kg BW.day(-1), additional protein supplementation does not affect body composition or performance in trained futsal players, regardless of protein type/source

Tissue Oxygenation in Response to Different Relative Levels of Blood-Flow Restricted Exercise

Blood flow restrictive (BFR) exercise elicits a localized hypoxic environment compatible with greater metabolic stress. We intended to compare the acute changes in muscle microvascular oxygenation following low-intensity knee extension exercise, combined with different levels of BFR. Thirteen active young men (age: 23.8 ± 5.4 years) were tested for unilateral knee extension exercise (30 + 15 + 15 + 15 reps at 20% one repetition maximum) on four different conditions: no-BFR (NOBFR), 40, 60, and 80% of arterial occlusion pressure (AOP). Deoxyhemoglobin+myoglobin concentration Deoxy[Hb+Mb], total hemoglobin [T(H+Mb)] and tissue oxygen saturation [TOI] were measured on the vastus lateralis muscle using near-infrared spectroscopy (NIMO, Nirox srl, Brescia, Italy). The magnitude of change in Deoxy[Hb+Mb]during exercise was similar between 60 and 80% AOP. Overall, compared to that seen during 60 and 80% AOP, NOBFR as well as 40% AOP resulted in a lower magnitude of change in Deoxy[Hb+Mb] (p &lt; 0.05). While the oxygen extraction decreased during each inter-set resting interval in NOBFR and 40% AOP, this was not the case for 60 or 80% AOP. Additionally, TOI values obtained during recovery from each set of exercise were similarly affected by all conditions. Finally, our data also show that, when performed at higher restrictive values (60 and 80%), BFR exercise increases total Deoxy[Hb+Mb] extraction (p &lt; 0.05). Taken together, we provide evidence that BFR is effective for increasing deoxygenation and reducing tissue oxygenation during low-intensity exercise. We also showed that when using low loads, a relative pressure above 40% of the AOP at rest is required to elicit changes in microvascular oxygenation compared with the same exercise with unrestricted conditions

A Novel Plant-Based Protein Has Similar Effects Compared to Whey Protein on Body Composition, Strength, Power, and Aerobic Performance in Professional and Semi-Professional Futsal Players

IntroductionThe effects of dietary protein on body composition and physical performance seemingly depend on the essential amino acid profile of the given protein source, although controversy exists about whether animal protein sources may possess additional anabolic properties to plant-based protein sources.PurposeTo compare the effects of a novel plant-based protein matrix and whey protein supplementation on body composition, strength, power, and endurance performance of trained futsal players.MethodsFifty male futsal players were followed during 8 weeks of supplementation, with 40 completing the study either with plant-based protein (N = 20) or whey protein (N = 20). The following measures were assessed: bone mineral content, lean body mass, and fat mass; muscle thickness of the rectus femoris; total body water; blood glucose, hematocrit, C-reactive protein, aspartate aminotransferase, alanine aminotransferase, creatine kinase, creatinine, and estimated glomerular filtration rate; salivary cortisol; maximal strength and 1-RM testing of the back squat and bench press exercises; muscle power and countermovement jump; VO2max and maximal aerobic speed. Subjects were asked to maintain regular dietary habits and record dietary intake every 4 weeks through 3-day food records.ResultsNo differences in any variable were observed between groups at baseline or pre- to post-intervention. Moreover, no time*group interaction was observed in any of the studied variables, and a time effect was only observed regarding fat mass reduction.ConclusionsSupplementing with either a novel plant-based protein matrix or whey protein did not affect any of the variables assessed in high-level futsal players over 8 wks. These results suggest that whey protein does not possess any unique anabolic properties over and above those of plant-based proteins when equated to an essential amino acid profile in the population studied. Furthermore, when consuming a daily protein intake &gt;1.6 g/kg BW.day−1, additional protein supplementation does not affect body composition or performance in trained futsal players, regardless of protein type/source

Biomechanical Performance Factors in the Track and Field Sprint Start: A Systematic Review

In athletics sprint events, the block start performance can be fundamental to the outcome of a race. This Systematic Review aims to identify biomechanical factors of critical importance to the block start and subsequent first two steps performance. A systematic search of relevant English-language articles was performed on three scientific databases (PubMed, SPORTDiscus, and Web of Science) to identify peer-reviewed articles published until June 2021. The keywords &ldquo;Block Start&rdquo;, &ldquo;Track and Field&rdquo;, &ldquo;Sprint Running&rdquo;, and &ldquo;Kinetics and Kinematics&rdquo; were paired with all possible combinations. Studies reporting biomechanical analysis of the block start and/or first two steps, with track and field sprinters and reporting PB100m were sought for inclusion and analysis. Thirty-six full-text articles were reviewed. Several biomechanical determinants of sprinters have been identified. In the &ldquo;Set&rdquo; position, an anthropometry-driven block setting facilitating the hip extension and a rear leg contribution should be encouraged. At the push-off, a rapid extension of both hips and greater force production seems to be important. After block exiting, shorter flight times and greater propulsive forces are the main features of best sprinters. This systematic review emphasizes important findings and recommendations that may be relevant for researchers and coaches. Future research should focus on upper limbs behavior and on the analysis of the training drills used to improve starting performance