Caste Specific Pheromone-Related Gene Expression in Honeybee Mandibular Glands

Dissertação de mestrado em Biocinética, apresentada à Faculdade de Ciências do Desporto e Educação Física da Universidade de CoimbraO presente trabalho teve como principal objetivo determinar a influência dos valores da curva força-velocidade dos flexores e extensores do joelho no remate de futebol. Foram avaliados sete atletas amadores de futebol (25,1 ± 1,97 anos), os quais foram submetidos a dois tipos de testes. Sendo o primeiro cinemático, através da filmagem de execução de um remate de futebol. Posteriomente, foi feita uma análise no programa kinovea, para determinar a velocidade de saida de bola, variáveis temporais e comportamento angular. Para a recolha dos dados isocinéticos foi utilizado um dinamómetro (Biodex Multijoint System 3 Pro 2) para cálculo da força máxima do membro inferior dos sujeitos. Na análise cinemática do futebol a variável velocidade máxima do joelho foi a que obteve maior correlação com a velocidade de saída de bola (r=0.96; P≤0.001). As variáveis, Distância do pé à bola e a Percentagem da posição do centro de gravidade obtiveram valores de correlação significativos (r=0.81 e r= 0.83; P≤ 0.01) correspondentemente. Por fim, as variáveis, ângulo da cintura pélvica e deslocamento angular do joelho em extensão, também mostraram correlações significativas (r=0.76 e r=0.70; P≤0.05) com a velocidade de saída de bola. Na correlação entre variáveis cinemáticas do remate de futebol com os testes isocinéticos, constatou-se que das três variáveis analisadas, os resultados das diferenças dos valores dos torques máximos em todas as velocidades de teste e o valor dos ângulos nos torques máximos obtiveram maior número de correlações significativas (ambas com 12 correlações cada para P≤0.05), já os valores dos torques máximos apenas obtiveram quatro correlações significativas para P≤0.05. Concluindo, a variável cinemática mais importante para o remate de futebol é a velocidade máxima do joelho, sendo que só é possível a sua análise a partir da utilização de novas tecnologias no treino. Relativamente aos testes isocinéticos, verificamos que a aplicação da curva força-velocidade nos testes isocinéticos apresenta mais informações do que os valores dos torques máximos. Palavras-chave: Futebol; Análise Cinemática; Testes Isocinéticos; Curva Força-Velocidade. iv Abstract The purpose of this study was to establish the values which influence the force-velocity curve of flexors and knee extensors on the soccer kick. Our subjects were seven amateur soccer players (25.1 ± 1.97 years), who underwent two types of tests. Therefore, the first Kinematic test was the avaluation of the soccer kick and maximal instep kicking with the preferred leg. Afterwards, an analysis on Kinovea program was made in order to calculate the ball speed, the angular behavior, and temporal variables. For the collection of data we used isokinetic dynamometers ((Biodex System 3 Pro Multijoint 2) to calculate the maximum strength of the lower member of the subjects. In kinematics football analysis, the variable maximum knee speed, was the one with the highest correlation with the ball speed (r = 0.96; P≤0.001). The variables, distance from the ball to the foot and the percentage of the center of gravity position obtained significant correlation (r = 0.81 and r = 0.83; P ≤ 0.01) correspondingly. Finally, the variables angle of the pelvis and the knee angular movement in extension, also showed a significant correlation (r = 0.76 and r = 0.70; P≤0.05) with the ball speed. As far as the correlation between kinematic variables football kicking with the isokinetic testing is concerned, it was found that the three variables in study, which were the results of the differences of the values of the maximum torque in all test speed and the value of the angles the maximum torques obtained more significant correlations (both with 12 correlations for each P≤0.05), since the values of the maximum torques only had four significant correlations for p≤0.05. In conclusion, the most important kinematics variable for the football kick is the maximum speed of the knee, and it is only possible to analyse by using new technologies in training. For isokinetic tests, we found that the application of the force-velocity curve in isokinetic tests gave us more information than the values of the maximum torque

Теоретические основы интенсификации работы грануляционных устройств с усовершенствованной гидродинамикой

Одним із способів зменшення габаритів грануляційного обладнання є вдосконалення гідродинамічних умов перебування в ньому дисперсної фази. Цього можна досягти, зокрема, за рахунок застосування вихрових і високотурбулізованних потоків. Представлена робота присвячена обґрунтуванню можливості створення алгоритму управління рухом дисперсної фази в робочому просторі грануляційного пристрою, на підставі якого буде визначена його оптимальна конструкція з мінімальними габаритами.Одним из способов уменьшения габаритов грануляционного оборудования является усовершенствование гидродинамических условий пребывания в нём дисперсной фазы. Этого можно достичь, в частности, за счет применения вихревых и высокотурбулизованных потоков. Представленная работа посвящена обоснованию возможности создания алгоритма управления движением дисперсной фазы в рабочем пространстве грануляционного устройства, на основании которого будет определена его оптимальная конструкция с минимальными габаритами

Distribution of Bemisia tabaci in different agro-ecological regions in Uganda and the threat of vector-borne pandemics into new cassava growing areas

Previous studies in sub-Saharan Africa have showed the spread of cassava mosaic disease (CMD) and cassava brown streak disease (CBSD) pandemics into different cassava growing regions by high Bemisia tabaci populations. Studies did indicate that there were stark differences in some whitefly species, yet they have not looked extensively across agroecologies. Members of B. tabaci species complex termed sub-Saharan Africa 1 (SSA1) and SSA2 have been linked to the spread of CMD and CBSD viruses. During the period of a severe CMD pandemic in the 1990s, SSA2 was the most predominant until the resurgence of SSA1, particularly SSA1-subgroup1 (SSA1-SG1) from the early 2000s to date. Cassava being a drought resilient crop has become an important food security crop and has been introduced into new areas and regions. Considering the role B. tabaci in the spread of cassava virus pandemics into neighboring regions, we investigated the genetic diversity and distribution of B. tabaci in nine different agro-ecological regions of Uganda in 2017. Adult whiteflies were collected from cassava and 33 other host plants from cassava-growing areas, those with limited cassava and areas with no cassava, where it is being introduced as a food security crop. The partial sequences of the mitochondrial cytochrome oxidase 1 (mtCO1) gene (657 bp) were used to determine the phylogenetic relationships between the sampled B. tabaci. Cassava B. tabaci SSA1 (-SG1, -SG2, -Hoslundia (previously called SSA1-SG1/2), -SG3), SSA2 and SSA3; non-cassava B. tabaci SSA6, SSA10, SSA11, SSA12, SSA13, MED-ASL, MED-Q1, MEAM1, Indian Ocean; and other Bemisia species, Bemisia afer and Bemisia Uganda1 were identified in the study. SSA3, one of the key B. tabaci species that occurs on cassava in West Africa, was identified for the first time in Uganda. The SSA1-SG1 was widely distributed, predominated on cassava and was found on 17 other host-plants. The ability of SSA1-SG1 to exist in environments with limited or no cassava growing poses the risk of continued spread of virus pandemics. Therefore, measures must be put in place to prevent the introduction of diseased materials into new areas, since the vectors exist

The molecular mechanisms that determine different degrees of polyphagy in the Bemisia tabaci species complex

The whitefly Bemisia tabaci is a closely related group of >35 cryptic species that feed on the phloem sap of a broad range of host plants. Species in the complex differ in their host‐range breadth, but the mechanisms involved remain poorly understood. We investigated, therefore, how six different B. tabaci species cope with the environmental unpredictability presented by a set of four common and novel host plants. Behavioral studies indicated large differences in performances on the four hosts and putative specialization of one of the species to cassava plants. Transcriptomic analyses revealed two main insights. First, a large set of genes involved in metabolism (>85%) showed differences in expression between the six species, and each species could be characterized by its own unique expression pattern of metabolic genes. However, within species, these genes were constitutively expressed, with a low level of environmental responsiveness (i.e., to host change). Second, within each species, sets of genes mainly associated with the super‐pathways “environmental information processing” and “organismal systems” responded to the host switching events. These included genes encoding for proteins involved in sugar homeostasis, signal transduction, membrane transport, and immune, endocrine, sensory and digestive responses. Our findings suggested that the six B. tabaci species can be divided into four performance/transcriptomic “Types” and that polyphagy can be achieved in multiple ways. However, polyphagy level is determined by the specific identity of the metabolic genes/pathways that are enriched and overexpressed in each species (the species' individual metabolic “tool kit”)

Data from: Asymmetric adaptation to indolic- and aliphatic-glucosinolates in the B and Q sibling species of Bemisia tabaci (Hemiptera: Aleyrodidae)

The role glucosinolates play in defending plants against delicate phloem feeders such as aphids and whiteflies is currently not clear as these herbivores may avoid bringing glucosinolates from the phloem sap into contact with myrosinase enzymes. Here, we investigated the effects of high levels of aliphatic- and indolic-glucosinolates on life history traits and detoxification gene expression in two sibling species, B and Q, of the whitefly Bemisia tabaci. High levels of aliphatic-glucosinolates decreased the average oviposition rate of both species, and reduced the survival and developmental rate of Q nymphs. High levels of indolic-glucosinolates decreased the oviposition rate and survival of nymphal stages of the B species and the developmental rate of both species. Molecular analyses revealed two major asymmetries between the B and Q species. First, specific GST genes (BtGST1 and BtGST2) were significantly induced during exposure to indolic-glucosinolates only in Q. This may reflect the genes putative involvement in indolic-glucosinolates detoxification and explain the species' good performance on plants accumulating indolic-glucosinolates. Second, the constitutive expression of eight of the ten detoxification genes analyzed was higher in the Q species than in the B species. Interestingly, four of these genes were induced in B in response to high levels of glucosinolates. It seems, therefore, that the B and Q species differ in their "optimal defense strategy". B utilizes inducible defenses which are profitable if the probability of experiencing the stress is small and its severity is low, while Q invests significant resources in being always "ready" for a challenge

CLKGR is present in the nucleus, and competitively inhibits CLK function.

<p><b>A.</b> CLKGR protein is expressed in large amounts in TIM-CLKGR flies. Western blot from fly heads collected at CT15 using an anti-CLK antibody. The assay was performed from TIM-CLKGR flies and control flies (<i>tim-gal4/+</i>). Arrows indicate CLK or the CLKGR fusion protein, which can be distinguished by their size. <b>B.</b> CLKGR is present in both the nuclei and cytoplasm in TIM-CLKGR flies. Western blot from nuclear and cytoplasmic extracts of control (<i>tim-gal4/+</i>) or TIM-CLKGR fly heads collected at CT15. TUBULIN staining is shown as negative control for the nuclear fraction separation and positive control for the cytoplasm fraction. HISTONE-3 staining is shown as positive control for nuclear separation and negative control for the cytoplasm separation. <b>C.</b> CLKGR expression can inhibit CLK-mediated activity in <i>Drosophila</i> S2 cells. <i>Drosophila</i> S2 cells were transfected with <i>vri-luciferase</i> reporter plasmid, pAc-CLK plasmid, a plasmid that express CLK or CLKGR under regulation of a copper inducible promoter (metallothionein; MT-CLK or MT-CLKGR respectively), and a plasmid used for controlling transfection efficiency (pCopia-Renilla). No copper or two different amounts of copper were utilized as indicated in the graph. Experiment was done at three separate biological repeats. Plot shows average values of biological duplicates of one representative experimental repeat. Error bars represent standard deviation. One-way Anova was performed to determine statistical significance. *<i>p</i><0.05. <b>D.</b> CLKGR can bind to CLK targets promoters. We induced CLKGR expression in S2 cells using the MT-CLKGR plasmid; in parallel to constant expression of CYCVP16 (from the pAc-CYCVP16 expressing plasmid). CYCVP16 and CLKGR together activate CLK-driven transcription suggesting they bind to CLK targets promoters. Experiment was done at three separate biological repeats. Plot shows average values of duplicates of one representing repeat. Error bars indicate standard deviation. CLK-target activity was measured using a <i>vri</i>-luciferase reporter and values were normalized to a transfection control (pCopia-Renilla). T-test was performed to determine statistical significance between time points. ***<i>p</i><0.001.</p

Synergistic Interactions between the Molecular and Neuronal Circadian Networks Drive Robust Behavioral Circadian Rhythms in <i>Drosophila melanogaster</i>

<div><p>Most organisms use 24-hr circadian clocks to keep temporal order and anticipate daily environmental changes. In <i>Drosophila melanogaster</i> CLOCK (CLK) and CYCLE (CYC) initiates the circadian system by promoting rhythmic transcription of hundreds of genes. However, it is still not clear whether high amplitude transcriptional oscillations are essential for circadian timekeeping. In order to address this issue, we generated flies in which the amplitude of CLK-driven transcription can be reduced partially (approx. 60%) or strongly (90%) without affecting the average levels of CLK-target genes. The impaired transcriptional oscillations lead to low amplitude protein oscillations that were not sufficient to drive outputs of peripheral oscillators. However, circadian rhythms in locomotor activity were resistant to partial reduction in transcriptional and protein oscillations. We found that the resilience of the brain oscillator is depending on the neuronal communication among circadian neurons in the brain. Indeed, the capacity of the brain oscillator to overcome low amplitude transcriptional oscillations depends on the action of the neuropeptide PDF and on the <i>pdf</i>-expressing cells having equal or higher amplitude of molecular rhythms than the rest of the circadian neuronal groups in the fly brain. Therefore, our work reveals the importance of high amplitude transcriptional oscillations for cell-autonomous circadian timekeeping. Moreover, we demonstrate that the circadian neuronal network is an essential buffering system that protects against changes in circadian transcription in the brain.</p></div

TIM-CLKGR flies display impaired function of peripheral oscillators.

<p><b>A.</b> Eclosion circadian gating is absent in TIM-CLKGR flies and in flies that express CLKGR under P{GawB}Mai60 driver (MAI60-CLKGR). The Mai60 driver express predominantly in the prothoracic gland. The eclosion ratio is calculated by determining the portion of flies that emerged in two hours intervals over the total amount of flies that emerged in 24 hours. The experiments were performed in TIM-CLKGR and MAI60-CLKGR flies and for the control lines UAS-CLKGR (UAS-<i>ClkGR/+</i>), TIMGAL4 (<i>tim-gal4/+</i>) and MAI60GAL4 (flies that carry the P{GawB}Mai60 insertion). Values are the means of 3 or 4 biological repeats (20 to 32 flies in each repeat) Error bars represent SEM. One-way Anova was performed to determine statistical significance of the differences between timepoints, for TIMGAL4, UAS-CLKGR and MAI60GAL4. <i>p</i><0.01. Experiment was performed in LD conditions <b>B.</b> Total sleep is not affected in TIM-CLKGR flies. Male flies were kept in 12∶12 LD conditions. Sleep was measured for 5 days. Sleep data was analyzed using pySolo software. Fly strains: TIM-CLKGR (n = 35), TIM-CLKGR(X2) (n = 32) and control flies TIMGAL4 (<i>tim-gal4/+</i>,n = 28) and UAS-CLKGR (UAS-<i>ClkGR/+</i>,n = 31). Values represent means and errors bars represent SEM. <b>C.</b> Expression of CLKGR leads to a dose dependent increase in the number of sleep episodes during the dark phase. Experimental conditions and genotypes as described in B. One-way Anova was performed to determine statistical significance of the differences between fly strains. <i>p</i><0.0001. <b>D.</b> Expression of CLKGR leads to a dose dependent decrease in the average length of the sleep episodes during the dark phase. Conditions and genotypes are as described in B. One-way Anova was performed to determine statistical significance of the differences between fly strains, <i>p</i><0.0001.</p

Behavioral characterization of flies expressing CLKGR.

<p>Behavioral analysis of flies maintained for 10 days in constant darkness (DD 1–10). Fly strains: TIM-CLKGR (<i>tim-gal4</i>; UAS-<i>ClkGR</i>), TIM-CLKGR(X2) (<i>tim-gal4</i>; UAS-<i>ClkGR/</i>UAS-<i>ClkGR</i>), TIMGAL4 (<i>tim-gal4/+</i>) and UAS-CLKGR (UAS-<i>ClkGR/+</i>). Period of rhythmic flies, rhythmic flies percentage and average power were calculated by chi square power p<0.05. SEM is shown in brackets.</p