Creation of a multiple-use recombinant inbred line population for the development of molecular markers in soft white winter wheat

Tese de doutoramento em Física (Pré-Bolonha), especialidade de Física Experimental, apresentada à Faculdade de Ciências e Tecnologia da Universidade de CoimbraPositron emission tomography based on resistive plate chambers (RPC-PET) has been proposed for both preclinical and clinical applications. We firstly present imaging results of needle-like and planar 22Na sources obtained with a prototype of a high-acceptance small-animal RPC-PET. The two detector modules utilized in this experiment had an effective front face of 6.4 x 6.4 cm^2 and consisted of 5 gas gaps and 6 glass electrodes with a total thickness of 5 mm. The data included lines of response (LORs) inclined up to 58º, and the depth of interaction (DOI) was accurately measured, demonstrating the parallax-free property inherent to RPC-PET. The maximum likelihood expectation-maximization (MLEM) reconstruction of the acquired data yielded an excellent and stable resolution of 0.4 mm full width at half maximum (FWHM). Concurrently, we pursued studies of a suggested whole-body single-bed RPC-PET. It has been shown by simulation that RPC-PET with an axial field-of-view (AFOV) of 2.4 m is feasible and yields an absolute sensitivity at least one order of magnitude superior to that of typical crystal-based PET scanners. In addition, RPC-PET offers an important time-of-flight (TOF) advantage and provides a potentially very-high spatial resolution at the detector level. In the second part of this work, a fully three-dimensional reconstruction algorithm capable of processing the very inclined LORs from large AFOV systems such as RPC-PET is demonstrated. It relies on the application of a TOF-based-kernel into the MLEM algorithm. With the 300 ps FWHM time resolution, already experimentally demonstrated, a rejection of 63% of the body-scattered events is obtained. We present reconstructed results from blind simulations corresponding to the anthropomorphic phantom, NCAT, with oncological lesions introduced into different locations within the human body. A comparison between 300 and 600 ps FWHM TOF reconstructed images is performed, with an increasing detectability being observed for a better TOF resolution. We finally compare issues related to image convergence speed. An alternative new approach, which consists in dividing the full-body data into nine different image regions that are reconstructed independently with graphical processing unit (GPU) assistance, provides a six times faster reconstruction compared with a GPU-based whole-body reconstruction. For a 300 ps FWHM RPC-PET scanner, this allows reaching a reconstructed image, that results from 1.6 x 10^10 annihilations within 7 minutes and upon injection of 2 mCi, just 4 minutes after the end of data acquisition. We conclude that RPC-PET is well oriented to compete with other commercial PET scanners in the global market.A tomografia por emissão de positrões baseada em detectores do tipo câmaras de placas resistivas (RPC-PET) foi proposta para aplicação em ensaios com pequenos animais e na prática clínica. Neste trabalho, apresentamos primeiramente resultados experimentais obtidos a partir de um protótipo RPC-PET de alta aceitação para pequenos animais. Foram obtidas imagens de fontes do radioisótopo 22Na, uma quase pontual e outra planar. Usámos dois módulos de detectores RPC com uma área activa de 6.4 x 6.4 cm^2 e uma espessura de 5 mm, constituída por 6 vidros empilhados e 5 espaços gasosos definidos entre eles. Os dados adquiridos incluíram linhas de coincidência (LORs) inclinadas até um ângulo de 58º, tornando essencial a medida precisa da profundidade de interacção. A identificação dos espaços gasosos onde ocorreram as avalanches permitiu demonstrar a ausência de erro de paralaxe nas medidas realizadas com o RPC-PET para pequenos animais. A partir da reconstrução dos dados processados com o algoritmo maximum likelihood expectation-maximization (MLEM), obtivemos uma resolução espacial com largura a meia altura (FWHM) de 0.4 mm, excelente e estável. Em paralelo, continuámos a estudar as potencialidades de um protótipo RPC-PET de corpo inteiro e cama única, orientado para pessoas. Já foi anteriormente demonstrado por simulação que um scanner RPC-PET com 2.4 m de campo de visão axial (AFOV) é viável e permitirá o aumento de sensibilidade de pelo menos uma ordem de grandeza em relação aos scanners PET com cristais. Duas outras virtudes do RPC-PET são a sua capacidade de medição do tempo de voo (TOF) dos fotões e a elevada resolução espacial ao nível do detector. Na segunda parte deste trabalho apresentamos um algoritmo de reconstrução, totalmente tridimensional, capaz de processar LORs muito inclinadas em sistemas com um AFOV longo, como é o caso do RPC-PET. Este algoritmo acrescenta um kernel ao algoritmo MLEM, baseado na informação de TOF. Com uma resolução temporal de 300 ps FWHM, já experimentalmente comprovada, é possível rejeitar 63% dos eventos dispersados no corpo humano. Exibimos imagens reconstruídas obtidas a partir de simulações do fantoma antropomórfico, NCAT, com lesões oncológicas situadas em diferentes locais do corpo humano. A comparação entre imagens conseguidas com resoluções temporais de 300 ps e 600 ps FWHM, permite observar uma detectabilidade acrescida associada à melhor resolução de TOF. Por último, são estudados os tempos de convergência da reconstrução. Um método inovador e alternativo, que consiste na divisão dos dados do corpo humano em nove regiões e na reconstrução independente desses dados com recurso a unidades de processamento gráfico (GPUs), permite uma reconstrução seis vezes mais rápida do que a reconstrução de corpo inteiro também com o auxílio de GPUs. A partir de dados de 1.6 x 10^10 aniquilações ocorridas durante uma aquisição de 7 minutos e para uma actividade injectada de 2 mCi, um scanner RPC-PET com uma resolução temporal de 300 ps FWHM permitirá obter uma imagem reconstruída apenas 4 minutos após o fim da aquisição. Podemos assim concluir que o RPC-PET está bem colocado para competir no mercado dos scanners PET comerciais

Single-cell profiling of myasthenia gravis identifies a pathogenic T cell signature.

Myasthenia gravis (MG) is an autoimmune disease characterized by impaired neuromuscular signaling due to autoantibodies targeting the acetylcholine receptor. Although its auto-antigens and effector mechanisms are well defined, the cellular and molecular drivers underpinning MG remain elusive. Here, we employed high-dimensional single-cell mass and spectral cytometry of blood and thymus samples from MG patients in combination with supervised and unsupervised machine-learning tools to gain insight into the immune dysregulation underlying MG. By creating a comprehensive immune map, we identified two dysregulated subsets of inflammatory circulating memory T helper (Th) cells. These signature ThCD103 and ThGM cells populated the diseased thymus, were reduced in the blood of MG patients, and were inversely correlated with disease severity. Both signature Th subsets rebounded in the blood of MG patients after surgical thymus removal, indicative of their role as cellular markers of disease activity. Together, this in-depth analysis of the immune landscape of MG provides valuable insight into disease pathogenesis, suggests novel biomarkers and identifies new potential therapeutic targets for treatment

Effect of Biodiversity Changes in Disease Risk: Exploring Disease Emergence in a Plant-Virus System

The effect of biodiversity on the ability of parasites to infect their host and cause disease (i.e. disease risk) is a major question in pathology, which is central to understand the emergence of infectious diseases, and to develop strategies for their management. Two hypotheses, which can be considered as extremes of a continuum, relate biodiversity to disease risk: One states that biodiversity is positively correlated with disease risk (Amplification Effect), and the second predicts a negative correlation between biodiversity and disease risk (Dilution Effect). Which of them applies better to different host-parasite systems is still a source of debate, due to limited experimental or empirical data. This is especially the case for viral diseases of plants. To address this subject, we have monitored for three years the prevalence of several viruses, and virus-associated symptoms, in populations of wild pepper (chiltepin) under different levels of human management. For each population, we also measured the habitat species diversity, host plant genetic diversity and host plant density. Results indicate that disease and infection risk increased with the level of human management, which was associated with decreased species diversity and host genetic diversity, and with increased host plant density. Importantly, species diversity of the habitat was the primary predictor of disease risk for wild chiltepin populations. This changed in managed populations where host genetic diversity was the primary predictor. Host density was generally a poorer predictor of disease and infection risk. These results support the dilution effect hypothesis, and underline the relevance of different ecological factors in determining disease/infection risk in host plant populations under different levels of anthropic influence. These results are relevant for managing plant diseases and for establishing conservation policies for endangered plant species

Different Domains of the RNA Polymerase of Infectious Bursal Disease Virus Contribute to Virulence

BACKGROUND: Infectious bursal disease virus (IBDV) is a pathogen of worldwide significance to the poultry industry. IBDV has a bi-segmented double-stranded RNA genome. Segments A and B encode the capsid, ribonucleoprotein and non-structural proteins, or the virus polymerase (RdRp), respectively. Since the late eighties, very virulent (vv) IBDV strains have emerged in Europe inducing up to 60% mortality. Although some progress has been made in understanding the molecular biology of IBDV, the molecular basis for the pathogenicity of vvIBDV is still not fully understood. METHODOLOGY, PRINCIPAL FINDINGS: Strain 88180 belongs to a lineage of pathogenic IBDV phylogenetically related to vvIBDV. By reverse genetics, we rescued a molecular clone (mc88180), as pathogenic as its parent strain. To study the molecular basis for 88180 pathogenicity, we constructed and characterized in vivo reassortant or mosaic recombinant viruses derived from the 88180 and the attenuated Cu-1 IBDV strains. The reassortant virus rescued from segments A of 88180 (A88) and B of Cu-1 (BCU1) was milder than mc88180 showing that segment B is involved in 88180 pathogenicity. Next, the exchange of different regions of BCU1 with their counterparts in B88 in association with A88 did not fully restore a virulence equivalent to mc88180. This demonstrated that several regions if not the whole B88 are essential for the in vivo pathogenicity of 88180. CONCLUSION, SIGNIFICANCE: The present results show that different domains of the RdRp, are essential for the in vivo pathogenicity of IBDV, independently of the replication efficiency of the mosaic viruses

The use of common bean (Phaseolus vulgaris ) traditional varieties and their mixtures with commercial varieties to manage bean fly (Ophiomyia spp .) infestations in Uganda

The bean fly (Ophiomyia spp.) is considered the most economically damaging field insect pest of common beans in Uganda. Despite the use of existing pest management approaches, reported damage has remained high. Forty-eight traditional and improved common bean varieties currently grown in farmers’ fields were evaluated for resistance against bean fly. Data on bean fly incidence, severity and root damage from bean stem maggot were collected. Generalized linear mixed model (GLMM) revealed significant resistance to bean fly in the Ugandan traditional varieties. A popular resistant traditional variety and a popular susceptible commercial variety were selected from the 48 varieties and evaluated in pure and mixed stands. The incidence of bean fly infestation on both varieties in mixtures with different arrangements (systematic random versus rows), and different proportions within each of the two arrangements, was measured and analysed using GLMMs. The proportion of resistant varieties in a mixture and the arrangement type significantly decreased bean fly damage compared to pure stands, with the highest decrease in damage registered in the systematic random mixture with at least 50 % of resistant variety. The highest reduction in root damage, obvious 21 days after planting, was found in systematic random mixtures with at least 50 % of the resistant variety. Small holder farmers in East Africa and elsewhere in the world have local preferences for growing bean varieties in genetic mixtures. These mixtures can be enhanced by the use of resistant varieties in the mixtures to reduce bean fly damage on susceptible popular varieties

Effect of within-species plant genotype mixing on habitat preference of a polyphagous insect predator

The effects of within-species plant genotype mixing on the habitat preference of a polyphagous ladybird were studied. Plant species diversity is often claimed to positively affect habitat preferences of insect predators, but the effects of within-species genotype diversity have not been extensively studied. In a field experiment with different barley (Hordeum vulgare) genotypes in mixed and pure stands, adult seven-spot ladybird Coccinella septempunctata, a polyphagous predator, preferred a specific combination of genotypes over the single genotypes alone before aphids had arrived in the crop, and again when aphids were emigrating. In laboratory experiments on adult ladybird orientation to odour from barley, ladybirds were attracted/arrested by the mixed odour of the same barley genotype mixture that was preferred in the field. Exposure of one barley genotype to volatiles from the other also caused the odour of the exposed plants to become more attractive to ladybirds. The results support the hypothesis that plant volatiles may attract or arrest foraging adult ladybirds, contributing to the selection of favourable habitats, and they show that within-species plant genotype mixing can shape interactions within multitrophic communities

Decelerating Spread of West Nile Virus by Percolation in a Heterogeneous Urban Landscape

Vector-borne diseases are emerging and re-emerging in urban environments throughout the world, presenting an increasing challenge to human health and a major obstacle to development. Currently, more than half of the global population is concentrated in urban environments, which are highly heterogeneous in the extent, degree, and distribution of environmental modifications. Because the prevalence of vector-borne pathogens is so closely coupled to the ecologies of vector and host species, this heterogeneity has the potential to significantly alter the dynamical systems through which pathogens propagate, and also thereby affect the epidemiological patterns of disease at multiple spatial scales. One such pattern is the speed of spread. Whereas standard models hold that pathogens spread as waves with constant or increasing speed, we hypothesized that heterogeneity in urban environments would cause decelerating travelling waves in incipient epidemics. To test this hypothesis, we analysed data on the spread of West Nile virus (WNV) in New York City (NYC), the 1999 epicentre of the North American pandemic, during annual epizootics from 2000–2008. These data show evidence of deceleration in all years studied, consistent with our hypothesis. To further explain these patterns, we developed a spatial model for vector-borne disease transmission in a heterogeneous environment. An emergent property of this model is that deceleration occurs only in the vicinity of a critical point. Geostatistical analysis suggests that NYC may be on the edge of this criticality. Together, these analyses provide the first evidence for the endogenous generation of decelerating travelling waves in an emerging infectious disease. Since the reported deceleration results from the heterogeneity of the environment through which the pathogen percolates, our findings suggest that targeting control at key sites could efficiently prevent pathogen spread to remote susceptible areas or even halt epidemics

Identification of Clinically Relevant Protein Targets in Prostate Cancer with 2D-DIGE Coupled Mass Spectrometry and Systems Biology Network Platform

Prostate cancer (PCa) is the most common type of cancer found in men and among the leading causes of cancer death in the western world. In the present study, we compared the individual protein expression patterns from histologically characterized PCa and the surrounding benign tissue obtained by manual micro dissection using highly sensitive two-dimensional differential gel electrophoresis (2D-DIGE) coupled with mass spectrometry. Proteomic data revealed 118 protein spots to be differentially expressed in cancer (n = 24) compared to benign (n = 21) prostate tissue. These spots were analysed by MALDI-TOF-MS/MS and 79 different proteins were identified. Using principal component analysis we could clearly separate tumor and normal tissue and two distinct tumor groups based on the protein expression pattern. By using a systems biology approach, we could map many of these proteins both into major pathways involved in PCa progression as well as into a group of potential diagnostic and/or prognostic markers. Due to complexity of the highly interconnected shortest pathway network, the functional sub networks revealed some of the potential candidate biomarker proteins for further validation. By using a systems biology approach, our study revealed novel proteins and molecular networks with altered expression in PCa. Further functional validation of individual proteins is ongoing and might provide new insights in PCa progression potentially leading to the design of novel diagnostic and therapeutic strategies