Durable rust resistance in wheat is effective against multiple pathogens

Tese de doutoramento em Ciências da Saúde, no ramo de Medicina Dentária, apresentada à Faculdade de Medicina da Universidade de CoimbraA osteonecrose maxilar associada aos bifosfonatos, que se apresenta como uma lesão pós-cirúrgica associada a incapacidade de cicatrização, é um dos efeitos secundários mais frequentemente observados nos doentes submetidos a terapêutica com bifosfonatos nitrogenados. Assim, esta patologia é diagnosticada maioritariamente em doentes com metastização óssea, sob terapêutica com bifosfonatos nitrogenados intra-venosos. A fisiopatologia da osteonecrose maxilar associada aos bifosfonatos tem sido relacionada com a supressão da remodelação óssea, com a infeção local e com a toxicidade dos bifosfonatos nos tecidos envolventes, que mantém e perpetua a exposição óssea. Os fatores de risco da osteonecrose maxilar associada aos bifosfonatos foram categorizados em fatores relacionados com a terapêutica, que incluem a administração intravenosa em doenças oncológicas e a utilização do bifosfonato mais potente, o zoledronato; e factores locais, que incluem as exodontias, a colocação de implantes, a cirurgia periapical e a cirurgia periodontal que envolva os tecidos ósseos. No entanto, no caso da cirurgia periapical, não foram encontrados estudos que relacionem este procedimento com a osteonecrose maxilar. No contexto da toxicidade do zoledronato, uma vez que os compostos de fosfato de cálcio têm a capacidade de o adsorver, nomeadamente quando utilizados como sistemas transportadores de bifosfonatos, e são passíveis de aplicação nas locas cirúrgicas, constituindo substitutos ósseos adequados, colocou-se a hipótese de estes compostos poderem, potencialmente, ter um efeito protetor nos tecidos que envolvem as locas cirúrgicas. Assim, constituíram objetivos deste trabalho a avaliação da cirurgia periapical como desencadeadora de osteonecrose maxilar na presença de zoledronato e, paralelamente, a avaliação do potencial protetor da aplicação de compostos de fosfato de cálcio na loca cirúrgica. Para cumprir os objetivos propostos neste trabalho, realizaram-se estudos de química, estudos in vitro e estudos in vivo. No que respeita aos estudos de química, avaliou-se a reação entre o zoledronato e os compostos de fosfato de cálcio. Através de espetroscopia do visível e de análise elementar, verificou-se que o zoledronato é adsorvido, em solução aquosa, pelos compostos bifásicos de fosfato de cálcio. A etiologia da osteonecrose maxilar descreve um efeito deletério dos bifosfonatos nos tecidos moles, especialmente nos fibroblastos, que apresentam uma função insubstituível na cicatrização das lesões cirúrgicas orais. No contexto dos estudos in vitro, estabeleceram-se culturas primárias de fibroblastos gengivais humanos, que constituíram um modelo para avaliar a citotoxicidade na presença de zoledronato e na presença da associação zoledronato/compostos bifásicos de fosfato de cálcio. Avaliou-se a atividade metabólica dos fibroblastos gengivais humanos através do ensaio de MTT (brometo de 3-(4,5-dimetiltiazol-2-il)-2,5-difeniltetrazol), a sua viabilidade através do ensaio de SRB (ensaio da sulforrodamina B), os tipos de morte e o ciclo celular dos fibroblastos gengivais humanos através de citometria de fluxo e a capacidade de migração através do scratch assay. Verificou-se que o zoledronato apresentou um efeito citotóxico significativo nos fibroblastos gengivais humanos, com diminuição da atividade metabólica e da viabilidade, com aumento das populações celulares em morte por apoptose e por necrose e pela diminuição da capacidade de migração. Com a associação zoledronato/compostos bifásicos de fosfato de cálcio, foi possível diminuir, ou mesmo anular, a toxicidade do zoledronato, que se manifestou pela ausência de diferenças em relação aos grupos controlo. Nos estudos in vivo, foi utilizado um modelo experimental reprodutível já estudado, que relaciona diretamente a administração crónica de bifosfonatos com o desenvolvimento de osteonecrose maxilar após exodontia. Este modelo animal serviu como base ao desenvolvimento de um modelo de cirurgia apical com administração crónica de bifosfonatos. Os grupos de animais tratados foram submetidos a administração intra-peritoneal de zoledronato nas quatro semanas antecedentes à intervenção cirúrgica e nas duas ou três semanas seguintes. As mandíbulas foram avaliadas macroscopicamente, através da medicina nuclear, radiologia e histologia. No contexto da medicina nuclear, foi utilizado o radiofármaco 99mTc-zoledronato, que foi obtido após o desenvolvimento e a optimização de um procedimento de marcação radioquímica do zoledronato, e respetivo controlo de qualidade. Verificou-se que, nos animais submetidos à terapêutica com zoledronato, em que se desenvolveu a exodontia, existiu maior captação de 99mTc-zoledronato, menor densidade radiográfica e alterações histológicas compatíveis com cicatrização diminuída. Com a aplicação dos compostos bifásicos de fosfato de cálcio, não se observaram diferenças em relação aos animais controlo. No caso dos animais submetidos à cirurgia apical, não se verificaram alterações significativas em relação aos animais controlo, nos animais submetidos à terapêutica com zoledronato, tanto na presença como na ausência da aplicação de compostos bifásicos de fosfato de cálcio. Este trabalho de investigação permitiu elucidar acerca dos desígnios que deram origem a esta tese. No modelo animal de cirurgia periapical desenvolvido, não foi observado um atraso significativo da cicatrização nem sinais da osteonecrose maxilar, nos tempos avaliados, concluindo-se que a terapêutica com zoledronato poderá não constituir um fator de risco nesta intervenção cirúrgica. Pelo contrário, no modelo animal de exodontia confirmou-se que, efetivamente, o zoledronato constitui um fator de risco, e que os compostos bifásicos de fosfato de cálcio, apresentam potencial efeito protetor da toxicidade inerente aos bifosfonatos. Esta conclusão foi sustentada pelos estudos de química, em que se verificou a adsorção do zoledronato, corroborada por supressão da toxicidade nos estudos in vitro e cicatrização favorecida no modelo animal de exodontia com administração crónica de zoledronato.Bisphosphonate-associated osteonecrosis of the jaw, a post-surgical non-healing wound condition, is one of the most often seen side effects in patients treated with nitrogencontaining bisphosphonates. Therefore, this pathology is primarily diagnosed in patients with metastatic bone disease, receiving intravenous administration of nitrogen-containing bisphosphonates. Bisphosphonate-associated osteonecrosis of the jaw physiopathology has been related with suppression of bone turnover, local infections or soft tissue toxicity. Recent studies associate the physiopathological mechanisms of the osteonecrosis of the jaw with toxic effects of bisphosphonates on different cell types, besides osteoclast, being the most important cause of soft tissues toxicity that contributes for the maintenance of bone exposure. Bisphosphonate-associated osteonecrosis of the jaw risk factors were categorized as drug-related factors, including intravenous administration in oncological diseases and the use of the most potent bisphosphonate, zoledronate; and local factors including, dental extractions, dental implant placement, periapical surgery and periodontal surgery involving osseous injury. However, there are no studies that relate periapical surgery with osteonecrosis of the jaw. Considering zoledronate toxicity, since calcium phosphate compounds are able to adsorb it, namely when used as drug delivery vehicle, and are also used in surgical wounds, as a bone substitute, it was hypothesised this compounds had a potential protective effect to the soft tissues surrounding surgical osseous wounds. Thus, the aim of this study was to assess periapical surgery as a trigger of osteonecrosis of the jaw in the presence of zoledronate and also the potential protective effect of calcium phosphate compounds when applied in the surgical wound. To fulfil the proposed objectives of this work were carried out studies of chemistry, in vitro and in vivo studies. Regarding chemical studies, it was evaluated the chemical reaction between zoledronate and calcium phosphate compounds. Through ultraviolet-visible spectroscopy and elemental analysis it has been found that zoledronate, in aqueous solution, was adsorbed by biphasic calcium phosphate compounds. Bisphosphonate-associated osteonecrosis of the jaw aetiology describes a deleterious effect of bisphosphonates on soft tissues, especially on fibroblasts, which play an important role in oral wound healing. Considering in vitro studies it was established a primary culture of human gingival fibroblasts that constituted a model to evaluate the cytotoxicity in the presence of zoledronate and of zoledronate/biphasic calcium phosphate compounds association. It was investigated the metabolic activity of human gingival fibroblasts through MTT (3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, cell viability through SRB (sulforhodamine B) assay, types of cell death and cell cycle through flow cytometry and migration ability of human gingival fibroblasts through scratch assay. It was verified that zoledronate had a strong cytotoxic effect in the human gingival fibroblasts, by the reduction of metabolic activity, cell viability, increase of cells in apoptosis and reduction of migration. With the association zoledronate/biphasic calcium phosphate compounds it was possible to reduce or abolish zoledronate toxicity, as it was demonstrated by the absence of differences related to control. In the in vivo study it was used a reproducible experimental model that directly relates chronic bisphosphonate administration with the development of osteonecrosis of the jaw with tooth extraction. This animal model also served as basis to the development of a osteotomy in periapical surgery model with chronic bisphosphonate administration. The animals were treated with zoledronate intraperitoneally, during the four weeks that preceded the surgeries and in the following two and three weeks. The mandibles were macroscopic evaluated, examined by nuclear medicine, radiology and histologically analysed. Concerning nuclear medicine it was used the radiopharmaceutical 99mTc-zoledronate, which was obtained after the development and optimization of a procedure for zoledronate radiochemical labelling, and respective quality control. It has been found that the animals with zoledronate, submitted to tooth extraction, had a higher 99mTc-zoledronate uptake, lower radiological density and histologic images compatible with a decreased healing. With biphasic calcium phosphate compounds application, there were no differences related to controls. In the animals submitted to osteotomy in periapical surgery there were no significant differences related to the controls, in both animals with zoledronate, with and without biphasic calcium phosphate compounds application. This research work allowed the clarification of the goals that led to this thesis. In the created animal model of osteotomy in periapical surgery, it was not observed a significant delay in the wound healing, neither osteonecrosis of the jaw, in the evaluated periods, therefore it was conclude that zoledronate therapeutic may not constitute a risk factor in this surgical approach. Contrarily, in the tooth extraction animal model, it was confirmed that zoledronate therapeutic constitute a risk factor, and it was found that biphasic calcium phosphate compounds presents a potential protector effect from bisphosphonates toxicity. This conclusion was supported by the chemical studies, in which it was observed adsorption of zoledronate, corroborated by the lower toxicity in the in vitro studies and by the improved cicatrisation in the tooth extraction animal model with chronic bisphosphonates administration

Pattern breaking: a complex systems approach to psychedelic medicine

Recent research has demonstrated the potential of psychedelic therapy for mental health care. However, the psychological experience underlying its therapeutic effects remains poorly understood. This paper proposes a framework that suggests psychedelics act as destabilizers, both psychologically and neurophysiologically. Drawing on the ‘entropic brain’ hypothesis and the ‘RElaxed Beliefs Under pSychedelics’ model, this paper focuses on the richness of psychological experience. Through a complex systems theory perspective, we suggest that psychedelics destabilize fixed points or attractors, breaking reinforced patterns of thinking and behaving. Our approach explains how psychedelic-induced increases in brain entropy destabilize neurophysiological set points and lead to new conceptualizations of psychedelic psychotherapy. These insights have important implications for risk mitigation and treatment optimization in psychedelic medicine, both during the peak psychedelic experience and during the subacute period of potential recovery

Major haplotype divergence including multiple germin-like protein genes, at the wheat Sr2 adult plant stem rust resistance locus

Background The adult plant stem rust resistance gene Sr2 was introgressed into hexaploid wheat cultivar (cv) Marquis from tetraploid emmer wheat cv Yaroslav, to generate stem rust resistant cv Hope in the 1920s. Subsequently, Sr2 has been widely deployed and has provided durable partial resistance to all known races of Puccinia graminis f. sp. tritici. This report describes the physical map of the Sr2-carrying region on the short arm of chromosome 3B of cv Hope and compares the Hope haplotype with non-Sr2 wheat cv Chinese Spring. Results Sr2 was located to a region of 867 kb on chromosome 3B in Hope, which corresponded to a region of 567 kb in Chinese Spring. The Hope Sr2 region carried 34 putative genes but only 17 were annotated in the comparable region of Chinese Spring. The two haplotypes differed by extensive DNA sequence polymorphisms between flanking markers as well as by a major insertion/deletion event including ten Germin-Like Protein (GLP) genes in Hope that were absent in Chinese Spring. Haplotype analysis of a limited number of wheat genotypes of interest showed that all wheat genotypes carrying Sr2 possessed the GLP cluster; while, of those lacking Sr2, some, including Marquis, possessed the cluster, while some lacked it. Thus, this region represents a common presence-absence polymorphism in wheat, with presence of the cluster not correlated with presence of Sr2. Comparison of Hope and Marquis GLP genes on 3BS found no polymorphisms in the coding regions of the ten genes but several SNPs in the shared promoter of one divergently transcribed GLP gene pair and a single SNP downstream of the transcribed region of a second GLP. Conclusion Physical mapping and sequence comparison showed major haplotype divergence at the Sr2 locus between Hope and Chinese Spring. Candidate genes within the Sr2 region of Hope are being evaluated for the ability to confer stem rust resistance. Based on the detailed mapping and sequencing of the locus, we predict that Sr2 does not belong to the NB-LRR gene family and is not related to previously cloned, race non-specific rust resistance genes Lr34 and Yr36

Major haplotype divergence including multiple germin-like protein genes, at the wheat Sr2 adult plant stem rust resistance locus

Background The adult plant stem rust resistance gene Sr2 was introgressed into hexaploid wheat cultivar (cv) Marquis from tetraploid emmer wheat cv Yaroslav, to generate stem rust resistant cv Hope in the 1920s. Subsequently, Sr2 has been widely deployed and has provided durable partial resistance to all known races of Puccinia graminis f. sp. tritici. This report describes the physical map of the Sr2-carrying region on the short arm of chromosome 3B of cv Hope and compares the Hope haplotype with non-Sr2 wheat cv Chinese Spring. Results Sr2 was located to a region of 867 kb on chromosome 3B in Hope, which corresponded to a region of 567 kb in Chinese Spring. The Hope Sr2 region carried 34 putative genes but only 17 were annotated in the comparable region of Chinese Spring. The two haplotypes differed by extensive DNA sequence polymorphisms between flanking markers as well as by a major insertion/deletion event including ten Germin-Like Protein (GLP) genes in Hope that were absent in Chinese Spring. Haplotype analysis of a limited number of wheat genotypes of interest showed that all wheat genotypes carrying Sr2 possessed the GLP cluster; while, of those lacking Sr2, some, including Marquis, possessed the cluster, while some lacked it. Thus, this region represents a common presence-absence polymorphism in wheat, with presence of the cluster not correlated with presence of Sr2. Comparison of Hope and Marquis GLP genes on 3BS found no polymorphisms in the coding regions of the ten genes but several SNPs in the shared promoter of one divergently transcribed GLP gene pair and a single SNP downstream of the transcribed region of a second GLP. Conclusion Physical mapping and sequence comparison showed major haplotype divergence at the Sr2 locus between Hope and Chinese Spring. Candidate genes within the Sr2 region of Hope are being evaluated for the ability to confer stem rust resistance. Based on the detailed mapping and sequencing of the locus, we predict that Sr2 does not belong to the NB-LRR gene family and is not related to previously cloned, race non-specific rust resistance genes Lr34 and Yr36

From Generative Models to Generative Passages: A Computational Approach to (Neuro) Phenomenology

This paper presents a version of neurophenomenology based on generative modelling techniques developed in computational neuroscience and biology. Our approach can be described as computational phenomenology because it applies methods originally developed in computational modelling to provide a formal model of the descriptions of lived experience in the phenomenological tradition of philosophy (e.g., the work of Edmund Husserl, Maurice Merleau-Ponty, etc.). The first section presents a brief review of the overall project to naturalize phenomenology. The second section presents and evaluates philosophical objections to that project and situates our version of computational phenomenology with respect to these projects. The third section reviews the generative modelling framework. The final section presents our approach in detail. We conclude by discussing how our approach differs from previous attempts to use generative modelling to help understand consciousness. In summary, we describe a version of computational phenomenology which uses generative modelling to construct a computational model of the inferential or interpretive processes that best explain this or that kind of lived experience

Genomics accelerated isolation of a new stem rust avirulence gene - wheat resistance gene pair

Stem rust caused by the fungus Puccinia graminis f. sp. tritici (Pgt) is a devastating disease of the global staple crop wheat. Although this disease was largely controlled by genetic resistance in the latter half of the 20th century, new strains of Pgt with increased virulence, such as Ug99, have evolved by somatic hybridisation and mutation. These newly emerged strains have caused significant losses in Africa and other regions and their continued spread threatens global wheat production. Breeding for disease resistance provides the most cost-effective control of wheat rust diseases. A number of race-specific rust resistance genes have been characterised in wheat and most encode immune receptors of the nucleotide-binding leucine-rich repeat (NLR) class. These receptors recognize pathogen effector proteins often known as avirulence (Avr) proteins. However, only two Avr genes have been identified in Pgt to date, AvrSr35 and AvrSr50 and none in other cereal rusts, which hinders efforts to understand the evolution of virulence in rust populations. The Sr27 resistance gene was first identified in a wheat line carrying an introgression of the 3R chromosome from Imperial rye. Although not deployed widely in wheat, Sr27 is widespread in the artificial crop species Triticosecale (triticale) which is a wheat-rye hybrid and is a host for Pgt. Sr27 is effective against Ug99 and other recently emerged Pgt strains. Here we identify both the Sr27 gene in wheat and the corresponding AvrSr27 gene in Pgt and show that virulence to Sr27 can arise experimentally and in the field through deletion mutations, copy number variation and expression level polymorphisms at the AvrSr27 locus

Discovery and characterization of two new stem rust resistance genes in Aegilops sharonensis

Stem rust is one of the most important diseases of wheat in the world. When single stem rust resistance (Sr) genes are deployed in wheat, they are often rapidly overcome by the pathogen. To this end, we initiated a search for novel sources of resistance in diverse wheat relatives and identified the wild goat grass species Aegilops sharonesis (Sharon goatgrass) as a substantial reservoir of resistance to wheat stem rust. The objectives of this study were to discover and map novel Sr genes in Ae. sharonensis and to explore the possibility of identifying new Sr genes by genome-wide association study (GWAS). We developed two biparental populations between resistant and susceptible accessions of Ae. sharonensis and performed QTL and linkage analysis. In an F6 recombinant inbred line and an F2 population, two genes were identified that mapped to the short arm of chromosome 1Ssh, designated as Sr-1644-1Sh, and the long arm of chromosome 5Ssh, designated as Sr-1644-5Sh. The gene Sr-1644-1Sh confers a high level of resistance to race TTKSK (one of the Ug99 lineage races), while the gene Sr-1644-5Sh conditions strong resistance to TRTTF, another widely virulent race found in Yemen. Additionally, GWAS was conducted on 125 diverse Ae. sharonensis accessions for stem rust resistance. The gene Sr-1644-1Sh was detected by GWAS, while Sr-1644-5Sh was not detected, indicating that the effectiveness of GWAS might be affected by marker density, population structure, low allele frequency and other factors