Fusarium head blight (FHB), caused mainly by Fusarium graminearum (teleomorph: Gibberella zeae), is one of the most damaging diseases of wheat. A ‘Brio’/‘TC 67’ spring wheat population was used to map quantitative trait loci (QTLs) for resistance to FHB, and to study the association of morphological and developmental characteristics with FHB resistance. Interval mapping (IM) detected a major QTL on chromosome 5AL for resistance to disease severity (type II resistance) and Fusarium-damaged kernels (FDK) under greenhouse and field conditions, respectively. Inconsistent QTL(s) was also detected on chromosome 5BS for disease severity and index using field data. The associations of plant height and number of days to anthesis were negative with disease incidence, severity, index, and deoxynivalenol (DON) accumulation data under field conditions. However, number of days to anthesis was positively correlated with disease severity (greenhouse) and FDK (field). Awnedness had a negative effect on FHB, namely the presence of awns resulted in less disease in the population. Spike threshability also affected FHB so that the hard threshable genotypes represented lower disease. Phylogenetic relationships of putative F. graminearum isolates from different sources were characterized using Tri101 gene sequencing data. Canadian and Iranian isolates clustered in F. graminearum lineage 7 (=F. graminearum sensu stricto) within the F. graminearum clade while the isolates received from CIMMYT, Mexico were placed in F. graminearum lineage 3 (=Fusarium boothii) within the Fg clade or Fusarium cerealis. The PCR assay based on the Tri12 gene revealed the presence of the NIV, 3-ADON, and 15-ADON chemotypes with 15-ADON being the predominant chemotype. While we did not find the NIV chemotype among the Canadian isolates, it was the predominant chemotype among the Iranian isolates. High variation in aggressiveness was observed among and within Fusarium species tested, with the isolates of F. graminearum sensu stricto being the most aggressive and the NIV chemotype being the least aggressive. The interactions between Fusarium isolates and wheat genotypes from different sources were investigated by inoculating isolates of F. graminearum sensu stricto and F. boothii on wheat genotypes. Significant differences were observed among the genotypes inoculated by single isolates. Results also showed significant interactions between Fusarium isolates and wheat genotypes. The F. boothii isolates from CIMMYT produced low disease symptom and infection on wheat genotypes regardless of the origin of the genotypes while F. graminearum sensu stricto isolates from Canada and Iran resulted in higher FHB scores.

Made available in DSpace on 2014-07-29T15:16:38Z (GMT). No. of bitstreams: 1 Dissertacao Deyze Alencar Soares.pdf: 632456 bytes, checksum: 33012995df8eabb3f4b7509fe372764d (MD5) Previous issue date: 2010-03-26 / Paracoccidioides brasiliensis, a thermodimorphic fungus, is the causative agent of the most prevalent systemic mycosis in Latin America, paracoccidioidomycosis. The phospholipase B (PLB) enzyme is considered an important virulence factor in this dimorphic fungus, involved in the immune response of the host-pathogen interaction. Our objective was to determine whether a P. brasiliensis (Pb18) PLB is involved in adhesion / internalization of yeast and evasion of host immune responses. The effect of PLB was analysed using specific inhibition of PLB (alexidine dihydrochloride) and pulmonary surfactant in an ex vivo model (Pb18) of alveolar macrophage (MHS cells) infection. PLB enzyme assays and real time RT-PCR (qRTPCR) analysis of genes differentially expressed in the process of evasion: plb1 (phospholipase B1), icl1 (isocitrate lyase) and sod3 (Cu, Zn dismutase) and immune responses: clec2 (C-type lectin domain 2), cd14 (cluster of differentiation 14), tlr2 (toll-like receptor 2), nfkb (nuclear factor kappa B), nkrf (NF-kappaB repressing factor), il1β (inteleukin-1β) and tnfα (tumor necrosis factor alpha) were carried out using selective inhibition of PLB activity and pulmonary surfactant. The levels of cytokines inteleukin 10 (IL-10), IL-12 and TNF-α) were also determined by ELISA. PLB activity under adhesion conditions of P. brasiliensis (Pb18) to alveolar macrophage cells was found at high levels up to 6 hours post-infection. In the conditions of exposure to pulmonary surfactant and alexidine dihydrochloride, PLB activity and the level of transcripts of genes related to phagocytosis and inflammatory response were measured. We found that PLB activity had an influence on the phagocytic activity of alveolar macrophages. Alexidine dihydrochloride (0,25 μM) selectively inhibited PLB activity by 66% and decreased significantly the adhesion and internalization of yeast on MHS cells. Genes involved in phagocytosis (trl2 and cd14) and inflammatory response (nrkf, tnfα and il1β) were down-regulated in the presence of the PLB inhibitor. In contrast, the PLB activity and internalization of fungal yeast cells increased significantly in the presence of pulmonary surfactant (100 μg/mL) and genes such as clec2, important for effective phagocytosis by MHS cells, and the pro-inflammatory inhibitor (nkrf) were up-regulated. Also, the pulmonary surfactant did not alter cytokine production, while alexidine dihydrochloride decreased the levels of IL-10 and increased the levels of IL-12 and TNF-α. In addition, through simultaneous analyses of gene expression for the pathogen, P. brasiliensis, we found upregulation of the genes sod3, icl1 and plb1, required for the evasion of alveolar macrophages. P. brasiliensis PLB is important for the binding and internalization of yeast at macrophage surfaces. The specific effect of inhibiting PLB enzyme activity indicates that adhesion may be facilitated indirectly via fatty acid release from phospholipids of the membrane of host cells. This is the first study to show that PLB activity may modulate immune responses to P. brasiliensis infection. / Paracoccidioides brasiliensis, fungo dimórfico, é o agente etiológico principal micose sistêmica da América Latina, paracoccidioidomicose. A enzima fosfolipase B (PLB) é considerada um importante fator de virulência nesse fungo dimórfico e está envolvida na resposta imune da interação patógeno-hospedeiro. Nosso objetivo foi determinar se a PLB de P. brasiliensis (Pb18) está envolvida na adesão e internalização de leveduras e na evasão da resposta imune hospedeira. O efeito da PLB foi analisado usando o inibidor seletivo de PLB (alexidine dihydrochloride) e o surfactante pulmonar (Survanta) em um modelo ex vivo de infecção de macrófagos alveolares (MHS) com Pb18. Ensaio enzimático de PLB e análise de genes diferencialmente expressos por RT-PCR em tempo real (qRT-PCR) no processo de evasão: plb1 (fosfolipase B1), icl1 (isocitrato liase) e sod3 (Cu, Zn dismutase); e na resposta imune: clec2 (lecitina tipo-C 2), cd14 (cluster de diferenciação 14), tlr2 (receptor toll-like 2), nfkb (fator nuclear kappaB), nkrf (repressor fator nuclear kappaB), il1β (interleucina- 1 beta) e tnfα (fator de necrose tumoral alfa) foram realizados usando o inibidor seletivo da atividade de PLB e surfactante pulmonar. Os níveis de citocinas interleucina 10 (IL-10), IL-12 e TNF- α) foram determinados por ELISA. A atividade de PLB usadas em baixas condições para a adesão de P. brasiliensis (Pb18) obteve altos níveis em 6 horas pós-infecção. Na presença do surfactante pulmonar e alexidine dihydrochloride, a atividade da PLB e os níveis de transcritos dos genes relacionados à fagocitose e à resposta inflamatória foram quantificados. A PLB teve influência na atividade fagocítica dos macrófagos. Alexidine dihydrochloride (0,25 μM) inibiu seletivamente a atividade PLB em 66% e diminuiu significativamente a adesão e internalização de leveduras por macrófagos alveolares (MHS). Genes envolvidos na fagocitose (trl2 e cd14) e resposta inflamatória (nrkf, tnfα e il1β) foram reprimidos na presença do inibidor de PLB. Em contraste, a atividade PLB e internalização de leveduras aumentou significativamente na presença do surfactante pulmonar (100 μg/mL) e genes assim como clec2, importante para uma fagocitose efetiva pelos macrófagos alveolares (MHS), e o inibidor pró-inflamatório (nkrf) foram induzidos. Entretanto, o surfactante pulmonar não alterou a produção de citocinas, enquanto que alexidine dihydrochloride diminuiu os níveis de IL-10 e aumentou os níveis de IL-12 e TNF-α. Em adição, nas análises simultâneas de expressão de genes, P. brasiliensis, houve indução dos genes sod3, icl1 e plb1, requeridos para a evasão dos macrófagos alveolares. A PLB de P. brasiliensis é importante na adesão e internalização de leveduras pelos macrófagos alveolares. O efeito específico da inibição da atividade da PLB indica que a adesão pode ser facilitada indiretamente via liberação de ácidos graxos dos fosfolipídeos de membrana das células hospedeiras. Esse é o primeiro estudo mostrando que a atividade da PLB pode modular a resposta imune à infecção pelo P. brasiliensis.

Die Durchfallerkrankung Giardiasis wird durch den Protisten Giardia duodenalis ausgelöst. Die Infektion erfolgt fäkal-oral, meist über kontaminiertes Trinkwasser. Der Parasit kolonisiert den oberen Bereich des Dünndarms und heftt sich an das Epithel, wodurch es die Krankheitsbeschwerden auslöst. Allerdings sind Details über die Mechanismen der Pathogenese unbekannt. Dazu kommt, dass der Ausgang einer Infektion fallspezifisch starken Schwankungen unterworfen ist, von selbst-limitierend bis chronisch und asymptomatischer Kolonisierung bis hin zur schweren Enteritis. Ein möglicher Pathomechanismus ist der Wegfall der Barrierefunktion des Dünndarmepithels, z.B. durch Beeinträchtigung von tight junctions oder Zelltod. In dieser Arbeit wurden Effekte von G. duodenalis auf in vitro Modellsysteme des humanen Dünndarmepithels untersucht. Dazu wurden hauptsächlich Daten über die Barrierefunktion sowohl von der weit verbreiteten Caco-2 Zelllinie, als auch über ein neu etabliertes humanes Dünndarmorganoidsystem, erhoben. Es konnte gezeigt werden, dass mehrere - mitunter in der Literatur als hochvirulent beschriebene - G. duodenalis Isolate zu keinerlei Beeinträchtigung der Barrierefunktion oder irgendeiner anderen untersuchten potenziellen Schädigung an zwei unterschiedlichen Caco-2 Zelllinien unter diversen Infektions- und Kulturbedingungen führte. Jedoch andererseits das neu entwickelte Dünndarmorganoidsystem mit pseudo-luminalem Medium TYI S 33 reproduzierbar die Zerstörung des Epithelmodells mit Zellverlust, Zelltod (apoptotisch und nicht-apoptotisch), Störung der tight junctions (Abbau und Dislokation von Claudinen und ZO-1) und den Verlust von Mikrovilli innerhalb ein bis zwei Tage nach Parasiteninfektion zeigen konnte. Zudem wurde das Auftauchen von ClCa-1-Signalen unter andauerndem Infektionsstress beobachtet, was die Differenzierung bzw. Metaplasie zu Becherzellen nahelegt, jedoch keine Wirtsreaktion auf die Gewebszerstörung zu sein scheint. / The protozoan parasite Giardia duodenalis is the etiological agent for the intestinal diarrheal disease giardiasis. Infections are acquired via the fecal-oral route, mostly via uptake of cysts from contaminated drinking water. The colonization of the hosts’ duodenum and upper jejunum and the attachment of Giardia trophozoites onto the epithelium is the cause of a variety of gastrointestinal complaints but the exact pathomechanisms are unknown. Furthermore, the outcome of Giardia infections varies greatly between individuals, ranging from self-limiting to chronic, and asymptomatic to severe enteritis. One proposed mechanism for the pathogenesis is the breakdown of intestinal barrier function, e.g. by tight junction impairment or induction of cell death. In this work, effects of G. duodenalis on in vitro models of the human small intestinal epithelium were investigated by studying mainly barrier-related properties and changes of widely used Caco-2 cells as well as newly established human small intestinal organoid-derived monolayers (ODMs). It could be shown that several isolates of G. duodenalis, some described as highly virulent, fail to induce barrier dysfunction or any other investigated pathological effect on two Caco-2 cell lines under various infection and culturing conditions. On the other side, by developing a new organoid-based model system and the use of luminal mock medium TYI-S-33, considerable epithelial disruption (including loss of cells), cell death (apoptosis and non-apoptotic), tight junction impairment (degradation and dislocation of claudins and ZO-1), and microvilli depletion reproducibly induced by G. duodenalis trophozoites between one and two days after infection could be observed. Moreover, emergence of ClCa-1 positive cells with ongoing parasite infections suggest epithelial differentiation or metaplasia towards goblet cells, which is furthermore not associated to tissue damage.