Gene induction during plant-microbe interactions : the role of chitinases during fungal infection and the investigation of mycorrhiza-induced genes in the model plant "M. truncatula"

In this thesis, the model legume Medicago truncatula was used for research on plant-microbe interactions. Unlike most other plants, legumes are able to form two distinct root symbioses. Together with soil-borne fungi of the Glomeromycota, they form the arbuscular mycorrhiza and with rhizobial bacteria, they form nitrogen fixing root nodules. Here, plant responses to these symbiotic microbes were investigated, and compared to the plant's defence response against antagonistic microbes. Plant chitinases have an important role in the interplay between plants and microbes; they have been shown to act as defence-related antifungal enzymes, but they seem to be involved in symbiotic processes as well. In previous work, genes encoding eight different chitinases were identified in M. truncatula. The main part of this thesis is dedicated to an indepth study of these genes with regard to their regulation and function. Additionally, the expression patterns of genes that are related to the arbuscular mycorrhizal interaction of M. truncatula were analysed in order to further explore this important symbiosis. In roots of the M. truncatula ecotype R108-1, expression of the gene Mtchit5, encoding a class V chitinase, was induced during nodule formation after infection with wild type rhizobia, but not in response to pathogenic fungi or arbuscular mycorrhizal fungi. Mtchit5 transcripts were first detectable in roots forming nodule primordial and accumulated during nodule ripening. The gene was induced in response to purified Nod factors and also in ineffective white nodules formed by a mutant rhizobial strain. Phylogenetic analysis of the deduced amino acid sequence revealed that the putative Mtchit5 chitinase forms a separate clade within class V chitinases of plants. These results, together with the additional finding that Mtchit5 expression is high in flowers, indicate that Mtchit5 is a putative early nodulin that is specifically induced by rhizobia in roots and may have a function in plant developmental processes. The gene Mtchit4, encoding a class IV chitinase, is induced during infection by pathogenic fungi in roots of M. truncatula but not during mycorrhiza formation. During nodule formation, Mtchit4 was strongly induced only in the M. truncatula ecotype Jemalong A17 after infection with wild-type rhizobium strains. Its expression was elevated in nodules formed with a Kantigen deficient rhizobium mutant, but not in response to purified Nod factors. The putative Mtchit4 chitinase is closely related to pathogenesis-related class IV chitinases from other plants, and it is assumed that Mtchit4 is a pathogenesis related protein. This is supported by an additional study that revealed a low overall expression of Mtchit4 throughout the plant, independent of the plant’s symbiotic status, and an in silico analysis of the Mtchit4 promoter sequence, which contains a variety of putative cis-elements related to plant defence. The expression of two genes encoding class I chitinases, Mtchit1a and Mtchit1c, were compared to the expression of Mtchit4 in leaves, roots and flowers and after infection with a mycorrhizal fungus. In contrast to the constitutively expressed Mtchit1c, the expression of Mtchit1a was similar in leaves or roots but low in flowers. Both chitinase genes were not affected by mycorrhizal infection. The differential expression patterns, together with sequence data and in silico promoter analyses, suggest that these genes encode pathogenesis related chitinases, that are specifically regulated in response to infection by pathogenic fungi. Mtchit3-3 is a class III chitinase gene that was specifically induced in mycorrhizal roots. The Mtchit3-3 promoter directs reporter gene expression to arbuscule containing cells, which is consistent with mycorrhiza-related elements found in the promoter sequence. Disruption of the Mtchit3-3 gene expression in root organ cultures stimulated spore germination of mycorrhizal fungi and in one fungal strain resulted in a higher probability of root colonization and spore formation. No effect on the abundance of arbuscules within colonized roots became apparent. Mtchit3-3-GFP fusion constructs revealed that the putative signal peptide could direct the Mtchit3-3 protein to the apoplast. It is suggested that the chitinase Mtchit3-3 is enzymatically active and might act on chitin in the fungal cell wall or fungal chitin-related signals during the symbiosis and it may be involved in communication processes between plant and AM. The class III chitinase genes Mtchit3-1 and Mtchit3-4 are induced in response to infection by pathogenic fungi in roots of M. truncatula. Mtchit3-4, but not Mtchit3-1, was also slightly induced during mycorrhiza formation. According to their gene and protein structures in comparison to chitinases in other plant species and additional in silico promoter analyses, it is proposed that Mtchit3-1 is a pathogenesis related chitinase while Mtchit3-4 may be related in a general way to fungal infections. The functionality of the arbuscular mycorrhizal symbiosis was measured by comparing the plant’s nutritional status and growth response of three mycorrhizal fungi from two different phylogenetic taxa, namely Glomus intraradices, Glomus mosseae and Scutellospora castanea. Mycorrhiza formation enhanced biomass accumulation and nutritional status of the plants in each case, although the response was not related to the colonization degree. To supplement the expression data of chitinase genes in relation to different fungal infections, the expression was measured in the roots of M. truncatula plants colonized with the three AMF. In addition, a selected set of other symbiosis related genes were tested that responded differently to the AMF colonization. It can be concluded that a subset of the genes that respond to colonization by the two Glomus species also responded to at least one fungus from the Gigasporaceae. These data indicate that different genes showing arbusculespecific gene expression in colonized roots are regulated by different mechanisms, depending on the fungal partner

Sinorhizobium meliloti -induced chitinase gene expression in Medicago truncatula ecotype R108-1: a comparison between symbiosis-specific classV and defence-related classIV chitinases

The Medicago truncatula (Gaertn.) ecotypes JemalongA17 and R108-1 differ in Sinorhizobium meliloti-induced chitinase gene expression. The pathogen-inducible classIV chitinase gene, Mtchit4, was strongly induced during nodule formation of the ecotype JemalongA17 with the S. meliloti wild-type strain 1021. In the ecotype R108-1, the S. meliloti wild types Sm1021 and Sm41 did not induce Mtchit4 expression. On the other hand, expression of the putative classV chitinase gene, Mtchit5, was found in roots of M. truncatula cv. R108-1 nodulated with either of the rhizobial strains. Mtchit5 expression was specific for interactions with rhizobia. It was not induced in response to fungal pathogen attack, and not induced in roots colonized with arbuscular mycorrhizal (AM) fungi. Elevated Mtchit5 gene expression was first detectable in roots forming nodule primordia. In contrast to Mtchit4, expression of Mtchit5 was stimulated by purified Nod factors. Conversely, Mtchit4 expression was strongly elevated in nodules formed with the K-antigen-deficient mutant PP699. Expression levels of Mtchit5 were similarly increased in nodules formed with PP699 and its parental wild-type strain Sm41. Phylogenetic analysis of the deduced amino acid sequences of Mtchit5 (calculated molecular weight = 41,810Da, isoelectric point pH7.7) and Mtchit4 (calculated molecular weight 30,527Da, isoelectric point pH4.9) revealed that the putative Mtchit5 chitinase forms a separate clade within classV chitinases of plants, whereas the Mtchit4 chitinase clusters with pathogen-induced classIV chitinases from other plants. These findings demonstrate that: (i) Rhizobium-induced chitinase gene expression in M. truncatula occurs in a plant ecotype-specific manner, (ii) Mtchit5 is a putative chitinase gene that is specifically induced by rhizobia, and (iii) rhizobia-specific and defence-related chitinase genes are differentially influenced by rhizobial Nod factors and Kantigen

Anale Inkontinenz als Folge angeborener Enddarmfehlbildungen:Experimentelle Untersuchungen zur Substitution des Musculus sphincter ani internus durch Tissue Engineering

Die Analatresie stellt eine schwerwiegende Missbildung des Enddarmabschnittes dar. Als Hauptproblem wird die anale Inkontinenz gesehen, bedingt durch eine nur rudimentär angelegte Schließmuskulatur. Bisher gibt es kein bioatifizielles Implantat als Schließmuskelersatz. So entstand die Idee der Kultivierung eines glattmuskulären Gewebeverbandes zur Unterstützung des insuffizienten, glattmuskulären inneren Analsphincters (IAS). Glatte Muskelzellen wurden aus dem IAS gewonnen, in vitro kultiviert, auf drei unterschiedliche Trägermaterialien überbracht und wieder reimplantiert. Die Zellen wurden in vitro im Hinblick auf Morphologie und Proliferation untersucht. Die in vivo Analyse legte den Schwerpunkt auf die Vaskularisation und die Integration der Implantate ins native Gewebe. Zudem wurde in beiden Phasen der Differenzierungsgrad der Zellen bestimmt. Die Membranpotentiale der Zellen wurden mit Hilfe eines spannungssensitiven Farbstoffs gemessen

Return to play management after concussion in football: recommendations for team physicians

A concussion is a rare but potentially serious injury of football players. Thus, an immediate and valid diagnosis, estimate of severity and therapeutic management is required. To summarise the published information on management of concussion with respect to a safe return to play (RTP), a literature search was conducted. Current guidelines on concussion in sports and significant studies on concussion in football were analysed. After concussion, management and RTP decision should remain in the area of clinical judgement on an individualised basis according to the current international guidelines. If a concussion is suspected, the player should not be allowed to RTP the same day. The RTP programme should follow a gradual step-wise procedure. A concussed player should not RTP unless he/she is asymptomatic and the neurological and neuropsychological examinations are normal. Untimely RTP bears an increased risk of sustaining another more severe brain injury and repetitive brain injury of long-term sequelae. In football, the management of concussion should primarily follow the recommendations proposed by the Concussion in Sports Group. Information and education of players and their medical and coaching team help to protect the players' health. Future studies on concussion should include validated and detailed information on RTP protocols

Recommendations for initial examination, differential diagnosis, and management of concussion and other head injuries in high-level football

Head injuries can result in substantially different outcomes, ranging from no detectable effect to transient functional impairments to life-threatening structural lesions. In high-level international football (soccer) tournaments, on average, one head injury occurs in every third match. Making the diagnosis and determining the severity of a head injury immediately on-pitch or off-field is a major challenge for team physicians, especially because clinical signs of a brain injury can develop over several minutes, hours, or even days after the injury. A standardized approach is useful to support team physicians in their decision whether the player should be allowed to continue to play or should be removed from play after head injury. A systematic, football-specific procedure for examination and management during the first 72 hours after head injuries and a graduated Return-to-Football program for high-level players have been developed by an international group of experts based on current national and international guidelines for the management of acute head injuries. The procedure includes seven stages from the initial on-pitch examination to the graduated Return-to-Football program. Details of the assessments and the consequences of different outcomes are described for each stage. Criteria for emergency management (red flags), removal from play (orange flags), and referral to specialists for further diagnosis and treatment (persistent orange flags) are provided. The guidelines for return to sport after concussion-type head injury are specified for football. Thus, the present paper presents a comprehensive procedure for team physicians after a head injury in high-level football

Approach to investigation and treatment of persistent symptoms following sport-related concussion: a systematic review

OBJECTIVE: To conduct a systematic review of the literature regarding assessment and treatment modalities in patients with persistent symptoms following sport-related concussion (SRC). DATA SOURCES: We searched Medline, Embase, SPORTSDiscus, PsycINFO, CINAHL, Cochrane library and ProQuest Dissertation & Theses Global electronic databases. STUDY ELIGIBILITY CRITERIA: Studies were included if they were original research, reported on SRC as the primary source of injury, included patients with persistent postconcussive symptoms (>10 days) and investigated the role of assessment or treatment modalities. RESULTS: Of 3225 articles identified in the preliminary search, 25 articles met the inclusion criteria. 11 articles were concerned with assessment and 14 articles with treatment of persistent symptoms following SRC. There were three randomised control trials and one quasi-experimental study. The remainder consisting of cross-sectional studies, historical cohorts and case series. SUMMARY: 'Persistent symptoms' following SRC can be defined as clinical recovery that falls outside expected time frames (ie, >10-14 days in adults and >4 weeks in children). It does not reflect a single pathophysiological entity, but describes a constellation of non-specific post-traumatic symptoms that may be linked to coexisting and/or confounding pathologies. A detailed multimodal clinical assessment is required to identify specific primary and secondary processes, and treatment should target specific pathologies identified. There is preliminary evidence supporting the use of symptom-limited aerobic exercise, targeted physical therapy and a collaborative approach that includes cognitive behavioural therapy. Management of patients with persistent symptoms is challenging and should occur in a multidisciplinary collaborative setting, with healthcare providers with experience in SRC

FIFA Women's World Cup 2011: Pre-Competition Medical Assessment of female referees and assistant referees

BACKGROUND: Precompetition screening was implemented for male referees during the 2010 Fédération Internationale de Football Association (FIFA) Word Cup. In contrast, female football referees have been neglected in this respect although they experience similar physical work loads compared to male referees. METHODS: The standardised football-specific Pre-Competition Medical Assessment (PCMA) was performed in 51 referees and assistant referees selected for the 2011 FIFA Women's World Cup. RESULTS: Family history for sudden cardiac death (SCD) was positive in four referees (7.8%), but cardiac examinations did not reveal any pathological findings. Training-unrelated ECG changes were identified in three referees (5.9%), all without correlates in echocardiography or clinical examination. Most common echocardiography findings (66.6%, n=34) were asymptomatic tricuspid and mitral regurgitations. CONCLUSIONS: During the present screening, no elite female referee was identified being at risk for SCD, and no referee had to be excluded from participating in the 2011 FIFA Women's World Cup

Rest and treatment/rehabilitation following sport-related concussion: a systematic review

AIM OR OBJECTIVE: The objective of this systematic review was to evaluate the evidence regarding rest and active treatment/rehabilitation following sport-related concussion (SRC). DESIGN: Systematic review. DATA SOURCES: MEDLINE (OVID), CINAHL (EbscoHost), PsycInfo (OVID), Cochrane Central Register of Controlled Trials (OVID), SPORTDiscus (EbscoHost), EMBASE (OVID) and Proquest DissertationsandTheses Global (Proquest) were searched systematically. ELIGIBILITY CRITERIA FOR SELECTING STUDIES: Studies were included if they met the following criteria: (1) original research; (2) reported SRC as the diagnosis; and (3) evaluated the effect of rest or active treatment/rehabilitation. Review articles were excluded. RESULTS: Twenty-eight studies met the inclusion criteria (9 regarding the effects of rest and 19 evaluating active treatment). The methodological quality of the literature was limited; only five randomised controlled trials (RCTs) met the eligibility criteria. Those RCTs included rest, cervical and vestibular rehabilitation, subsymptom threshold aerobic exercise and multifaceted collaborative care. SUMMARY/CONCLUSIONS: A brief period (24-48 hours) of cognitive and physical rest is appropriate for most patients. Following this, patients should be encouraged to gradually increase activity. The exact amount and duration of rest are not yet well defined and require further investigation. The data support interventions including cervical and vestibular rehabilitation and multifaceted collaborative care. Closely monitored subsymptom threshold, submaximal exercise may be of benefit. SYSTEMATIC REVIEW REGISTRATION: PROSPERO 2016:CRD42016039570