Objectives: The objectives of this study were to a) examine the distribution of chondrocalcinosis (CC), b) determine the risk factors of CC, and c) examine the radiographic phenotype of osteoarthritis (OA) associated with CC. Methods: Data from the Genetics of Osteoarthritis and Lifestyle (GOAL) study were used to describe the radiographic distribution of CC, and to conduct a case-control study in which cases with CC were compared with controls without CC. All participants had already completed a detailed questionnaire, been examined by a research metrologist, had radiographs of knees, hands, and pelvis, and had given urine and blood samples. All radiographs had been scored for structural radiographic changes of OA, and for the presence of CC. Frontal plane knee alignment was measured on all knee radiographs. The prevalence (95% confidence interval (CI)) of CC was calculated. The odds ratio (OR) and 95% CI were calculated for risk factors of CC, and for structural changes associated with CC in joints with OA. This was adjusted for age, gender, body mass index (BMI), and OA as appropriate, using logistic regression. Results: 3170 participants were included in this study. There were 431 cases with CC. The overall prevalence (95%CI) of CC in the GOAL population was 13.7% (12.5% - 14.9%). In the GOAL population, knee was the commonest site of CC. However, 42% of participants with CC did not have any knee involvement. There was evidence for a generalized predisposition to CC. For example, CC at one joint associated with CC at distant joints. Joints with CC clustered together more than would be expected by chance alone. At knees, wrists and hips, bilateral CC was more likely to associate with CC at distant joints than unilateral CC – also supporting the existence of a systemic predisposition to CC. After adjusting for confounding factors, there was an association between CC and increasing age, lower current BMI, and OA. The association between OA at one joint and CC at the same joint was present for all joints except for the hip. There was no association between CC and gender, diuretic intake, and selected single nucleotide polymorphisms in enzymes involved in pyrophosphate (PPi) metabolism. CC associated with peri-articular calcification, vascular calcification, low cortical bone mineral density (BMD) but not with low cancellous BMD. Self-reported arthroscopy, meniscectomy, knee injury, occupational knee joint loading and knee mal-alignment in the 3rd decade of life associated with knee CC. However, after adjusting for confounding factors including OA, there was no association between either self-reported or radiographically assessed current knee mal-alignment and knee CC. In joints with OA, the additional presence of CC at the same joint associated with a different radiographic phenotype of structural arthropathy. For example, in knees with OA, knee CC associated with attrition. In hips with OA, hip CC associated negatively with osteophytes, joint space narrowing, and sclerosis at the right hip but not at the left. Similarly, in wrists with OA, wrist CC associated with sclerosis in the right but not in the left wrist; in scapho-trapezioid joints (STJs) with OA wrist CC associated with sclerosis on both sides; in metacarpophalangeal joints with OA, wrist CC associated with cysts in the right but not in the left hand; and in 1st carpometacarpal joint with OA, wrist CC associated with cysts in the left but not in the right hand. In knees with OA, the additional presence of CC at distant joints associated with knee attrition. Those with knee CC + OA were excluded from this analysis to remove any local effects of CC. CC at distant joints did not associate with a distinct structural OA phenotype in other joints examined. Conclusion: These findings suggest that CC results form a systemic predisposition, and that it commonly occurs at other joints in the absence of knee involvement. Established risk factors of CC such as age, OA, and previous arthroscopy and/or meniscectomy were validated in this study. Several novel risk factors of CC e.g. low current BMI, low cortical BMD, and vascular calcification were identified. Several novel associations of knee CC i.e. early life knee malalignment, self-reported knee injury, and occupational knee loading were also recognised. There was convincing evidence to suggest that in joints with OA, the additional presence of CC modifies the OA phenotype, and that this varies from joint to joint.

Breast carcinoma is the most frequent type of cancer affecting women. Among the recently described molecular and phenotypic classes of breast cancer, human epidermal growth factor receptor 2 (HER2)-positive tumours are associated with a poor prognosis. HER2 status is currently assessed in routine breast cancer reporting using immunohistochemistry (IHC) in addition to in situ hybridisation (ISH) in borderline cases. The ability of HER2 gene status to predict response to targeted therapy (Trastuzumab) is well documented. However, prognostic information provided by IHC expression categories and prognostic value added by using ISH in borderline cases remains unclear. HER2 plays an important role in cancer progression being targeted to provide predictive and prognostic information. Moreover, HER2 is related to cancer resistance against a variety of therapies; however, trastuzumab has proved successful in treatment of this subgroup. Nevertheless, patients may acquire resistance to this drug after a period of treatment, which indicates that other molecular mechanisms might influence success of this therapy. Dimerisation between members of the HER family may contribute to resistance against treatments due to different combinations that trigger different downstream pathways. This is promoted by ligands, which are expressed as transmembrane precursor protein molecules and have a conserved epidermal growth factor-like domain. Through resistance to trastuzumab, other drugs are being developed to interact in different domains of HER2 protein. The study of interaction between receptors/ligands will characterise specifically their signalling pathway and understand which strategy to acquire. The main aim of this thesis was to assesses the status of HER2 protein (IHC), HER2 gene (chromogenic ISH) and HER heterodimers (in situ proximity ligation assay (PLA)), including HER2/EGFR, HER2/HER3 and HER2/HER4, in two BC series prepared in tissue microarray format; a series of consecutive primary operable BC cases (n = 1858) including HER2+ trastuzumab naïve cases (TrN, n = 221), the second series of HER2+ trastuzumab adjuvant treated cases (TrT, n = 143). Therefore determining the biological characterisation of these biomarkers by associating against clinicopathological parameters, survival outcome and understand the trastuzumab therapy value. There was excellent overall concordance between HercepTest negative (scores 0/1+) and positive (3+) with CISH positive/negative (defined as HER2/Chr17 copy number ratio of ≥ 2; p < 0.001). Kaplan-Meier analysis for breast cancer specific survival (BCSS) and disease free interval (DFI) revealed statistically significant differences between HER2 positive and negative cases detected by HercepTest and CISH (p < 0.001). Interestingly, it was identified that HercepTest 2+ non-amplified cases were not significantly different with those amplified 2+ or 3+ cases with respect to their behaviour (BCSS and DFI). The results revealed an inverse association between the HER heterodimerisation status and hormone receptor status (p < 0.001), and a significantly worse outcome amongst cases revealing high levels of all heterodimers (p < 0.001). Among ER+ cases, the heterodimers high levels were significantly associated with worse prognosis (p < 0.001) overall. However amongst the two HER2+ populations dimerisation status did not show an association with patient outcome. The overall concordance between HercepTest and CISH analysis for HER2 status was excellent. All HercepTest 2+ cases identified were observed to have poor outcomes similar to those HercepTest 3+ cases regardless of gene amplification status. In the current clinical environment cases exhibiting IHC 2+ and non-amplified gene HER2 status will not be offered targeted HER2 therapy but do exhibit aggressive clinical behavioural characteristics. Even though those patients with high levels of the HER2 truncated form, p95HER2, have shown poor outcome, this biomarker does not reveal any extra findings comparing with the HER2 expression results. Beside HER2, EGFR is the only monomer that reveals prognostic value amongst the breast cancer patients. Tumours exhibiting high levels of HER heterodimerisation have an adverse prognosis, however in the context of HER2+ breast cancer no association with clinical outcome was observed regardless of use of trastuzumab treatment. HER2/HER3 heterodimerisation status assessed in multivariate analyses has shown that this protein-protein interaction is associated with a poor prognostic outcome, which needs further investigation and assessment of clinical utility to use in the future in breast cancer treatment decision. Further quantification analysis of dimer/ligand complex using PLA of other HER family members may be useful to identify subset of patients associated with distinct outcome, response to treatment and relationships with HER signalling related biomarkers.

PCOS is one of the leading causes of infertility worldwide affecting 1 in 10 women of a reproductive age. One of the fundamental abnormalities in women with PCOS can be seen within hormonal irregularities, which may include hyperandrogenemia hyperinsulinemia and hyper secretion of luteinising hormone (LH); and it is hypothesised that a defect in steroid secreting ovarian theca cells is involved due to their contribution in non-PCOS hormonal synthesis. Hyperinsulinemia has been associated with hyper-androgenemia through in vitro studies of cultured PCOS theca, where it has been suggested that insulin increases progesterone and androstenedione secretion when compared to normal theca cells. Furthermore the augmented effects of LH and insulin have been seen to increase ovarian androgen synthesis in non-PCOS theca cultures whilst also increasing the expression of steroidogenic enzymes specific to the PI3-K pathway. Many theories exist toward the etiology of hyper androgenemia within PCOS. Very few approaches however, consider dysfunction in multiple tissue types that may contribute to hormonal imbalances. It is well established that an association between obesity and PCOS exists and it is often the first therapeutic target for re-establishing reproductive function in obese PCOS patients. Furthermore PCOS patients tend to show distinct gynoid body fat distribution, which is reported to aggravate PCOS symptoms. It was therefore valid to examine the involvement in adipocyte function and its contribution to androgen levels within peos. This is further supported through the link between metabolic disorders such as insulin resistance and hyperinsulinemia, and their associations to obesity. Our study employed isolated preadipocyte and thecal cultures with close regulation of the influential factors LH and insulin. In doing so, we analysed androgen synthesis through activation and expression of steroidogenic enzymes CYP17 within both normal and polycystic ovaries. This allowed us to examine whether protein/hormonal concentrations vary across non-PCOS and peos cultures. This also allowed us to examine the possibility of a novel pathway leading to localised adipocyte synthesis as well as pinpointing whether dysfunction existed within the insulin-signalling pathway of thecal androgen steroidogenesis. The work in this thesis shows that adipocytes derived from non-PCOS and PCOS women, maintained in vitro differ on the basis of their morphology, rates of differentiation and proliferation. Furthermore, they reacted differently under conditions designed to mimic PCOS in vitro (increased insulin and LH), with reduced non-PCOS proliferation, and increased non-PCOS androgen secretion on insulin treatment. We also found increased steroidogenic CYP 17 expression in PCOS cultures under insulin stimulation. However PCOS adipocytes androstenedione secretion remained unaffected by insulin stimulation and secreted constant levels of androstenedione similar to that seen by insulin stimulated non-PCOS adipocytes. Our examination of non-PCOS and PCOS primary thecal cultures showed CYP17 expression is increased in pcas theca under basal conditions and that increases in insulin and LH leads to increases in in vitro theca proliferation. These conditions were also seen to lead to significant increases in androstenedione secretion over non-PCOS thecal cultures, and the results suggest it to be acting through the PI3-K pathway. These results therefore point to a specific area of dysfunction that should be further targeted for examination. Furthermore, they suggest that an adipocyte dysfunction exists within PCOS patients that may significantly contribute to hyperandrogenemia through localized synthesis of androgens.

Progesterone has been shown to be neuroprotective in a number of central nervous system injury models, including cerebral ischaemia. There is still a lack of understanding behind progesterone’s neuropotective properties, and the purpose of this project is to clarify some of these issues. Osmotic mini-pump infusion was hypothesised to more effective in delivering progesterone to the target organ of the brain, when compared to a bolus intraperitoneal injection. Progesterone pharmacokinetic profiles were compared between different dosing regimes. Intraperitoneal progesterone injection had a short half-life in both plasma and brain, while osmotic mini-pumps delivered higher concentrations of progesterone in plasma and particularly in brain, over a longer period, which supports the hypothesis. It was hypothesised that progesterone will reduce NO production and cell death in in vitro. Progesterone reduced nitric oxide production after challenging microglia with LPS, which supports our hypothesis and the nuclear progesterone receptor was found not to have a major role in nitric oxide attenuation. Neither of the microglial cell lines, BV-2 and HAPI cells produced elevations in NO formation in ischaemic conditions. The in vitro oxygen and glucose deprived model of ischaemia, reduced viability in both microglial and neuronal cells. Also, high pharmacological concentrations of progesterone exacerbated ischaemic injury, which does not support the hypothesis of progesterone in reducing cell death. Progesterone administration, via osmotic mini-pump infusion, was hypothesised to have a better outcome compared to vehicle treatment. After the onset of experimental stroke, progesterone delivery via osmotic mini-pump with loading dose was found to be beneficial in terms of neurological deficit score in adult male mice, which supports the hypothesis. Also, we hypothesise that co-morbidity can affect the efficacy of progesterone treatment in outcomes. Aged animals have an increased sensitivity to experimentally induce stroke and did not display, in the outcomes measured, any benefit from progesterone treatment. NOD/ShiLtJ mice had severe symptoms, resulting in high mortality after surgery and are not recommended as a model of diabetes for experimental stroke. Hypertensive BPH/2 mice are a potential hypertensive model and had better functional outcomes after treatment with intraperitoneally administered progesterone, compared to non-treated hypertensive animals in our small preliminary study. This supports our hypothesis that co-morbidity can affect the efficacy of progesterone treatment in outcomes. The gold-standard for assessing intervention effects across studies within and between subgroups is to use meta-analysis based on individual animal data. We hypothesise meta-analysis would reveal progesterone to reduce lesion volume, but also discover other effects in different subgroups of animals. Progesterone significantly reduced lesion volume, it also appeared to increase the incidence of death following experimental stroke. Furthermore, this negative effect appears to be particularly apparent in young ovariectomised female animals. These findings support the hypothesis that progesterone reduces lesion volume and progesterone having other effects in different subgroups. This investigation has clarified some issues and expanded our understanding on the neuroprotective properties of progesterone. However, these findings indicate further investigation is still required before progesterone can be considered for use in clinical trials as a neuroprotectant in stroke.

Submitted by Daniella Sodre (daniella.sodre@ufpe.br) on 2015-04-13T13:41:14Z No. of bitstreams: 2 DISSERTAÇÃO Wanessa Botelho Cabral.pdf: 1347764 bytes, checksum: e922b207be73f5f6dd67b374c36e0e16 (MD5) license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) / Made available in DSpace on 2015-04-13T13:41:14Z (GMT). No. of bitstreams: 2 DISSERTAÇÃO Wanessa Botelho Cabral.pdf: 1347764 bytes, checksum: e922b207be73f5f6dd67b374c36e0e16 (MD5) license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Previous issue date: 2014-01-22 / CAPES; CNPq / Os agrotóxicos são largamente utilizados no mundo para proteger as plantações contra pragas e doenças, e na saúde pública no controle de vetores que causam doenças ao homem. Contudo, a exposição a esses produtos pode representar graves riscos à saúde humana e ao meio ambiente. Relatos da literatura sobre ação genotóxica das formulações comerciais isoladas de agrotóxicos é bastante escaça, uma vez que a maioria das pesquisas se preocupa em estudar o efeito das misturas desses compostos. Desta forma, o presente trabalho realizou um biomonitoramento in vivo, utilizando camundongos Mus musculus, para avaliar a ação genotóxica aguda, através da ingestão isolada dos agrotóxicos Ciromazina (Trigard 700 WP) e Maconzeb (Dithame NT) em baixas doses. Foram realizados os testes do micronúcleo e do ensaio cometa, para avaliar respectivamente a mutagenicidade e a genotoxicidade desses pesticidas. Sendo utilizados 80 camundongos, divididos em oito grupos contendo 10 animais (cinco machos e cinco fêmeas). As administrações foram feitas por gavagem e cada animal recebeu 1 mL da solução, de acordo com o grupo a que pertencia: o grupo controle negativo recebeu apenas o veículo de diluição (água destilada); para a Ciromazina foram usados três grupos experimentais: C1 com a concentração de 2,5.10−4 mg/Kg, o C2 com a concentração de 5.10−4 mg/Kg, e C3 com a concentração de 5.10-3 mg/Kg; para o Mancozeb foram usados também três grupos experimentais: C1 com a concentração de 3,76.10−4 mg/Kg, C2 com a concentração de 7,52.10−4 mg/Kg, e C3 com a concentração de 7,52.10-3 mg/Kg; para o grupo controle positivo foi administrado via injeção intraperitonial 20 mg/kg p.c. de ciclofosfamida. Na análise estatística, foram realizados dois testes: uma ANOVA multifatorial (Análise de variância), para comparar a ação genotóxica entre os grupos experimentais; e o teste de Mann-Whitney para verificar a influência do sexo no aparecimento do dano genético dentro de um mesmo grupo tratado. O agrotóxico Ciromazina demonstrou ser mutagênico e altamente genotóxico na concentração do grupo C2, além de apresentar uma diferença significativa entre os sexos no grupo C1. Já o pesticida Mancozeb mostrou ser genotóxico na concentração do grupo C2, e foi constatado também que houve diferença significativa entre os sexos dos individuos do grupo C3. Através desta pesquisar evidenciamos que mesmo em baixas concentrações estes pesticidas oferecem grandes riscos à saúde humana e ao meio ambiente.

The aim of this master´s thesis was the evaluation of options that should lead to the elimination of the harmful effects of the white grubs of Melolontha hippocastani to the gradational area of Moravian Sahara (part of forest maintenance LS Stážnice, the Bzenec district). The effectiveness of chosen preparations (Force 1,5 G and Actara 25 WP) on the white grubs of Melolontha hippocastani was observed and conducted under controlled conditions in the ´container experiment´ with 380 seedlings of Pinus sylvestris. This experiment was repeatedly conducted five times over a period of 3 years. The results show that the most effective preparation against the white grubs is Actara 25 WG (200 up to 400 g/ha/600 l water) in the form of watering. This preparation in the form of spraying was ineffective. Inconsistent results were reached with the preparation Force 1,5 G. At the same time, the vertical movements of white grubs during their development stages in the soil were being observed over the period of 2015–2018. This observation took place in the drill holes of the following proportions 50 x 50 x 60–140 cm. The results confirmed that the depth of wintering of the white grubs and their movements during their vegetative state depends on the soil´s temperature and moisture, and the available sources of food to the current instar of the white grub.

Der Südozean ist die Schlüsselregion zur Kenntnis der neogenen Klimaentwicklung. In diesem Zusammenhang ist die Untersuchung von planktonischen Gemeinschaften in Hinblick auf die Umweltentwicklung von großer Bedeutung. In antarktischen neogenen Sedimenten sind Radiolarien reichlich, in großer Diversität und einheitlich gut erhalten. Sie sind nicht nur perfekte Testobjekte für paläobiologischen Studien, sondern auch eine wichtige Quelle für eine verbesserte Biostratigraphie – bisher behinderte nämlich das Fehlen einer verlässlichen Geochronologie detaillierte Studien. Es wurde ein Datensatz aller Taxa von mehreren Standorten des Südozeans gesammelt: für alle der 98 Proben je ca. 7000 Radiolarien. Die Fauna enthält ca. 500 Arten (inclusive 120 neuer Taxa). Die Untersuchung der Makroevolutionsgeschichte dieser Fauna zeigt, dass eine wesentliche ökologische Umwälzung, ohne Aussterbeereignis, bezogen auf den Verlauf der Artengleichheit und den Anstieg der Gattung Antarctissa, bei ca. 8 Ma erfolgte. Dann, 3 My später, folgte ein wesentlicher Diversitätsverlust. Obwohl das ökologische Ereignis eventuell mit einer Änderung der Primärproduzenten assoziiert sein kann, ist der auslösende Faktor des Diversitätsabfalls unbekannt. Außerdem zeigt der Vergleich zwischen der Diversitätsgeschichte dieser Fauna und einer Paläodiversitätsrekonstruktion südozeanischer Faunen, beruhend auf der Neptune Datenbank, daß der generelle Verläufe mit den Probenteilungsmethodologien nachgezeichnet wird, jedoch diese Methodologien aufgrund grober Verzerrungen nicht geeignet für Detailstudien sind. Schließlich wurde eine biostratigraphische Analyse für die komplette Fauna vom Obermiozän bis Pliozän durchgeführt. Obwohl diese Analyse noch vorlaüfig ist, zeigt sich eine Angleichung des aktuellen Altersmodells um mehr als 1 My. Diese Studie zeigt auch 94 neue, sichere Ereignisse, die für die stratigraphische Einordnung der Antarktischen Sedimente genutzt werden können. / The Southern Ocean is the key to understand the Neogene climate evolution. Unfortunately the lack of a robust geochronological framework has hindered precise studies. Equally of interest is understanding how planktonic communities changed in relation with the evolution of these environments. Radiolarians are abundant in Antarctic Neogene sediments, diversified and consistently well-preserved. They should constitute not only an ideal testing ground for paleobiological studies but also a major resource for improved biostratigraphy. In this study, a quantitative, taxonomically-complete dataset have been collected in various sites of the Southern Ocean, using 98 samples and ca. 7000 specimens per sample. Ca. 500 species were uncovered in this fauna, including 120 new to science. The study of the macroevolutionary history of this fauna reveals that a significant, extinctionless ecological turnover, linked to a decrease in the evenness of the species'' abundances and the rise of genus Antarctissa to dominance, occured at ca. 8 Ma, followed 3 My later by a significant diversity loss. Although the ecological event can be tentatively associated with a regional change in the composition of primary producers, the triggering event of the diversity loss is yet to be found. The whole-fauna diversity history was compared to paleodiversity reconstructions computed using subsampling methodologies from the occurrences gathered in the Neptune database. The comparison shows that the main trends are retrieved by the subsampling procedures but also that substantial distortions make them poorly suited for detailed studies. Finally a biostratigraphical analysis was conducted on this whole-fauna dataset for the late Miocene - Pliocene sequence. Although this analysis is still very much preliminary, it shows a coherent readjustement of the current age models by more than 1 My. This study also shows that 94 events seem reliable enough to be used to correlate Southern Ocean sites together.

Parasitische Würmer wie Filarien können das Immunsystem ihres Wirtes modulieren. Diese Immunmodulation schützt nicht nur den Parasiten, sondern kann auch den Wirt vor allergischen Reaktionen schützen. In der vorliegenden Arbeit konnte belegt werden, dass Filariencystatin, ein sezernierter Proteinaseinhibitor des Fadenwurms Acanthocheilonema viteae Th2-assoziierte Entzündungen und die daraus resultierende Atemwegserkrankung im murinen Asthmamodell supprimiert. Diese Effekte von Cystatin beruhen wahrscheinlich auf der Induktion von IL-10 sezernierenden Makrophagen. Dafür spricht, dass sowohl die Depletion von Makrophagen, als auch die Blockierung von IL-10 den schützenden Effekt von Cystatin aufhoben. In einem murinen Dermatitismodell konnte durch Cystatinapplikation die Entzündung der Haut ebenfalls unterbunden werden. Die Verbesserung der atopischen Dermatitis führte zu einem Anstieg von TGF-Beta in den Ekzemregionen. Zusammenfassend belegen die Daten, dass ein einzelnes rekombinantes Helminthenprotein den anti-allergischen Effekt einer Gesamtwurminfektion nachahmen kann. Des weiteren wurde untersucht, ob die Immunmodulation von Helminthen an die Biologie des Wurmes gebunden ist. Der Einfluss einer gastrointestinalen Wurminfektion mit Heligmosomoides polygyrus wurde in Modellen mucosa-assoziierter allergischer Atemwegsentzündung und nicht-mukosa-assoziierter, kutanen Dermatitis untersucht. Im Asthmamodell konnte eine Reduzierung der Inflammation beobachtet werden, im Dermatitis Modell keinerlei Verbesserung des klinischen Bildes. Die Wurminfektion führte zu erhöhten Werten von Foxp3+ regulatorischen T-Zellen (Treg) in peribronchialen Lymphknoten, im Gegensatz dazu konnten keine Treg Zellen in ekzematöser Haut nachgewiesen werden. Diese Ergebnisse sind ein wichtiger Aspekt beim Einsatz von Helminthen zur Behandlung allergischer Erkrankungen, da die Lokalisation des Wurmes ein entscheidender Faktor in Richtung Verbesserung oder Verschlimmerung allergischer Erkrankungen zu sein scheint. / Parasitic worms, like filariae, have the intriguing capacity to modulate immune responses directed against them. This immunomodulation protects the parasite and was found to mediate a negative correlation between parasite infections and the prevalence of allergic disease. In the present study filarial cystatin (Av17), a secreted protease inhibitor of the filarial nematode Acanthocheilonema viteae, was found to suppress Th2-related inflammation and allergic airway responsiveness in mice. These effects of Av17 on allergic airway inflammation were probably due to induction of IL-10 secreting macrophages. Depletion of macrophages as well as blocking of IL-10 by application of anti-IL-10 receptor antibodies, prevented the curative effects and restored allergic airway inflammation. Treatment with cystatin in a model of allergic skin disease, suppressed inflammation, too. The protective effect on atopic dermatitis was accompanied by an increase of TGF-Beta in the eczema region. Hence, the data demonstrate that treatment with a single recombinant helminth protein can exert the anti-allergic effects of helminth infections. Furthermore this study investigated if immunomodulation by helminths is dependent on the biology of the worm. Infection with the gastrointestinal nematode Heligmosomoides polygyrus, was analyzed in models of mucosa-associated airway disease and non-mucosal, cutaneous atopic dermatitis. Infected mice showed reduced signs of allergic airway inflammation but no differences in phenotypical signs of dermatitis. Worm infection was associated with elevated numbers of Foxp3+ regulatory T cells (Treg) in peribronchial lymph nodes in the asthma model, whereas Treg cells were basically absent in eczematous skin. These findings might be an important aspect for future considerations of helminths for treatment of allergic diseases, as localisation of the parasites might be a crucial factor leading to amelioration or aggravation of allergic disease.

Submitted by Automa??o e Estat?stica (sst@bczm.ufrn.br) on 2016-08-26T23:09:02Z No. of bitstreams: 1 VitorAlcantaraDeAlmeida_DISSERT.pdf: 2275305 bytes, checksum: 861287dd67240f715c731a69f2fa5aec (MD5) / Approved for entry into archive by Arlan Eloi Leite Silva (eloihistoriador@yahoo.com.br) on 2016-08-30T23:32:12Z (GMT) No. of bitstreams: 1 VitorAlcantaraDeAlmeida_DISSERT.pdf: 2275305 bytes, checksum: 861287dd67240f715c731a69f2fa5aec (MD5) / Made available in DSpace on 2016-08-30T23:32:12Z (GMT). No. of bitstreams: 1 VitorAlcantaraDeAlmeida_DISSERT.pdf: 2275305 bytes, checksum: 861287dd67240f715c731a69f2fa5aec (MD5) Previous issue date: 2016-04-29 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior (CAPES) / A presente disserta??o descreve uma extens?o para a plataforma Frama-C e o plugin WP:o WPTrans. Essa extens?o permite a manipula??o, atrav?s de regras de infer?ncia, dasobriga??es de prova geradas pelo WP, com a possibilidade das mesmas serem enviadas,em qualquer etapa da modifica??o, a solucionadores SMT e assistentes de prova. Algumasobriga??es de prova podem ser validadas automaticamente, enquanto outras s?o muitocomplexas para os solucionadores SMT, exigindo uma prova manual pelo desenvolvedor,atrav?s dos assistentes de prova. Contudo, a segunda abordagem geralmente requer dousu?rio uma experi?ncia significativa em estrat?gias de prova. Alguns assistentes oferecemcomunica??o com provadores autom?ticos, entretanto, esta liga??o pode ser complexaou incompleta, restando ao usu?rio apenas a prova manual. O objetivo deste plugin ? interligaros dois tipos de ferramentas de modo preciso e completo, com uma linguagemsimples para a manipula??o. Assim, o usu?rio pode simplificar suficientemente as obriga??es deprova para que possam ser validadas por qualquer outro solucionador SMT.N?o obstante, a extens?o ? interligada diretamente ao WP, facilitando a instala??o doplugin no Frama-C. Esta extens?o tamb?m ? uma porta de entrada para outras poss?veisfuncionalidades, sendo as mesmas discutidas neste documento. / The platform Frama-C is a tool dedicated to analysis of source code of software written in C, with the possible types of analysis provided by plugins attached to the platform. One of its plugins is WP, used for deductive veri cation of C code with ACSL, a formal speci cation language. This dissertation describes the extension of this plugin, named WPTrans. This extension allows generated proof obligations from WP to be manipulated through inference rules and sent, at any stage of the proof, to automatic (mainly SMT solvers) and interactive theorem provers. Some proof obligations may be proved automatically, while others can be too complex to be solved by automatic theorem provers, requiring the users of Frama-C and WP to handle them manually. This approach usually requires a signi cant experience in proof strategies. Some interactive theorem provers provide communication with automatic provers. However, this connection can be complex and incomplete, leaving the user with the manual proof option only. The strength of WPTrans is to combine the features of automatic and interactive theorem provers in a precise and complete way, with a simple manipulation language. Thus, the user can simplify the proof obligations enough in order for its proof to be concluded with an SMT solver, letting the proof be partially manual and partially automatic. Nevertheless, the plugin is directly linked do WP, facilitating the installation of the extension in Frama-C. This tool is also a gateway to other possible features, which we discuss herein.

Diese Arbeit untersucht die Reaktion der Stieleiche auf pflanzenparasitären Nematoden mittels RNA-Sequenzierung und Analyse von stabilen Isotopen und Fettsäuren. Einblicke in Rhizosphäreninteraktionen wurden über mutualistische Partner (Ektomykorrhizapilze, Rhizosphärenhelferbakterien), fungivore Collembolen und multitrophische Gemeinschaften gewonnen. Die Struktur und Biomasse der Mikroorganismen sowie die Fitness der Eichen wurden erfasst. Die Effekte wurzelfressender Nematoden auf die Eiche wurden durch das endogene rhythmische Wachstum des Baumes reguliert. Die Nematoden lösten eine stärkere Reaktion während des Sprosswachstumsschubs aus, u.a. Aktivierung von Abwehrmechanismen und Hemmung der Photosynthese, wohingegen beim Wurzelwachstumsschub pathogen bezogene Signale unterdrückt waren. Die Anwesenheit des Pilzsymbionten schwächte die Pflanzenabwehr und verbesserte die Stresstoleranz, was indirekt das Wachstum der Mikroorganismen förderte. Die Helferbakterien begünstigten den Mykorrhizapilz, was wiederum das Pflanzenwachstum stimulierte und dem negativen Effekt der Nematoden entgegenwirkte. Parasitäre Nematoden und fungivore Collembolen beeinflussten die Verteilung des pflanzlichen Kohlenstoffes unabhängig voneinander; Nematoden verringerten und Collembolen verbesserten die Allokation von Photoassimilaten in Gram-postiven Bakterien. Zudem war steigende trophische Diversität der Bodenfauna in der Rhizosphäre entscheidend für die Balance innerhalb der mikrobiellen Gemeinschaft, welche das Pflanzenwachstum fördert. Diese Arbeit stellt die Bedeutung der endogenen Ressourcenzuteilung von Pflanzen für unterirdische biotische Wechselbeziehungen heraus. Diese Pflanzenstrategie als bedeutender Faktor für Rhizosphärenprozesse sollte in zukünftige Studien Berücksichtigung finden. Die Einbeziehung der Hauptakteure in der Rhizosphäre ermöglicht zudem ein realistischeres Bild von Nematoden-Pflanzen Interaktionen und damit ein effektiveres Management. / This thesis investigated the response of Pedunculate oak to the plant-parasitic nematode Pratylenchus penetrans, using RNA-sequencing, stable isotope labelling and fatty acid analyses. Insight into rhizosphere interactions was gained by employing beneficial biotic partners (ectomycorrhizal fungi, rhizosphere helper bacteria), fungal grazers (Collembola) and multitrophic environments. Microbial biomass and community structure as well as oak fitness were assessed. The effects of root-feeding nematodes on oak were largely governed by the endogenous rhythmic growth of the tree. The nematodes triggered a stronger response during shoot flush, e.g. activation of multi-layered defence mechanisms and repression of photosynthesis, as compared to root flush where pathogen-related signalling was repressed. With the presence of the mycorrhizal symbiont plant defence was attenuated and stress tolerance enhanced, indirectly promoting the growth of rhizosphere microorganisms. The helper bacteria fostered the ectomycorrhizal fungus, which in turn stimulated plant growth, counteracting the negative effects of nematodes. Plant-parasitic nematodes and Collembola grazers had independent roles in plant carbon allocation patterns, with nematodes hampering whilst Collembola enhancing the flux of recent photoassimilates to Gram-positive bacteria. Lastly, increasing trophic diversity of the soil fauna in the rhizosphere of oaks was crucial for the maintenances of a microbial community equilibrium that promotes plant growth. In sum, this study highlights the importance of endogenous resource allocation pattern of plants in determining the outcome of belowground biotic interactions. Therefore such plant traits should be considered as important drivers for rhizosphere processes in future studies. Moreover, taking into account the rhizosphere main players in studies on parasitic nematode-plant interactions will result in a more realistic picture and thus more effective nematode management.