This thesis is an investigation of detecting and quantifying airborne inoculum of Sclerotinia sclerotiorum (Lib.) de Bary to improve the Sclerotinia rot of carrot (SRC) forecast model. A quantitative polymerase chain reaction (qPCR) assay was developed to specifically detect and quantify DNA from airborne ascospores of S. sclerotiorum. The qPCR assay was evaluated on air samples collected using a Burkard Sampler, and showed that ascospores of S. sclerotiorum were specifically detected among a pool of foreign DNA. The concentration of detected ascospores was related to the observed incidence of SRC to suggest a preliminary threshold of 2 to 4 ascospores m-3 of air for SRC development. Evaluation of an Andersen Sampler, the blue plate test (BPT) and the qPCR assay showed that the latter two methods were equally effective in detecting and quantifying ascospores of S. sclerotiorum and consistently detected greater numbers of ascospores than an Andersen Sampler. Three days are required to confirm the presence of S. sclerotiorum using the BPT, while results from the qPCR assay can potentially provide results within five hours of air sampling. The choice of detection method depends on the available resources and need for a quick result. Analysis of data from nine years of air sampling using the BPT indicated that a single air sampling site is sufficient to detect ascospores when counts are low, increasing to two sites during periods when ascospores are detected near threshold levels and crop and environmental conditions are conducive to disease. Chitosan and canopy trimming were evaluated to manage SRC under field conditions. Chitosan reduced area under the disease progress curve (AUDPC) by 55 and 42% in 2009 and 2011, respectively, which was comparable to a standard fungicide. Trimming enhanced chitosan efficacy, reducing AUDPC by 88 and 82% in 2009 and 2011, respectively. Trimming as a stand-alone treatment reduced AUDPC by 66% in 2011. Under controlled environmental conditions, chitosan inconsistently enhanced defense responses against S. sclerotinia. The results show that chitosan has potential to be integrated into SRC management systems, particularly when combined with foliar trimming in years with moderate to high disease risk. / National Research Council of Canada; University of Guelph; Department of Plant Agriculture; Ontario Ministry of Agriculture, Food and Rural Affairs

Chytridiomycosis is an emerging cutaneous fungal disease that contributes to recent global declines and extinction of amphibian species, caused by infection of the skin with a fungus known as Batrachochytrium dendrobatidis (Bd). Many species of frogs secrete antimicrobial peptides onto their skin that are capable of killing Bd. This thesis is an investigation of the effect of corticosteroids on cutaneous innate immunity in frogs, in the context of infection with Bd. The general hypothesis was that injections of glucocorticoids would impair the cutaneous synthesis of these antimicrobial peptides, thereby increasing susceptibility to Bd infection. The objective of the first experiment was to measure and compare gene expression levels of cutaneous AMP’s in frogs treated with glucocorticoids with sham-treated controls. Wild-caught Lithobates pipiens were acclimatized and administered either the corticosteroid methylprednisolone or saline every 48 hours. Norepinephrine-elicited cutaneous secretions were collected prior to the first injection of corticosteroid or saline, and then every 8 days for 40 days. Gene expression of the AMP’s brevinin and ranatuerin in the cutaneous secretions was quantified relative to the reference genes EF1-α and RPL8 using reverse transcription quantitative polymerase chain reaction (RT-qPCR). Corticosteroid treatment was associated with a significant (P<0.027) increase in brevinin gene expression, which was most notable at 24-40 days of corticosteroid administration. Ranatuerin expression followed a similar but nonsignificant trend. The second experiment was a pilot study intended to establish a Bd challenge protocol in L. pipiens. Frogs were immersed in water containing 0, 104, 105 or 106 zoospores of Bd strain JEL 423. Cutaneous swabs were collected prior to challenge and tested for Bd by qPCR; unexpectedly, some tested positive, indicating pre-challenge infection. The analysis was complicated by an identified cross-reactivity of the assay with other fungi. The findings of the first experiment refuted the hypothesis, and suggested that corticosteroids promote rather than impair AMP gene expression in the skin of L. pipiens, under these experimental conditions. Further, the second study demonstrated that none of the frogs showed clinical abnormalities or died, despite exposure to Bd zoospores and despite molecular and histologic evidence of cutaneous Bd infection in some frogs. / NSERC Discovery Grant

La qPCR est actuellement la technique de référence en matière de quantification d'ADN. Elle peut être définie comme une amplification exponentielle, cyclique et ciblée de la séquence d'ADN cible. Le caractère exponentiel de la qPCR est à la fois à l'origine de la sensibilité de la méthode mais aussi d'une potentielle variabilité inter-échantillons. Cette variabilité est compensée par le caractère cyclique de la méthode qui entraine une synchronisation de la réaction pour tous les échantillons à chaque cycle. L'amplification ciblée de la séquence choisie traduit quand à elle la spécificité de la méthode. Néanmoins, cette dernière propriété de la PCR reste la moins vérifiée. La spécificité de la qPCR est indiscutable lorsque la cible à détecter se trouve en quantité suffisante (>100 copies environ). En revanche, pour des quantités plus faibles ou en présence d'inhibiteurs, la spécificité diminue ou disparaît (limites de détection et de quantification). Cette perte de spécificité retentit de façon significative sur la précision et la reproductibilité du dosage à réaliser. Cependant, si l'hybridation non spécifique est inéluctable dans ces conditions, l'amplification non spécifique consécutive peut être limitée, voire supprimée au moyen d'amorces de PCR soit plus spécifiques, soit moins susceptibles de générer des différences inter-échantillons. La RT-qPCR, grâce à une étape initiale de transcription inverse (conversion d'un ARN en un ADN complémentaire) permet la quantification des ARN. Cependant, la transcription inverse reste moins reproductible que la PCR, générant des différences inter-échantillons délétères en diagnostic comme en recherche. Au cours de ces travaux, je propose des méthodes originales améliorant de façon significative différentes étapes de ces techniques. Premièrement, je propose une amélioration de la standardisation de l'étape de transcription inverse capable de diminuer de façon significative la variabilité inter-échantillons ; l'utilisation d'un volume constant d'extrait d'ARN pour chaque échantillon améliore considérablement la précision de la quantification d'ARN messagers. Deuxièmement, je propose une modification des amorces par incorporation de résidus d'acides nucléiques bloqués (LNA) ; cette modification permet d'augmenter la spécificité des amorces aux limites de la détection. Enfin, je propose une méthode simple et économique permettant la mesure directe de la température de fusion (Tm) dans les conditions réelles de la PCR. Ce paramètre, considéré comme capital pour la réalisation des dosages par qPCR, est généralement obtenu par des méthodes prédictives peu précises. De plus, cette méthode doit permettre de déterminer précisément les paramètres thermodynamiques des amorces (∆G, ∆H et ∆S) et ainsi avoir accès d'une part au pourcentage réel d'amorces hybridées en fonction de la température et d'autre part d'identifier la susceptibilité des amorces aux inhibiteurs.

There is much evidence that herbivory is a key innovation for the tremendous success of insect. In this thesis I have investigated different aspects of host plant utilization and phenotypic plasticity using the polyphagous comma butterfly, Polygonia c-album. Even though external conditions affect a phenotypic plastic response, the outcome is often influenced by a genetic background which may differ among populations. In Paper I we suspected the genetic background to seasonal polymorphism to be X-linked. However, results from interspecific hybridization between two populations suggested that diapause response is instead inherited in a mainly autosomally additive fashion, with a possible influence of sexual antagonism on males. In Paper II we showed that female oviposition preference is not a plastic response influenced by larval experience, but has a genetic background coupled to host plant suitability. Further, there is a strong individual correlation between larval host plant acceptance and female host plant specificity (Paper III). We believe this to be a larval feed-back genetically linked to female host specificity: offspring to ‘choosy’ specialist mothers benefit by remaining on the original host while offspring to less discriminating generalist mothers should risk inspecting the surroundings, thus compensating for potential poor female choice. In the larval mid-gut, genes are differentially expressed depending on host plant diet (Paper IV). Therefore, we expected to find fitness consequences of host plant switch. However, although growth rate was affected in a few treatments, larvae were generally surprisingly good at adjusting to new diets (Paper V). To conclude, host plant choice in both female and larval life stage is connected to performance. Combined with increased understanding about the plastic response to diet intake and seasonal polymorphism we have gained further insights into the processes of local adaptations and speciation in the Lepidoptera. / At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 3: Submitted Manuscript; Paper 5: Manuscript

Les nématodes à kystes sont l'un des bioagresseurs causant le plus de dégâts sur les cultures de pommes de terre. La résistance trouvée chez l'accession spl88S.329.18, issue de l'espèce Solanum sparsipilum est caractérisée par un déterminisme oligogénique avec un QTL à localisé sur le chromosome V (GpaVspl) et un QTL mineur localisé sur le chromosome XI(GpaXIspl). Pour obtenir une résistance de haut niveau, l'effet du QTL GpaVspl, doit êtrecomplémenté par celui du QTL à effet faible GpaXIspl. Par génomique comparative, le locusGpaV a été localisé dans un intervalle compris entre 16 et 60 kb sur les génomes de la tomateet des espèces apparentées à la pomme de terre, Solanum demissum et Solanum phureja. Deuxgènes ont été annotés dans cet intervalle sur les génomes de la tomate et de S. demissum : lepremier appartient à la famille des TIR-NBS-LRR (TNL), famille de gènes de résistanceclassiques, et le second appartient à la famille des " mitochondrial, transcription terminationfactor " (mTERF), dont l'implication dans des mécanismes de résistance n'a jamais étédémontrée.Les objectifs de ma thèse étaient d'identifier le(s) gène(s) responsable(s) de la résistance àG. pallida, conférée par le locus GpaVspl, et d'étudier sa régulation. Suite à la publication de laséquence du génome de S. phureja, en 2011, nous avons mis en évidence que le locus GpaVétait dupliqué chez S. phureja et que cette duplication était également présente chezS. sparsipilum. Les quatre gènes annotés au locus GpaVspl ont été nommésSpl_mTERF18430, Spl_TNL18429, Spl_mTERF18453 et Spl_TNL18428.L'effet des deux gènes Spl_mTERF18430 et Spl_TNL18428 sur la résistance à G. pallida aété analysé via des expériences de transformation génétique suivies par des tests de résistancesur les plantes transformées. Un effet partiel du gène Spl_TNL18428 sur la résistance àG. pallida a été mis en évidence par complémentation de plantes sensibles. Aucun effetsignificatif n'a été détecté pour le gène Spl_mTERF18430. Des expériences d'extinctiongénique suggèrent que le deuxième gène TIR-NBS-LRR, Spl_TNL18429, qui est égalementexprimé dans les racines et qui présente un fort pourcentage d'identité de séquence avec legène Spl_TNL18428, pourrait également être impliqué dans la résistance à G. pallida.L'expression du gène rapporteur GFP, placé sous le contrôle du promoteur du gèneSpl_TNL18428, est fortement induite dans les cellules situées autour du syncytium. Cecirenforce l'hypothèse d'une implication du gène Spl_TNL18428 dans la résistance à G. pallida,car la localisation de l'expression de la GFP est similaire à celle de la nécrose, qui estcaractéristique de la réaction développée par les plantes résistantes autour du syncytium induitpar les nématodes.En tenant compte des données bibliographiques récentes, montrant que plusieurs gènes NBSLRRpeuvent être indispensables à l'expression d'une résistance, nos résultats suggèrent queles deux gènes Spl_TNL18428 et Spl_TNL18429 sont nécessaires à l'expression de larésistance à G. pallida

Με την ηλικία, νόσοι που σχετίζονται με δομικά ελαττώματα των οστών που οφείλονται σε κατάγματα ή εκφυλισμούς, αναμένονται να αυξηθούν σε συχνότητα. Επιπλέον, η αύξηση του προσδόκιμου ζωής επιβάλλει τη χρήση βελτιωμένων συνθετικών υλικών για την αντικατάσταση νοσούντων οστών, για παράδειγμα κατά τη χρήση μεταλλικών ράβδων σε περιπτώσεις βλαβών μη-ένωσης και στις χειρουργικές επεμβάσεις αντικατάστασης ισχίου. Τα υπάρχοντα υλικά σχετίζονται με υπο-βέλτιστη οστεοενσωμάτωση και προβληματική μακροπρόθεσμη επιβίωση του σύνθετου εμφυτεύματος. Για το λόγο αυτό, η βελτίωση των υλικών επικάλυψης και των μηχανικών ιδιοτήτων των νέων, κυτταρικά συμβατών, συστατικών είναι επιτακτική. Για να αντιμετωπιστεί αυτό το πρόβλημα, υλικά νέας γενιάς είναι διαθέσιμα, ενδεχομένως με καλύτερες ιδιότητες ως υπόστρωμα προσκόλλησης για τα κύτταρα των οστών. Ο σκοπός της παρούσας εργασίας ήταν να εκτιμηθεί η ικανότητα ενός νέου, ειδικά κατασκευασμένου υλικού απο νανοσωλήνες άνθρακα ως προς τη διατήρηση της σωστής έκφρασης των χαρακτηριστικών γονιδίων των οστεοβλαστών, με έμφαση στην έκφραση των γονιδίων που εμπλέκονται στις αλληλεπιδράσεις οστεοβλαστών-υποστρώματος και έτσι προωθούν την σταθερή προσκόλληση των κυττάρων στο υπόστρωμα. Παράλληλα, ερευνήσαμε και την επίδραση της μηχανικής καταπόνησης στην έκφραση των γονιδίων αυτών σε κύτταρα που καλλιεργήθηκαν σε νανοσωλήνες άνθρακα. Χρησιμοποιήσαμε δύο ανεξάρτητες απομονώσεις οστεοβλαστών διαφοροποιημένων από ανθρώπινα μεσεγχυματικά βλαστικά κύτταρα μυελού των οστών, δηλαδή προχωρήσαμε σε δύο ανεξάρτητα πειράματα. Και στα δύο, για να γίνει ο πειραματισμός όσο εγγύτερα στις πραγματικές συνθήκες, καλλιεργήσαμε τους οστεοβλάστες υπο στατικές συνθήκες όσο και υπό συνθήκες μηχανικής καταπόνησης, για την προσομοίωση "in vivo" συνθηκών, και συγκρίθηκε η γονιδιακή έκφραση οστεοβλαστών που καλλιεργήθηκαν σε πλαστικό έναντι επιφανειών επικαλυμμένων με νανοσωλήνες άνθρακα. Απομονώσαμε το RNA από τους οστεοβλάστες μετά από την καλλιέργειά τους για 3 και 24 ώρες και προσδιορίσαμε, χρησιμοποιώντας την τεχνική real time RΤ-PCR, την έκφραση των ακόλουθων γονιδίων σε επίπεδο mRNA: κολλαγόνο-α1, αλκαλική φωσφατάση, οστεοποντίνη, βινκουλίνη και ιντεγκρίνες α4, αV, β1 και β3. Συνολικά, τα αποτελέσματα της ανάλυσης του κυτταρικού mRNA έδειξαν ότι η γονιδιακή έκφραση μετά από 3 ώρες καλλιέργειας είναι πολύ μεταβλητή, και οριστικά συμπεράσματα δεν θα μπορούσαν να εξαχθούν. Ωστόσο, αφού δίνεται η ευκαιρία στα κύτταρα να προσκολληθούν σταθερά, στις 24 ώρες, κατέστη σαφές ότι: α) η κυτταρική ταυτότητα των διαφοροποιημένων οστεοβλαστών διατηρείται, με βάση το γεγονός ότι η έκφραση αυτών των χαρακτηριστικών γονιδίων, που σχετίζονται με την προσκόλληση, συντηρείται σωστά, αν και σε διάφορα επίπεδα, β) σε στατικές συνθήκες, το επίπεδο της έκφρασης των εξετασθέντων γονιδίων είναι κατά τι χαμηλότερο σε οστεοβλάστες που καλλιεργηθήκαν σε επικαλυμμένη επιφάνεια με νανοσωλήνες άνθρακα σε σύγκριση με τα κύτταρα που καλλιεργηθήκαν σε πλαστικό, και γ) σε σύγκριση με τις στατικές συνθήκες, το μηχανικό ερέθισμα ενισχύει την έκφραση αυτών των γονιδίων οστεοβλαστών όταν καλλιεργούνται σε νανοσωλήνες άνθρακα, για την επίτευξη υψηλών επιπέδων mRNA έκφρασης των γονιδίων κυτταρικής προσκόλλησης. Τα αποτελέσματα της τελευταίας ανάλυσης της γονιδιακής έκφρασης είναι επίσης συμβατά με τις συνολικές ποσότητες RNA που λαμβάνονται, υποστηρίζοντας έμμεσα τη σταθερή προσκόλληση και επιβίωση των οστεοβλαστών σε νανοσωλήνες άνθρακα υπο συνθήκες μηχανικής καταπόνησης. Συμπεραίνουμε λοιπόν ότι το νέο υπόστρωμα από νανοσωλήνες άνθρακα που αναλύθηκε σε μηχανικές συνθήκες διέγερσης που προσομοιάζουν, κατά το δυνατόν, συνθήκες καταπόνησης in vivo, συνιστά ένα κατάλληλο κυτταρικό υπόστρωμα, συμβατό με την επιβίωση των οστεοβλαστών, τη διαφοροποίηση, την ανάπτυξη και την σταθερή προσκόλλησή τους στο υπόστρωμα νανοσωλήνων. Η εργασία αυτή υποστηρίζει την πιθανότητα της χρήσης αυτών των νέων υλικών στο μέλλον για την επικάλυψη σκελετικών προσθέσεων, με σκοπό την απόκτηση βέλτιστης οστεοενσωμάτωσης. / As population ages, diseases related to bone structural defects due to fracture or degeneration are expected to increase in frequency. In addition, the increase in life expectancy necessitates better composite materials for replacement of diseased/fractured bones, for example during the use of metal rods for non-union defects and in hip replacement surgery. The existing materials are associated with sub-optimal osseointegration and problematic long-term survival of the composite graft. For this reason, improvement of coating materials and engineering of novel cell-compatible components is imperative. To address this problem, new-generation materials are available, with possibly better bone cell adherence properties. The Aim of this work was to evaluate the ability of a novel, specially-constructed carbon nanotube material to sustain proper expression of characteristic osteoblast genes, with emphasis on the expression of genes that are functionally involved in osteoblast-matrix interactions and promote firm cell adherence to substrate. We used two independent isolates of osteoblasts differentiated from human bone marrow mesenchymal stem cells, ie we proceeded to two independent experimental runs. In both, to make the experimentation more context-relevant, we grew the osteoblasts in static as well as under mechanical strain, to simulate in vivo conditions, and also compared gene expression in osteoblasts grown on plastic versus carbon nanotube-coated surface. We isolated RNA from the osteoblasts at 3 hours and 24 hours after seeding them on the culture vessels and determined, using real-time RT-PCR techniques, the level of expression of the following genes at the mRNA level: α1-collagen, alkaline phosphatase, osteopontin, vinculin, and integrins α4, αV, β1 and β3. All in all, the results on cell mRNA analysis indicated that gene expression at 3h post-plating is too variable and no firm conclusions could be drawn. However, once the cells are given a chance to firmly adhere, at 24h, it became clear that: a) osteoblast cell identity is maintained, based on the fact that the expression of these characteristic matrix- and adhesion-related genes is properly maintained, albeit in various levels, b) in static conditions, the level of expression of the examined genes is lower in cells grown on nanotube-coated surface compared to cells grown on plastic, and c) in comparison to static conditions, mechanical stimulation enhances expression of these genes in osteoblasts grown on nanotubes, to attain robust levels of cell adherence gene mRNA expression. The results of the latter gene expression analysis are also compatible with total RNA quantities obtained, indirectly arguing firm osteoblast adhesion/survival on nanotubes under mechanical strain conditions. We therefore conclude that the novel carbon nanotubes assayed herein in lifelike mechanical stimulation conditions, constitute an appropriate cell-bearing surface, compatible with osteoblast survival, differentiation, growth and firm adherence to substrate. This work raises the possibility of using this novel material in the future to coat skeletal prostheses, in order to obtain improved osseointegration.

No description available.

Colorectal cancer (CRC) is a common malignancy which develops due to old age and lifestyle factors, low percent of patients afflicted by a genetic disorders. Half of all colorectal cancer patients are diagnosed after metastasis. The high rate of the late detection, emphasizes on the requirement of convenient and inexpensive diagnostic methods for comprehensive screening programs. The aim of this study was to discover proto-oncogenes mutation and assessment of tumor suppressor genes expression. Formalin fixed paraffin embedded (FFPE) histologically verified colorectal cancer samples were used. APC, KRAS and BRAF mutations were investigated using polymerase chain reaction (PCR) fragments and direct sequencing. Gene expression assessment of MLH1, MGMT and CDKN2A were achieved via quantitative polymerase chain reaction (qPCR). In the present study we could detect a novel transversion heterozygous mutation in APC gene codon 1365 in three patients. BRAF codon 600 mutation were detected in one patient. KRAS codon 12 mutation was discovered in one sample and also a novel transition mutation in codon 15 was detected in 6 patients. In 80% of cases, MLH1 and MGMT expression were undetectable, in remaining 20%, MLH1 expression were reduced, but MGMT showed both reduced and increased expression compared to control. In 100% of patients CDKN2A expression was undetectable. The rate of mutations in predetermined hotspot codons and amount of uncommon mutations into APC, BRAF and KRAS in Nepalese patients indicates the requirement of further investigation in CRC patients from that part of the world. Also, the expression rate of MLH1, MGMT, CDKN2A and deficiency of an information source emphasizes the necessity of whole genome CRC expression profiling data to comparison and conclusion. / <p>-</p> / -

Orientador: Fernanda da Cruz Landim-Alvarenga / Resumo: As tecnologias de reprodução assistida, tais como a fertilização in vitro (FIV), transferência de embriões, transgenia e clonagem, ainda não tem o impacto comercial desejado devido a baixa produção embrionária. Apenas 30 a 40% dos blastocistos desenvolvidos são obtidos de oócitos após a MIV, fertilização e cultivo dos embriões, embora 80% dos oócitos maturados in vitro sejam fertilizados com sucesso. O soro fetal bovino (SFB) é o suplemento mais utilizado no cultivo de embriões in vitro, uma vez que melhora o desenvolvimento dos blastocistos. Apesar disso, sua presença está relacionada a alterações do metabolismo embrionário, perda de qualidade e indução de modificações na expressão de vários genes embrionários. Na tentativa de minimizar os efeitos deletérios do SFB, várias citocinas e fatores de crescimento têm sido acrescentados aos meios de cultivo embrionários in vitro, com a intenção de mimetizar as condições de cultivo in vivo. O presente experimento tem como objetivo avaliar e comparar os efeitos da adição do SFB e de meio condicionado por células mesenquimais estromais (MSCs) durante o cultivo embrionário. Os parâmetros analisados foram a viabilidade embrionária, apoptose e o perfil transcricional de genes relacionados à qualidade dos embriões. Não foi observado uma diferença (P≥0,05) na clivagem dos blastocistos, porém observou-se que a taxa de produção de embriões utilizando SFB no CIV foi maior (P≤0,05) quando comparada com MC, mas não diferiu (P≥0,05) do grupo pro... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Endometrial mesenchymal stromal cell (eMSCs) secretes bioactive molecules such as cytokines and growth factors which are released as soluble factors or through extracellular vesicles (EVs). Conditioned medium (CM) of the MSCs maintains the immunomodulation and regenerative potential properties of the cells that produced it. This study investigated the use of CM by eMSC plus BSA as alternative to FBS in embryo culture medium. The developmental ability and quality of bovine embryos were determined by assessing their cell number and gene expression. The percentage of embryos that underwent cleavage was similar (P>0.05) among the groups but blastocyst formation was higher (P<0.05) in FBS group. The total cell number was higher in CM group, but not statistically different from the others (P>0.05). The relative mRNA expression of ELOVL6 was higher in the CM group, CASP3 in the BSA group, ACSL3 and VEGF in the FBS group. Taken together, these data suggest that CM can be used as an alternative supplement to FBS. We observed a different gene expression profile, suggesting the CM inhibited an increase in the relative mRNA levels for CASP3. Moreover, the CM favored the total number cells, inhibited the percentage of cells in apoptosis and produces better quality embryo. / Mestre

Estudos preliminares visando a identificação de possíveis agentes virais associados à síndrome multissistêmica do definhamento dos suínos (SMDS) revelaram uma possível associação inversa entre a presença de TTSuV1 e a ocorrência da SMDS. Com base neste achado, foi formulada a hipótese de que o TTSuV1 poderia ser capaz de inibir a multiplicação do PCV2, impedindo assim o desenvolvimento da SMDS. Buscando esclarecer esta questão, seria necessário desenvolver um sistema eficiente de replicação para este vírus, até o presente ainda não disponível. Em vista disso, foi desenvolvido um método de detecção de infecções por TTSuV em cultivos celulares para a avaliação de possíveis linhagens a serem potencialmente utilizadas para isolamento e multiplicação destes vírus. Genomas de TTSuVs foram detectados em células de linhagem de origem suína e não suína assim como em um dos lotes de tripsina. Os soros utilizados como suplemento para o meio de cultivo não apresentaram genomas de TTSuV. Desta forma, o lote de tripsina contaminado pode ser considerado uma importante fonte de contaminação, principalmente em células de origem não suína. Com o objetivo de avaliar uma possível associação entre os TTSuVs e a ocorrência da SMDS, a frequência de detecção e quantificação de genomas de TTSuV1 e TTSuV2 em tecidos e soros de suínos com e sem SMDS foram determinadas. A análise feita nos diferentes tecidos de suínos revelou uma aparente correlação inversa entre a presença do genoma de TTSuV1 e a ocorrência da SMDS. Quanto ao TTSuV2 em tecidos de suínos com e sem a SMDS, nenhuma diferença estatística foi observada. A distribuição do genoma de TTSuV1 e TTSuV2 nos diferentes tecidos examinados não revelou um órgão alvo específico. A frequência de detecção e a carga viral de TTSuV1 e 2 nas amostras de soro de suínos com e sem a SMDS não apresentaram diferença significativa. No entanto, a carga viral de TTSuV2 foi mais alta do que a carga viral de TTSuV1 nos soros de todos os grupos de animais estudados. Estes resultados indicam uma alta frequência de detecção de ambas as espécies de TTSuV em amostras de tecidos e soros de suínos com e sem a SMDS. / Preliminary studies aiming the identification of possible viral agents associated with the postweaning multisystemic wasting syndrome (PMWS) revealed a possible negative association between TTSuV1 and occurrence of PMWS. Based on this finding was hypothesized that TTSuV1 might be able to inhibit the PCV2 multiplication, preventing the development of PMWS. To better clarify this, would be require an efficient system of replication for this virus, which has not been reported in the literature. In view of this, a method for detection of TTSuV infections in cell culture was developed to assess possible cell lineages to be potentially used for virus isolation and multiplication. TTSuV genomes were detected in cell lineages of porcine and nonporcine origin as well as a batch of trypsin. Sera used as media supplement was not found to contain TTSuV genomes. Thus, the contaminated batch of trypsin can be considered an important source of contamination, especially in cells of non-porcine origin. In order to evaluate a possible association between the TTSuVs and the occurrence of PMWS, the frequency of detection and quantification of TTSuV1 and TTSuV2 genomes in tissues and sera from pigs with and without PMWS were determined. The analysis in the different tissues of pigs reveal an apparent inverse correlation between the frequency of detection of TTSuV1 genomes and the occurrence of PMWS. Regarding TTSuV2 in tissues of PMWS and non-PMWS-affected animals no significant differences was observed. The distribution of TTSuV1 and TTSuV2 genomes in tissues did not reveal any particular target organ. The frequency of detection and viral load of TTSuV1 and TTSuV2 in sera samples were no significant statistically among animals PMWS-affected and healthy pig. The mean of TTSuV2 viral load was significantly highest than TTSuV1 in sera of all groups studied. These results indicate a high frequency of detection of both TTSuV species in tissues and sera samples from PMWS-affected and healthy pig.