The transforming growth factor beta (TGFβ)/activin signaling pathway has a number of documented roles during wound healing and is becoming increasingly appreciated as a vital component of multi-tissue regeneration. The leopard gecko (Eublepharis macularius) is able to spontaneously, and repeatedly, regenerate its tail following tail loss. We thus examined the expression and localization of several key components of the TGFβ/activin signaling pathway during tail regeneration of the leopard gecko. We observed a marked increase in phosphorylated-Smad2 expression among regenerating tissues corresponding to the location of the regenerate blastema. Interestingly, we observe that during early regeneration there appears to be an absence of TGFβ family member TGFβ1 and instead a strong upregulation of activin-βA. We also observe the expression of EMT transcription factors Snail1 and Snail2 in blastemal tissue. These observations combined with other data provide strong support for the importance of unique and non-overlapping expression patterns of different TGFβ ligands during multi-tissue regeneration

Molecular markers for seed quality traits including erucic acid content genes, seed coat color genes in Brassica napus and seed coat color genes in B. rapa were developed. A single base change in the Bn-FAE1.1 gene in the A genome and a two-base deletion in the Bn-FAE1.2 gene in the C genome produce the nearly zero content of erucic acid observed in canola. The single base change was detected as single nucleotide polymorphic (SNP) marker with an ABI SNaPshot kit. A multiplexing primer set was designed by adding a polyT to the 5´ primer end to increase SNP detection throughput through sample pooling. The two-base deletion in the C genome gene was detected as a sequence characterized amplified region (SCAR) marker in an ABI 3100 Genetic analyzer. To increase the throughput, one genome specific primer was labeled with four fluorescence dyes and combined with 20 different primers to produce PCR products with different fragment sizes. These multiplexed high throughput molecular markers have been successfully implemented in our canola/rapeseed breeding programs. Trigenic inheritance was observed for seed coat color in B. napus. Three Sequenced Related Amplified Polymorphism (SRAP) markers very closely linked to the three different seed coat color genes were developed. Chromosome-walking technology was used to convert the SRAP marker into a SCAR marker and a SNP marker. Subsequently, the first seed coat color gene (Bn1) marker was converted into a SCAR marker, and the second seed coat color gene (Bn2) marker was converted into a SNP marker. Digenic inheritance was observed for seed coat color genes in B. rapa. A SRAP marker was identified as being tightly linked to the major seed coat color gene (Br1). The SRAP marker was sequenced and extended sequences were obtained using chromosome-walking technology. The flanking sequences of the SRAP marker contained 24 SNPs and a 12-bp deletion position that allowed the marker to be converted into a co-dominant SNP marker and a co-dominant SCAR marker, respectively. The SCAR marker was detected in the ABI 3100 genetic analyzer with four fluorescently labeled M13 primers integrated with different SCAR primers, which permitted pooling of PCR samples for high throughput detection.

Biological tissues were studied with synchrotron infrared (IR) microspectroscopy, a technique that allows the spatially resolved determination and mapping of multiple components in situ at high spatial resolution. The first project involved studying brain tissue from TgCRND8 mice, a transgenic model of Alzheimer’s disease (AD). AD is the main cause of dementia in the ageing population, marked by the deposition of plaques composed of the Aβ peptide. Dense-cored and diffuse plaques were IR mapped and the results correlated with histochemistry and immunostaining. Spectral analysis confirmed that congophilic plaque cores were composed of highly aggregated protein in a β-sheet conformation. The amide I maximum of plaque cores was 1623 cm-1; there was no evidence of the high frequency (1680-1690 cm-1) peak seen in in vitro Aβ fibrils and attributed to anti-parallel β-sheet. A significant elevation in phospholipids was found around dense-cored plaques in TgCRND8 mice ranging in age from 5 to 21 months. This was due to an increase in cellular membranes from dystrophic neurites and glial cells around the core, but could also contribute to Aβ aggregation through the interaction of newly secreted Aβ with phospholipids. In contrast, diffuse plaques were not associated with infrared detectable changes in protein secondary structure or relative concentrations of other tissue components. In addition, focally elevated deposits of creatine, a molecule with a crucial role in energy metabolism, were discovered in AD brain tissue with IR microspectroscopy. The creatine deposits may be a previously undiscovered disease marker. A second project was part of a larger Natural Sciences and Engineering Research Council Collaborative Health Research Project (NSERC-CHRP) to test the hypothesis that treatment with anti-oxidants, L-2-oxo-thiazolidine-4-carboxylate (OTC) and quercetin, following spinal surgery may reduce oxidative stress, inflammation, and scarring. The effect of OTC and quercetin on scar tissue formation was evaluated in rats that had undergone laminectomy. Synchrotron IR microspectroscopy data were collected on scar tissue from OTC, quercetin and saline (control) treated animals, sacrificed at 3 and 21 days post-surgery. Spectral differences could be correlated with the stages of wound healing.

Molecular markers for seed quality traits including erucic acid content genes, seed coat color genes in Brassica napus and seed coat color genes in B. rapa were developed. A single base change in the Bn-FAE1.1 gene in the A genome and a two-base deletion in the Bn-FAE1.2 gene in the C genome produce the nearly zero content of erucic acid observed in canola. The single base change was detected as single nucleotide polymorphic (SNP) marker with an ABI SNaPshot kit. A multiplexing primer set was designed by adding a polyT to the 5´ primer end to increase SNP detection throughput through sample pooling. The two-base deletion in the C genome gene was detected as a sequence characterized amplified region (SCAR) marker in an ABI 3100 Genetic analyzer. To increase the throughput, one genome specific primer was labeled with four fluorescence dyes and combined with 20 different primers to produce PCR products with different fragment sizes. These multiplexed high throughput molecular markers have been successfully implemented in our canola/rapeseed breeding programs. Trigenic inheritance was observed for seed coat color in B. napus. Three Sequenced Related Amplified Polymorphism (SRAP) markers very closely linked to the three different seed coat color genes were developed. Chromosome-walking technology was used to convert the SRAP marker into a SCAR marker and a SNP marker. Subsequently, the first seed coat color gene (Bn1) marker was converted into a SCAR marker, and the second seed coat color gene (Bn2) marker was converted into a SNP marker. Digenic inheritance was observed for seed coat color genes in B. rapa. A SRAP marker was identified as being tightly linked to the major seed coat color gene (Br1). The SRAP marker was sequenced and extended sequences were obtained using chromosome-walking technology. The flanking sequences of the SRAP marker contained 24 SNPs and a 12-bp deletion position that allowed the marker to be converted into a co-dominant SNP marker and a co-dominant SCAR marker, respectively. The SCAR marker was detected in the ABI 3100 genetic analyzer with four fluorescently labeled M13 primers integrated with different SCAR primers, which permitted pooling of PCR samples for high throughput detection.

Biological tissues were studied with synchrotron infrared (IR) microspectroscopy, a technique that allows the spatially resolved determination and mapping of multiple components in situ at high spatial resolution. The first project involved studying brain tissue from TgCRND8 mice, a transgenic model of Alzheimer’s disease (AD). AD is the main cause of dementia in the ageing population, marked by the deposition of plaques composed of the Aβ peptide. Dense-cored and diffuse plaques were IR mapped and the results correlated with histochemistry and immunostaining. Spectral analysis confirmed that congophilic plaque cores were composed of highly aggregated protein in a β-sheet conformation. The amide I maximum of plaque cores was 1623 cm-1; there was no evidence of the high frequency (1680-1690 cm-1) peak seen in in vitro Aβ fibrils and attributed to anti-parallel β-sheet. A significant elevation in phospholipids was found around dense-cored plaques in TgCRND8 mice ranging in age from 5 to 21 months. This was due to an increase in cellular membranes from dystrophic neurites and glial cells around the core, but could also contribute to Aβ aggregation through the interaction of newly secreted Aβ with phospholipids. In contrast, diffuse plaques were not associated with infrared detectable changes in protein secondary structure or relative concentrations of other tissue components. In addition, focally elevated deposits of creatine, a molecule with a crucial role in energy metabolism, were discovered in AD brain tissue with IR microspectroscopy. The creatine deposits may be a previously undiscovered disease marker. A second project was part of a larger Natural Sciences and Engineering Research Council Collaborative Health Research Project (NSERC-CHRP) to test the hypothesis that treatment with anti-oxidants, L-2-oxo-thiazolidine-4-carboxylate (OTC) and quercetin, following spinal surgery may reduce oxidative stress, inflammation, and scarring. The effect of OTC and quercetin on scar tissue formation was evaluated in rats that had undergone laminectomy. Synchrotron IR microspectroscopy data were collected on scar tissue from OTC, quercetin and saline (control) treated animals, sacrificed at 3 and 21 days post-surgery. Spectral differences could be correlated with the stages of wound healing.

Injury to the central nervous system (CNS) results in the formation of a highly inhibitory glial scar consisting mainly of chondroitin sulfate proteoglycans (CSPGs). CSPGs are comprised of a protein core with covalently attached chondroitin sulfate glycosaminoglycan (CS-GAG) side chains. CSPGs and CS-GAGs have been implicated in the regenerative failure of the CNS, though the mechanism underlying inhibition is unclear. Sulfation affects both the physical and chemical characteristics of CS-GAGs and, therefore, it has been hypothesized that certain sulfation patterns are more inhibitory than others. To investigate this hypothesis, specific chondroitin sulfate sulfotransferases (CSSTs), the enzymes responsible for CS-GAG sulfation, were knocked down in vitro using siRNA. C4ST-1, C4ST-2, and C46ST were chosen as targets for gene knockdown in this study based on their expression in neural tissue and the extent of inhibition caused by their respective CS-GAG. It was hypothesized that transfection of primary rat astrocytes with siRNAs designed to prevent the expression of C4ST-1, C4ST-2, and C46ST would decrease specific sulfation patterns of CSPGs, resulting in improved neurite extension in a neurite guidance assay. Through optimization of siRNA dose, astrocyte viability was maintained while successfully knocking down mRNA levels of C4ST-1, C4ST-2, and C46ST and significantly reducing total levels of secreted CS-GAGs. However, no increase in the incidence of neurite extension was observed using conditioned media collected from siRNA transfected astrocytes compared to non-transfected controls. These data suggest that sulfation does not contribute to CSPG-mediated neurite inhibition, though further investigation is necessary to confirm these findings. Significantly, this work has established a paradigm for investigating the role of CSPG sulfation patterns in CNS regeneration.

Fundação Carlos Chagas Filho de Amparo a Pesquisa do Estado do Rio de Janeiro / Os estuários são ambientes altamente dinâmicos e concentram a maior parte da população mundial em seu entorno. São ambientes complexos que necessitam de uma gama de estudos. Nesse contexto, este trabalho visa contribuir para o entendimento dos estuários lagunares, tendo como objetivo comparar duas ferramentas geofísicas acústicas no mapeamento de uma porção submersa do Mar de Cananéia que está inserido no Sistema Estuarino Lagunar de Cananéia-Iguape (SP). Os equipamentos utilizados nesta pesquisa são o Sonar de Varredura Lateral e o Sistema Acústico de Classificação de Fundo RoxAnn, através da parametrização de amostras de fundo. A comparação do padrão acústico do Sonar de Varredura Lateral com as amostras de fundo da região permitiu o reconhecimento de 6 tipos distintos de padrões acústicos e a relação positiva com o diâmetro médio do grão foi de 50%. A comparação da resposta acústica do Sistema Acústico de Classificação de Fundo RoxAnn com o diâmetro médio do grão foi igualmente de 50%. Isto deve-se ao fato de que os valores produzidos pelo eco 1 e pelo eco 2 deste equipamento mostram que, por ser um mono-feixe e por analisar valores de intensidade do retorno acústico, o equipamento em questão pode responder a outros fatores ambientais que não seja somente o diâmetro médio do grão. Ao comparar a resposta acústica do Sonar de Varredura Lateral com o Sistema Acústico de Classificação de fundo RoxAnn obteve-se um resultado positivo de 93%. Isto pode ser explicado pelo fato de o Sonar de Varredura Lateral gerar uma imagem acústica do fundo. Em locais onde tem-se amostra e os valores do eco 1 e do eco 2 do Sistema Acústico de Classificação de Fundo RoxAnn são altos, pode-se associar a esses locais a influência da compactação dos sedimentos finos através da análise das imagens do Sonar de Varredura Lateral. Por meio da comparação destes dois métodos foi possível estabelecer um intervalo de valores para o eco 1 que pode ser associado ao diâmetro médio do grão. Assim, valores entre 0.170 a 0.484 milivolts podem ser associados a sedimentos finos com granulometria até areia fina. Valores entre 0.364 a 0.733 podem ser associados a sedimentos de granulometria entre areia fina a média. Valores acima de 0.805 milivolts até 1.585 milivolts podem ser associados a sedimentos mais grossos como carbonatos biodetríticos ou areias grossas. E, por fim, valores acima de 2.790 milivolts podem ser associados a afloramentos rochosos. / Estuaries are highly dynamic environments which concentrate the largest part of the world`s population in it`s surroundings. They are complex environments which need a variety of studies. The objective of this work is to contribute to a better understanding of the estuarine system, having as a main objective the comparison between two geophysical acoustic methods in the mapping of a submersed portion of the Cananéia Sea, located in the Estuarine System of Cananéia Iguape (SP). The equipment used in this research were the Side Scan Sonar and the RoxAnn Acoustic Ground Discrimination System, through the correlation of the geological bottom samples. The comparison of the acoustic pattern between the Side Scan Sonar and the geological bottom samples enabled the recognition of 6 distinct acoustic patterns and the positive correlation between the medium diameters of the sediments was 50%. The correlation of the acoustic feedback of the RoxAnn Acoustic Ground Discrimination System and the medium diameter of the sediments was equally 50%. This is due to the fact that the data produced by the echo 1 and echo 2 of this system show that because it is a single beam and because it analyses acoustic intensity feedback data, the equipment may also respond to other environmental factors which are not only related to the medium diameter of sediments. When comparing the acoustic feedback taken from the Side Scan Sonar and the RoxAnn Acoustic Ground Discrimination System a positive result of 93% was obtained. This may be explained due to the fact that the Side Scan Sonar generates an acoustic ground image. In areas where one has high value samples from echo 1 and echo 2 and also from the RoxAnn Acoustic Ground Discrimination System, one may correlate to these places the influence of fine sediment compression through the analysis of the images taken from the Side Scan Sonar. By comparing these two methods it was possible to establish an interval of values for echo 1 which can be associated to the medium diameter of the sediments. In this way, values between 0,170 to 0.484 milivolts can be associated to fine sediments with granulometry variating up to very fine sediments. Values between 0.364 to 0.733 can be associated to fine sediments with granulometry variating from medium to fine sediments. Values above 0.805 milivolts to 1.585 milivolts can be associated to coarser sediments such as biodetritic carbonates or thick sediments. Finally, values above 2.790 milivolts can be associated to rocky emergences.

Introdução: As alopecias cicatriciais são subdivididas em primárias e secundárias. Nas alopecias cicatriciais primárias o folículo piloso é o alvo principal da destruição; diferentemente das secundárias onde a destruição folicular não é o evento patológico primário. Objetivo: Revisar os achados histológicos de pacientes com diagnóstico de alopecia cicatricial primária, em uma fase tardia e reclassificá-los em seus respectivos subtipos. Casuística e Métodos: Os espécimes de biópsia de pacientes com diagnóstico histológico prévio de alopecia cicatricial foram revisados e submetidos a colorações por hematoxilina e eosina (HE), ácido periódico-Schiff (PAS) e Weigert, a fim de reclassificá-los de acordo com os diferentes subtipos de alopecia. Foram utilizados para a revisão histológica critérios de infiltrado inflamatório acrescidos de coloração para fibra elástica. Resultados: Os 37 casos de alopecia cicatricial primária foram reclassificados em: lupus eritematoso discóide (16), líquen plano pilar (4), pseudopelada de Brocq (12), foliculite decalvante (3), foliculite abscedante/dessecante (1), e alopecia não-específica (1). Conclusão: Mesmo em uma fase tardia, pauci ou não inflamatória, o exame histológico, utilizando colorações de rotina, PAS e coloração para fibra elástica, permitiu o diagnóstico mais acurado das alopecias cicatriciais primárias / BACKGROUND: Scarring alopecias are classified into primary and secondary according to the initial site of inflammation. In primary scarring alopecias, the hair follicle is the main target of destruction. The term secondary cicatricial alopecia implies that follicular destruction is not the primary pathologic event. AIMS: To review the histopathologic diagnoses of cases of cicatricial alopecia in order to classify them according to the North American Hair Research Society. PATIENTS AND METHODS: Patients with biopsy specimens diagnosed as cicatricial alopecia seen from 2000 to 2005 at the Dermatologic Department of Hospital das Clínicas, São Paulo University Medical School had hematoxylin and eosin, Periodic Acid-Schiff (PAS) and Weigert stained slides reevaluated and sub-typed into different primary cicatricial alopecias. RESULTS: Thirty-seven cases of primary cicatricial alopecias were reclassified as: discoid lupus erythematosus (16), lichen planus pilaris (4), pseudopelade of Brocq (12), folliculitis decalvans (3), dissecting folliculitis (1), and non-specific scarring alopecia (1). CONCLUSIONS: Even in late, pauci or noninflammatory phases, an approach with systematic evaluation of a constellation of criteria in routine hematoxylin and eosin stain, PAS and Weigert stain permitted an accurate diagnosis of cicatricial alopecias

Os caracóis terrestres são animais capazes de produzir através de glândulas localizadas em toda superfície do seu corpo, uma secreção glicoproteica denominada muco, que dentre outras funções, apresenta poder antibacteriano, que pode auxiliar nos processos de reparação de feridas de origens diversas. Desta forma, o objetivo da presente pesquisa foi o de avaliar macroscópica e histologicamente, os efeitos reparadores do muco dos escargots Achatina fulica e Achatina achatina monochromatica, em lesões provocadas na pele de camundongos da linhagem "hairless"; verificar sua potencialização após a ingestão de uma ração contendo em sua formulação uma planta com propriedades cicatrizantes comprovadas, o Confrei (Symphytum officinale); e analisar bioquimicamente a composição do muco destes caracóis. Foram selecionados caracóis das espécies Achatina fulica (n=30) e Achatina monochromatica monochromatica (n=30), retirado seu muco através de estimulação manual da glândula podal e verificação de seu efeito reparador. Utilizaram-se camundongos da linhagem "hairless" (n=75), submetidos à intervenção cirúrgica e tratados com muco de ambas as espécies de caracóis. As características macroscópicas da lesão foram registradas em protocolo e avaliadas. Fragmentos de pele foram submetidos à biópsia aos 3, 5 e 7 dias de experimento, fixados em Paraformoldeído, processados e incluídos em parafina. Os cortes foram corados com Hematoxilina-Eosina e Tricrômio de Mason. Macroscopicamente, os animais tratados mostraram edemas menores, maior presença de crosta e maior contração das bordas das feridas ao final do experimento, quando comparados ao grupo controle. Histologicamente, os grupos tratados também revelaram melhor reparação da lesão, apresentando edemas menos intensos, grandes quantidades de tecido de granulação e infiltrados inflamatórios. O grupo tratado com muco de caracóis alimentados com ração contendo Symphytum officinale foi o que apresentou melhores resultados. / Land snails are animals able to produce through glands located all over their body surface, a glycoprotein secretion called mucus. This mucus, within other roles, shows an antibacterial power that can help in the healing processes of wounds from several sources. Therefore, the aim of this study was to evaluate through macroscopic and histological techniques the repairing effects of the mucus in the snails Achatina fulica and Achatina achatina monochromatica in lesions intentionally caused to hairless linage mice skin; to investigate mucus potential effect after snails have been fed with a diet formulation containing a plant with proved scar properties, the Comfrey (Symphytum officinale); and to study the biochemical composition of mucus from these snails. Snails of Achatina fulica (n=30) and Achatina monochromatica monochromatica (n=30) species were sorted, the mucus extracted through manual stimulation of the podal gland and the repairing effect studied. Hairless linage mice (n=75) used in this study went through surgery and then treated with mucus from both species of snails. The macroscopic characteristics of the lesion were recorded and assessed. Fragments of skin were taken to biopsy at 3, 5 and 7 days of trial, fixed in paraformaldehyde, processed and embedded in wax. Sections were stained with Hematoxilin-Eosin and Tricromio of Mason. Macroscopically, treated mice showed minor swelling, higher presence of scabs and higher contraction of wound edges at the end of the trial, when compared with control. Histologically, groups that received treatment also showed better lesion repair, presenting less intense swellings, large amount of scar tissue and inflammatory infiltrates. The group treated with mucus from snails that received a diet containing Symphytum officinale presented the best results.

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No. of bitstreams: 2 Dissertação Raque Bartz Kneib.pdf: 709774 bytes, checksum: ed9b7474064e09348586e51c9221a9fa (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2014-07-31 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / A batata é o terceiro principal alimento produzido no mundo, e é um dos cultivos mais promissores para suprimir a fome em países em desenvolvimento. Cerca de 40 viroses infectam a cultura da batata, entre estas, o Potato virus Y (PVY) é, atualmente, o que causa maior impacto negativo na cultura. O PVY é transmitido de diversas formas, todas com controle e manejo deficitários, tornando a resistência genética a medida de controle mais eficaz. Esta resistência é controlada pelos genes Ry encontrados em espécies silvestres de batata. Marcadores do tipo SCAR (Sequence Characterized Amplified Region), os quais amplificam a região que contém o gene Ry, foram desenvolvidos e estão disponíveis para uso pelos programas de melhoramento de batata. Este trabalho foi realizado com o objetivo de aumentar a eficiência dos programas de melhoramento de batata em relação à resistência ao PVY fazendo uso da seleção assistida por marcadores moleculares. A dissertação foi dividida em dois capítulos. No primeiro, visando estimar a dosagem alélica do gene Ry e selecionar clones com múltiplas cópias do gene Ry para serem usados como genitores no programa de melhoramento de batata, foram genotipadas sete famílias clonais, envolvendo cinco genitores com resistência ao PVY, utilizando os marcadores SCAR RYSC3 e M45. Para o gene Ryadg, com base no marcador RYSC 3, dos cinco genitores resistentes ao PVY avaliados, quatro, C1883-22-97, C2372-02-02, C2388-01-02, e MB9846-1, são simplex, e o genitor C2389-01-02 é duplex. Para o gene Rysto, com base no marcador M45, dois genitores, C2388-01-02 e MB9846-1, são simplex, e o genitor C2389-01-02 é duplex. Para os genitores C1883-22-97 e C2372-02-02, as frequências observadas de presença do gene Rysto, nas progênies avaliadas, não se adequaram estatisticamente a nenhuma das constituições genotípicas propostas. No segundo capítulo, com objetivo de identificar germoplasma de batata com resistência extrema ao vírus Y associada aos componentes da aparência e do rendimento de tubérculos, além verificar as associações entre esses caracteres, foram avaliados clones portadores do gene Ry, oriundos de oito combinações genéticas distintas. As combinações genéticas C2389-01-02 x Asterix, e, BRS Ana x C1883-22-97, mostraram maior potencial para serem exploradas visando agregar a resistência ao vírus Y aos caracteres desejáveis de rendimento e de aparência de tubérculo. O grau de correlação entre caracteres indica fortíssima e direta associação entre aparência geral e aspereza de tubérculo; forte e direta associação da intensidade da cor de película com aspereza e aparência geral de tubérculo, entre número e massa média de tubérculo, entre massa média e cor da película, e entre massa total e número total de tubérculos; e forte e inversa associação de formato com cor da polpa, cor da película e massa média de tubérculos. Os resultados encontrados no nosso estudo mostraram que os marcadores moleculares RYSC3 e M45 são uma importante ferramenta para ser usada pelos programas de melhoramento de batata visando acelerar o processo de desenvolvimento de cultivares com resistência ao PVY. / Potato is the third main food crop in the world, and is one of the most promising crop to suppress hunger in developing countries. From the 40 viruses which infect potato growing fields, Potato virus Y (PVY) is currently considered the most important one. PVY is transmitted by several ways, all of them with limited control, begin genetic resistance the main way to control this important disease controlled by Ry genes. Molecular markers, which amplify the region containing Ry gene have been developed and are available to be used by potato breeding. This work was developed with the aim of increasing the efficiency of potato breeding programs for resistance to PVY making use of molecular assisted selection. The dissertation is divided into two chapters. In the first chapter, in order to estimate allelic dosage of Ry genes in clones used as genitors in the breeding program, progenies from seven families were genotyped with the markers RYSC3 and M45. For Ryadg gene, based on the marker RYSC, from the five PVY resistant parents analysed, four are simplex, C1883-22-97, C2372-02-02, C2388-01-02 and MB9846-1, and one is duplex, C2389- 01-02. For Rysto gene, based on the marker M45, both parents, C2388-01-02 and MB9846-1, are simplex, and the parent C2389-01-02 is duplex. For the C1883-22-97 and C2372-02-02, it was not possible to estimate the allelic dosage of the Rysto gene. In the second chapter, in order to identify germplasm with extreme resistance to PVY associated to desirable agronomic traits, clones carrying the Ry gene, belonging from eight different genetic combinations, were evaluated for characteres related to tuber appearance and yield. The genetic combinations C2389-01-02 x Asterix and BRS Ana x C1883-22-97 showed good potential to be exploited in order to add resistance to virus Y to traits related to yield and tuber appearance. The correlation between traits indicates very strong and direct association between tuber general appearance and roughness of tuber; strong and direct association of the intensity of the periderm color and tuber general appearance; between number of tubers per plant and average tuber weight; between average tuber weight and intensity of the periderm color and between the total mass of tubers per plant and total number of tubers per plant. Also, strong and inverse associations between tuber shape and flesh color and between periderm color and average tuber weight were detected. The results of our study showed that the molecular markers RYSC3 and M45 are important tools to be used by potato breeding programs to accelerate the development of cultivars with resistance to PVY.