The South West Botanical Province of Western Australia is one of the most floristically rich areas of the world with over, 8,000 species present, the majority of which (70%), are endemic to this region. Coupled with this high level of endemism, many taxa are threatened which makes them vulnerable to habitat alterations, modifications and destruction. Significant habitat alteration in many areas has resulted in 27 species becoming extinct in the South West Botanical Province, while an additional 327 species are classified as rare and endangered. In the context of stemming this loss of biodiversity, research in cryopreservation was undertaken to provide offsite protection and conservation of somatic germplasm.Cryostorage techniques were evaluated in this study to determine the key factors which may affect the ability of somatic tissues of Haemodoraceae species to survive, recover and grow following liquid nitrogen (LN) immersion and storage. Using Anigozanthos viridis as a comparator in most experiments, the base vitrification protocol was established, which involved: (1) preculturing shoot apices on 0.4 M sorbitol for 48 h; (2) incubation in a vitrification cryoprotective solution (PVS2) for 25 min at 0 degrees celsius; (3) LN immersion; (4) recovery to active growth through warming (immersion in a 40 degrees celsius water bath). Using this procedure the highest post-LN survival of shoot apices for A. viridis was 41.4 plus or minus 6.1% Four additional taxa were successfully cryopreserved with this base protocol (Anigozanthos manglesii, A. rufus, Conostylis wonganensis and A. rufus x A. pulcherrimus); a fifth taxon, Macropidia fuliginosa, however, proved unresponsive.To improve on post-LN survival, further research established that four of the six study taxa responded to the following amendments to the basic protocol: (1) longer preculture duration; (2) preculture on ++ / 0.8 M glycerol rather than sorbitol, (3) utilisation of PVS2 solutions with reduced DMSO content; and (4) incorporation of an additional loading phase (2 M glycerol plus 0.4 M sucrose for 20 mins at 0 degrees celsius).Macropidia fuliginosa, a species with poor recovery after LN exposure, was successfully cryostored using somatic embryos. Treatments which resulted in the highest survival (67.3% 5.7 plus or minus %) included preculture with 0.4 M sorbitol, and incubation in PVS2 for 30 min. Further experimentation indicated that preculture for two days on 0.8 M glycerol (replacing 0.4 M sorbitol) was more beneficial for achieving high post-LN survival.Post-LN survival was significantly correlated to the use of polyalcohols when the total number of hydroxyl (-OH) groups (regardless of molarity) present was the same as that found in 0.4 M sorbitol. It was hypothesised that hydroxyl number is more important than molarity in membrane stabilisation, during dehydration and cooling. Post-LN survival was also found to be significantly influenced by stereochemical arrangement of the -OH groups of polyalcohol molecules used in the preculture media. Finally, post-LN survival was also found to be significantly influenced by the size of the molecule, with smaller polyalcohols with more -OH groups on one flank of the carbon chain being superior as cryoprotective agents.The influence of plant growth regulators on post-LN survival and recovery growth was also investigated. The survival of shoot apices was not correlated to cytokinin or auxin treatments administered in culture media prior to cryostorage. However, in the recovery medium, a combination of cytokinin and 0.5 mu M GA(subscript)3 in the medium was found to be the most efficacious for obtaining healthy plantlets.Genetic fidelity was then examined using Amplified Fragment Length Polymorphism (AFLP). Plantlets of one done kept ++ / or maintained under the following conditions: (1) standard tissue culture conditions; (2) cold storage and (3) cryostorage, over a 12 month duration, showed no detectable genetic changes.Further, shoot apex viability evaluated at regular intervals (after 0, 3, 6 and 12 months of LN storage) suggested that medium term storage of samples cryopreservation did not reduce shoot apex viability over this time span.This study has provided a better understanding of the factors influencing post-LN survival and recovery and, as a result, the cryopreservation protocols have been refined. Consequently, the prospects for conserving threatened Haemodoraceae species from Western Australia through cryostorage is now significantly improved.

Submitted by Socorro Pontes (socorrop@ufersa.edu.br) on 2017-03-22T14:51:38Z No. of bitstreams: 1 AlanaAB_DISSERT.pdf: 4740716 bytes, checksum: e3666ea7a25906742164df387e99208b (MD5) / Made available in DSpace on 2017-03-22T14:51:38Z (GMT). No. of bitstreams: 1 AlanaAB_DISSERT.pdf: 4740716 bytes, checksum: e3666ea7a25906742164df387e99208b (MD5) Previous issue date: 2016-11-01 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The cryopreservation of somatic tissue derived from collared peccaries represents an interesting step in the obtainment and conservation of cells for nuclear transfer (cloning). In this sense, tissue vitrification protocols need to be optimized to ensure maximum preservation of viable cell characteristics. Therefore, the aims of this study were to characterize histologically the peripheral auricular integumentary system (Stage 1) and evaluate different cryoprotectants in the solid-surface vitrification of somatic tissue of collared peccaries (Stage 2). Thun, ear fragments (9.0 mm3) were collected of sixteen animals derived from the Multiplication Center of Wild Animals (CEMAS/UFERSA). In the first stage, tissue samples were evaluated for the characterization of layers, its components and proliferative activity. For the second stage, tissue fragments were cryopreserved by solidsurface vitrification using Dulbecco modified minimum essential medium supplemented with 10% fetal bovine serum and different cryoprotectants and concentrations [dimethylsulfoxide (DMSO, 3.0 M), ethylene glycol (EG; 3.0 M) and association DMSO/EG (1.5 M; 1.5 M) in the absence and presence of sucrose (0.25M)]. After two weeks, warmed and non-vitrified (control) fragments were analyzed using histological techniques. Thus, for both steps, tissue samples were evaluated using hematoxylin-eosin and Gomori Trichrome, quantification of regions argyrophilic nucleolar organizer (AgNORs) and viability by MTT assay (3-[4,5- dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide). Also, in the first stage, fragments were analyzed by transmission electronic microscopy. Thus, in the first stage, sizes of 104.2 μm and 222.6 μm were observed in the epidermis and dermis, with a volumetric ratio of 36.6% and 58.7%, respectively. Moreover, in the epidermis were evidenced the basal layer (22.5 μm), intermediate (53.5 μm) and cornea (28.2 μm), with mean values of 65.3 epithelial cells, 43.4 melanocytes and 14.8 perinuclear halos. Already the dermis has 127 fibroblasts with 2.5 AgNORs by nucleolus. Additionally, the metabolic activity was 0.243. In the second stage, the 3.0 M EG with sucrose was able to maintain normal tissue characteristics compared with non-vitrified (control), especially for the volumetric ratio of epidermis (61.2 vs. 58.7) and dermis (34.5 vs. 36.6), number of fibroblast (90.3 vs. 127.0), and AgNOR ratio (0.09 vs. 0.17), respectively. In conclusion, the peripheral auricular integumentary system derived from collared peccaries possessed some variations compared to other mammals, as the number of layers and thickness of the epidermis, number of epithelial cells, melanocytes and proliferative parameters. Moreover, 3.0 M EG with 0.25 M sucrose resulted in a better cryoprotectant composition in the vitritification for somatic tissue of collared peccaries / A criopreservação de tecido somático de catetos representa uma etapa interessante na obtenção e conservação de células para a transferência nuclear (clonagem). Nesse sentido, protocolos de vitrificação tecidual necessitam ser otimizados para garantir a máxima conservação das características viáveis das células. Portanto, os objetivos do presente trabalho foram caracterizar histologicamente o sistema tegumentar da região auricular periférica (Etapa 1) e avaliar diferentes crioprotetores na vitrificação em superfície sólida de tecido somático de catetos (Etapa 2). Para tanto, fragmentos auriculares (9,0 mm3) foram recuperados de dezesseis animais oriundos do Centro de Multiplicação de Animais Silvestres (CEMAS/UFERSA). Na primeira etapa, amostras teciduais foram avaliadas quanto à caracterização das camadas, seus componentes e atividade proliferativa. Para a segunda etapa, fragmentos teciduais foram criopreservados por vitrificação em superfície sólida em meio essencial mínimo modificado por Dulbecco suplementado com 10% de soro fetal bovino e diferentes crioprotetores e concentrações [dimetilsulfóxido (DMSO; 3,0 M), etilenoglicol (EG; 3,0 M) e associação DMSO/EG (1,5 M; 1,5 M) na ausência e presença de sacarose (0,25 M)]. Após duas semanas, fragmentos aquecidos e não criopreservados (controle) foram analisados usando técnicas histológicas. Assim, para ambas as etapas, amostras teciduais foram avaliadas usando colorações hematoxilina-eosina e tricômico de Gomori, quantificação de regiões argirofílicas organizadoras nucleolares (AgNORs) e viabilidade pelo ensaio de MTT (brometo de 3-[4,5-dimetil-tiazol-2-il]-2,5-difeniltetrazólio). Ainda, na primeira etapa, fragmentos foram analisados por microscopia eletrônica de transmissão. Assim, na primeira etapa, tamanhos de 104,2 μm e 222,6 μm foram observados para epiderme e derme, com uma proporção volumétrica de 36,6% e 58,7%, respectivamente. Além disso, na epiderme, foram evidenciadas as camadas basal (22,5 μm), intermediárias (53,5 μm) e córnea (28,2 μm), com valores médios de 65,3 de células epidermais, 43,4 melanócitos e 14,8 de halos perinucleares. Já a derme apresentou 127 fibroblastos com 2,5 AgNORs por nucléolo. Adicionalmente, a atividade metabólica foi de 0,243. Na segunda etapa, a combinação de 3,0 M de EG com sacarose foi adequada em manter as características teciduais normais quando comparado com os fragmentos não vitrificados, especialmente para a proporção volumétrica da epiderme (61,2 vs. 58,7) e derme (34,5 vs. 36,6), número de fibroblastos (90,3 vs. 127,0) e razão de AgNOR (0,09 vs. 0,17), respectivamente. Em conclusão, o sistema tegumentar auricular periférico de catetos possuiu algumas variações em relação a outros mamíferos, quanto ao número de camadas e espessura da epiderme, quantidade de células epidermais, melanócitos e parâmetros proliferativos. Além disso, 3,0 M de EG com 0,25 M de sacarose resultaram na melhor composição de crioprotetores na vitrificação de tecido somático de catetos / 2017-03-21

Submitted by Socorro Pontes (socorrop@ufersa.edu.br) on 2017-03-07T12:56:11Z No. of bitstreams: 1 MagdaLTS_DISSERT.pdf: 1454861 bytes, checksum: 4612469437c8dd924c24af03a052758d (MD5) / Made available in DSpace on 2017-03-07T12:56:11Z (GMT). No. of bitstreams: 1 MagdaLTS_DISSERT.pdf: 1454861 bytes, checksum: 4612469437c8dd924c24af03a052758d (MD5) Previous issue date: 2016-03-30 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The maintenance of metabolic activities during the in vitro culture of somatic cells of wild animals, especially collared peccary (Pecari tajacu), is an interesting step in the conservation of these cells for use in nuclear transfer (cloning). In this context, it is necessary to optimize the in vitro culture conditions of somatic cell by establishment of some appropriate supplementations to the media, in order to ensure the maximum preservation of the viable cell characteristics. Therefore, this study aimed to optimize the composition of the culture means of somatic cell derived from ear tissue of collared peccaries, evaluating two concentrations of fetal bovine serum (E1: FBS; 10% vs. 20%) and epidermal growth factor (E2: EGF, 5 ng/mL vs. 10 ng/mL). Thus, tissue fragments from 18 adult animals were submitted to primary culture and subcultures for 40 days until the fourth passage and the resulting cells were analyzed for morphology, adhesion, subconfluence, proliferative activity for developing growth curve for seven days and determining the population doubling time (PDT), viability by trypan blue and functional/metabolic activity by assay 3-[4,5-dimethyl-thiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT). Moreover, in the E1, comparisons as cell adhesion were performed with cells cultured in the presence of bovine serum albumin (BSA, 0.5% and 1.0%). All data were analyzed by ANOVA followed by post hoc test. In the E1, no difference (P>0.05) was observed between the concentrations of FBS for the number of adhered [SFB10: 39/39 vs. SB20: 35/39] and subconfluent fragments [SFB10: 39/39 vs. SB20: 35/39], day all adhered [SFB10: 3.5 vs. SFB20: 3.0], with growth [SFB10: 7.4 vs. SFB20: 7.2] and subconfluent samples [SFB10: 11.8 vs. SFB20: 11.8]. However, significant values were observed in cells cultured in the presence of 20% FBS for viability [SFB10: 85.6% vs. SFB20: 98.2%], PDT [SFB10: 155.4 h vs.77.25 h] and MTT assay [SFB10: 0.57-0.57 vs. SFB20: 0.82-0.99 (D5-D7)]. Additionally, comparisons of supplementation of BSA and FBS confirm the potential FBS cell adhesion. Thus, 20% FBS was used in the following experiment. In the evaluation of the presence of EGF in culture, no difference was observed in the evaluated parameters for the number of attached [EGF0: 31/31 vs. EGF5: 31/31 vs. EGF10: 31/31] and subconfluent fragments [EGF0: 31/31 vs. EGF5: 31/31 vs. EGF10: 31/31] day all adhered [EGF0: 4.9 vs. EGF5: 7.0 vs. EGF10: 3.5] growth [EGF0: 7.2 vs. EGF5: 8.2 vs. EGF10: 7.9] and subconfluent samples [0 EGF: 12.6 vs. EGF5: 16.6 vs. EGF10:12.6], viability [EGF0: 84.3% vs. EGF5: 88.8% vs. EGF10: 87.0%], PDT [EGF0: 69.6 h vs. EGF5: 64.8 h vs. EGF10: 65.3 h] and MTT assay [EGF0: 1.26-1.38 vs. EGF5: 1.06-1.14 vs. EGF10: 1.13-1.16 (D5-D7)]. In all experiments, the growth curves showed clear log and lag phases of development. In conclusion, 20% FBS is suitable for the recovery of somatic cells in vitro; however, EGF does not improve the quality of growing these cells / A manutenção das atividades metabólicas durante o cultivo in vitro de células somáticas de animais silvestres, especialmente catetos (Pecari tajacu), representa uma etapa interessante na conservação dessas células para aplicação na transferência nuclear (clonagem). Nesse contexto, faz-se necessário a otimização das condições de cultivo in vitro de células somáticas pelo estabelecimento de algumas suplementações adequadas aos meios, visando garantir a máxima preservação das características celulares viáveis. Portanto, o presente trabalho teve como objetivo otimizar a composição do meio de cultivo de células somáticas derivadas de tecido auricular de catetos, avaliando duas concentrações de soro fetal bovino (E1: SFB; 10% vs. 20%) e fator de crescimento epidermal (E2: EGF; 5 ng/mL vs. 10 ng/mL). Para tanto, fragmentos teciduais de 18 animais adultos foram submetidos ao cultivo primário e subcultivos por 40 dias, até a quarta passagem e as células resultantes foram analisadas quanto à morfologia, aderência, subconfluência, atividade proliferativa pela elaboração de curva de crescimento por sete dias e determinação do tempo de duplicação da população (PDT), viabilidade por azul de tripano e atividade funcional/metabólica pelo ensaio de brometo de 3-[4,5-dimetil-tiazol-2-il]-2,5-difeniltetrazólio (MTT). Além disso, no E1, comparações quanto à adesão celular foram realizadas com células cultivadas na presença de albumina sérica bovina (BSA, 0,5% e 1,0%). Todos os dados foram analisados por ANOVA seguido por teste post-hoc. No E1, nenhuma diferença (P>0,05) foi observada entre as concentrações de SFB para o número de fragmentos aderidos [SFB10: 39/39 vs. SB20: 35/39] e subconfluentes [SFB10: 39/39 vs. SB20: 35/39], dia de todas as amostras aderidas [SFB10: 3,5 vs. SFB20: 3,0], com crescimento [SFB10: 7,4 vs. SFB20: 7,2] e subconfluentes [SFB10: 11,8 vs. SFB20: 11,8]. Contudo, valores significativos foram observados em células cultivadas na presença de SFB a 20% quanto à viabilidade [SFB10: 85,6% vs. SFB20: 98,2%], PDT [SFB10: 155,4 h vs.77,2 h] e ensaio de MTT [SFB10: 0,57-0,57 vs. SFB20: 0,82-0,99 (D5-D7)]. Adicionalmente, comparações da suplementação do BSA e SFB confirmaram o potencial de adesão celular do soro. Assim, SFB a 20% foi empregado no experimento seguinte. Já na avaliação da presença de EGF no cultivo, nenhuma diferença foi observada nos parâmetros avaliados para o número de fragmentos aderidos [EGF0: 31/31 vs. EGF5: 31/31 vs. EGF10: 31/31] e subconfluentes [EGF0: 31/31 vs. EGF5: 31/31 vs. EGF10: 31/31], dia de todas as amostras aderidas [EGF0: 4,9 vs. EGF5: 7,0 vs. EGF10: 3,5], em crescimento [EGF0: 7,2 vs. EGF5: 8,2 vs. EGF10: 7,9] e subconfluentes [EGF0: 12,6 vs. EGF5: 16,6 vs. EGF10: 12,6], viabilidade [EGF0: 84,3% vs. EGF5: 88,8% vs. EGF10: 87,0%], PDT [EGF0: 69,6 h vs. EGF5: 64,8 h vs. EGF10: 65,3 h] e ensaio de MTT [EGF0: 1,26-1,38 vs. EGF5: 1,06-1,14 vs. EGF10: 1,13-1,16 (D5-D7)]. Em todos os experimentos, as curvas de crescimento apresentaram nítidas fases log e lag de desenvolvimento. Em conclusão, o SFB a 20% é adequado para a recuperação de células somáticas in vitro; contudo, o EGF não melhora a qualidade do cultivo dessas células / 2017-03-07