<p>This thesis concerns the history and the intrafamilial delimitations of the plant family Colchicaceae. A phylogeny of 73 taxa representing all genera of Colchicaceae, except the monotypic <i>Kuntheria</i>, is presented. The molecular analysis based on three plastid regions—the <i>rps16</i> intron, the <i>atpB-rbcL</i> intergenic spacer, and the <i>trnL-F</i> region—reveal the intrafamilial classification to be in need of revision. The two tribes Iphigenieae and Uvularieae are demon-strated to be paraphyletic. The well-known genus <i>Colchicum</i> is shown to be nested within <i>Androcymbium</i>, <i>Onixotis</i> constitutes a grade between <i>Neodregea</i> and <i>Wurmbea</i>, and <i>Gloriosa</i> is intermixed with species of <i>Littonia</i>. Two new tribes are described, Burchardieae and Tripladenieae, and the two tribes Colchiceae and Uvularieae are emended, leaving four tribes in the family. At generic level new combinations are made in <i>Wurmbea</i> and <i>Gloriosa</i> in order to render them monophyletic. The genus <i>Androcymbium</i> is paraphyletic in relation to <i>Colchicum</i> and the latter genus is therefore expanded. An investigation of the distribution of colchicine within the expanded Colchicaceae is conducted to evaluate the potential of colchicine as a synapomorphy of the re-circumscribed family. The results demonstrate presence of colchicine in all genera previously not examined in Colchicaceae and in the genus <i>Burchardia</i>, earlier reported to lack colchicine. Hence, demonstrating colchicine to be a synapomorphy for the family. An attempt to date the phylogeny of the order Liliales together with a dispersal-vicariance (DIVA) analysis indicates that the split between Colchicaceae and Alstromeriaceae-Luzuriagaceae represents a vicariance event following the disintegration of the Australian-Antarctican-South American link, ~34 million years ago. Further, the DIVA analysis indicates that Colchicaceae originated in Australia, first reached Asia and North America, and later Africa, from where they expanded to Europe and also dispersed back to Australia. </p>

This thesis concerns the history and the intrafamilial delimitations of the plant family Colchicaceae. A phylogeny of 73 taxa representing all genera of Colchicaceae, except the monotypic Kuntheria, is presented. The molecular analysis based on three plastid regions—the rps16 intron, the atpB-rbcL intergenic spacer, and the trnL-F region—reveal the intrafamilial classification to be in need of revision. The two tribes Iphigenieae and Uvularieae are demon-strated to be paraphyletic. The well-known genus Colchicum is shown to be nested within Androcymbium, Onixotis constitutes a grade between Neodregea and Wurmbea, and Gloriosa is intermixed with species of Littonia. Two new tribes are described, Burchardieae and Tripladenieae, and the two tribes Colchiceae and Uvularieae are emended, leaving four tribes in the family. At generic level new combinations are made in Wurmbea and Gloriosa in order to render them monophyletic. The genus Androcymbium is paraphyletic in relation to Colchicum and the latter genus is therefore expanded. An investigation of the distribution of colchicine within the expanded Colchicaceae is conducted to evaluate the potential of colchicine as a synapomorphy of the re-circumscribed family. The results demonstrate presence of colchicine in all genera previously not examined in Colchicaceae and in the genus Burchardia, earlier reported to lack colchicine. Hence, demonstrating colchicine to be a synapomorphy for the family. An attempt to date the phylogeny of the order Liliales together with a dispersal-vicariance (DIVA) analysis indicates that the split between Colchicaceae and Alstromeriaceae-Luzuriagaceae represents a vicariance event following the disintegration of the Australian-Antarctican-South American link, ~34 million years ago. Further, the DIVA analysis indicates that Colchicaceae originated in Australia, first reached Asia and North America, and later Africa, from where they expanded to Europe and also dispersed back to Australia.

Thesis (Ph. D.)--University of Chicago, Department of Chemistry, August 2003. / Includes bibliographical references. Also available on the Internet.

Colchicine and paclitaxel are two common drugs used in chemotherapy to halt tumor growth. In the present study IAR 20 cells were treated for 24 and 48 hr with colchicine and paclitaxel alone, in combination or no drug which served as a control. Through the use of Western blotting, we determined that the treatments affected expression due of several proteins including bcl-2, bax, p53 and caspase-8. The changes observed in protein expression due to the treatments correlated to the photomicrographs of the cells in culture and cell viability, indicating that the drugs were activating and initiating apoptosis. Interestingly, morphological changes such as membrane blebbing and cell swelling (indicators of apoptosis) were observed in the treated cultures and even more important the combined treatment yielded both changes in morphology. Also, activity assays were performed to study the effects the treatments had on the activities of SAPK/JNK and caspase-3, known activators of apoptosis. High activities of SAPK/JNK and caspase-3 in 48 hr treatments directly influenced cell viability in that the treatments with the highest activities yielded the lowest cell numbers, indicating that apoptosis was occurring. Based on these findings it was concluded that combined treatments of colchicine and paclitaxel are not advantageous in hepatocytes and could provide some insight into the treatment of liver cancer. Additionally, it appeared the drugs were initiating apoptosis in a p53 independent manner. / Department of Biology

A poliploidia induzida é uma ferramenta valiosa para o melhoramento genético de plantas, resultando em genótipos com características superiores aos diploides correspondentes. Considerando a importância da cultura de eucalipto para o setor florestal e para a economia brasileira, o presente trabalho teve como objetivo estabelecer um protocolo de poliploidização in vitro para eucalipto, utilizando a espécie Eucalyptus urophylla ST Blake. Segmentos nodais contendo gemas laterais de plântulas desenvolvidas in vitro foram utilizados como explantes para os testes de indução. Os tratamentos avaliados consistiram na imersão e agitação dos explantes em soluções de colchicina nas concentrações: 0; 0,125% e 0,250% em tempos de exposição de 12 e 24 horas e posterior inoculação in vitro em meio de cultura para multiplicação. Após 60 dias, as brotações emitidas foram analisadas por citometria de fluxo e constatou-se indivíduos diploides, tetraploides e mixoploides. Estas foram micropropagadas para a obtenção de mudas clonais ao final do processo. As mudas obtidas foram avaliadas por meio de análises morfológicas organográficas e anatômicas para verificar possíveis alterações resultantes da poliploidização in vitro. Os tratamentos promoveram a indução de 16 poliploides, sendo 11 mixoploides, quatro tetraploides e um octaplóide. A eficência máxima de indução obtida foi de 28% de poliploides no tratamento com 0,125% de colchicina por 12 horas sob agitação, com a obtenção tanto de mixoploides como de tetraploides puros. O protocolo geral de micropropagação utilizado, apesar de algumas perdas durante as etapas, garantiu a obtenção de altos percentuais de mudas clonais inteiramente tetraploides, comprovando a eficiência da técnica no isolamento de setores poliploides. A análise morfo-anatômica das mudas mostrou diferenças significativas em relação às características estomáticas, apontando este tipo de análise como indicador de poliploidização na separação preliminar dos indivíduos. A estrutura anatômica das folhas, no entanto, não sofreu modificações significativas, com exceção da espessura e área foliar da epiderme adaxial. A análise de componentes principais realizada para algumas variáveis morfo-anatômicas demonstrou que, apesar da análise estatística não ter apontado diferenças significativas, existe uma tendência em agrupar os genótipos diploides e tetraploides, diferenciando-os principalmente em relação às características estomáticas, espessura foliar, área do mesofilo, parênquimas paliçádico e lacunoso e espaço intercelular. Os resultados obtidos neste trabalho instigam a continuidade do estudo em diversas possibilidades de abordagens, como o estudo do desenvolvimento das mudas em condições de campos, anatomia da madeira e avaliação do comportamento meiótico. / The induced polyploidy is a valuable tool for genetic improvement of plants, resulting in genotypes with superior characteristics to their corresponding diploid. Considering the Eucalyptus culture importance to the forest sector and Brazilian economy, the aim of this study was establish an in vitro polyploidy protocol for eucalyptus, using the species Eucalyptus urophylla ST Blake. Nodal segments containing lateral buds developed from in vitro seedlings were used as explants for induction tests. The treatments tested consisted of soak and shake the explants in colchicine solutions at the concentrations: 0; 0.125% and 0.250% in soaking by periods of 12 or 24 hours and subsequent in vitro inoculation in culture medium for multiplication. After 60 days, the emitted shoots were classified by flow cytometry in levels of ploidy (diploid, tetraploid and mixoploids) and were subsequently micropropagated to obtain clonal seedlings at the end of the process. The clonal seedlings were evaluated using morphological and anatomical analysis to check the possible changes resulting from the in vitro polyploidy. The treatments promoted the induction of sixteen polyploids, being eleven mixoploids, four tetraploid and one octaploid. The maximum induction efficiency was obtained in 28% of polyploidy in the treatment with 0.125% of colchicine by soaking period of 12 hours, having obtained both mixoploids and pure tetraploids. The general protocol of micropropagation used, although presenting some losses during the steps, guaranted the inducing of high percentages of enterelly tetraploid, proving the efficiency of micropropagation in the isolation of polyploid sectors. The morpho-anatomical analysis of the poliploidy seedlings showed significant differences in relation to stomatal characteristics, indicating this type of analysis as polyploidy indicator in the preliminary separation of individuals. The anatomical structure of the leaves, however, did not change significantly, except for the thickness and leaf area of the adaxial epidermis. The principal component analysis (PCA) performed for some morphological and anatomical variables demonstrated that despite the statistical analysis did not have pointed out significant differences, there is a tendency to group the diploid and tetraploid genotypes, differentiating them mainly in relation to stomatal characteristics, leaf thickness, mesophyll area, palisade and spongy parenchyma areas and intercellular space. The results of this work instigate to continue the study in several possibilities of approaches, such as the study of the development of seedlings in fields conditions, wood anatomy and study of meiotic behavior.

No description available.

No description available.

Colchicine, an antimicrotubule agent, has enhanced the androgenic response of cruciferous and graminaceous species. Our objectives were to determine if colchicine treatments enhanced androgenic embryo production in diploid potato, and if colchicine treatments during anther culture affected the ploidy of anther-derived plants. An additional study, analysis of genetic identity among regenerated anther-derived plants based on RAPD markers, was also conducted. Anther culture of a hybrid between <i>Solanum chacoense</i> and <i>S. phureja</i> (clone CP2) was conducted with five colchicine treatments (0, 25, 50, 100, and 200 mg/L) for 24 h. Anthers containing late uninucleate pollen were cultured on modified liquid LS medium. A mean of 0.25 embryos per anther was obtained; however, there was no significant difference among the colchicine treatments, which ranged from a mean of 0.17 to 0.28 embryos per anther for 200 mg/L colchicine and control, respectively. In total 56 plants were regenerated of which 11% were monoploid. The second experiment using the same colchicine treatments was conducted with 54 hybrids derived by crossing six anther-derived doubled monoploids with three heterozygous pollinators. A mean of 1.78 embryos per anther was obtained; again, there was no significant difference among the colchicine treatments, which ranged from 1.27 to 2.75 for 50 mg/L colchicine and control, respectively. In total 679 plants were regenerated. The 312 plantlets for which ploidy was determined, 75% were monoploid. The third experiment was conducted to study various durations (0, 90 sec vacuum infiltration, 24, 48, and 72 h) of high colchicine treatment (200 mg/L) applied to <i>S. phureja</i> family DM 13-14 202 x ID 5. There was nearly significant difference among treatments (α = 0.05). Mean embryos per anther ranged from 0.96 to 1.90 for 48 h and 90 sec vacuum infiltration, respectively. In an additional study, 26 regenerated anther-derived monoploid plants, from the DM 13-14 202 x ID 5 family, from the second experiment, were genetically characterized based on RAPD markers. Forty-three loci were scored from 13 primers for groups of monoploids derived from the same flasks of anther culture. From one flask, two pairs of monoploids were found to be genetically identical. From a second flask 6 of 7 were genetically identical, and from a third 3 of 7 were genetically identical. The presence of genetically identical individuals within three monoploid populations indicates the prevalence of secondary embryogenesis during anther culture such that a single embryogenic microspore can generate many anther-derived plants. / Master of Science

The dorsal dentate gyrus (DDG) of the hippocampus plays a role in the expression of different forms of flexible behaviour mainly due to its ability to sustain neurogenesis throughout life. In the present thesis, we examined the role that the DDG and its adjacent areas, both collectively referred to as dorsal medial hippocampus (DMH), play in flexible, adaptive behaviour and cognitive processing. We used the neurotoxin, colchicine, to induce lesions of the DDG, which were found to affect neighbouring areas. Thus these lesions will be referred to as lesions of the DMH. In the first experiment, rats were tested for (1) perseverative behaviour before and after receiving chronic methamphetamine (METH) treatment, (2) METH-induced locomotor activity and stereotypy in an open field, and (3) working memory in a T-maze. The results showed that rats with lesions of the DMH exhibited perseveration and supersensitivity to the locomotor- and stereotypy-inducing effects of METH (0, 0.1, 0.3, 1 mg/kg i.p.) as well as increased long-term METH sensitization. Rats with DMH lesions also showed significant working memory deficits. Taken together, these results reveal specific forms of behavioural inflexibility in rats with lesions of the DMH that are mainly associated with perseveration, drug-related behaviours, including stimulant motor supersensitivity and drug sensitization, and impaired working memory functions.

Matrix metalloproteinases (MMPs) play a significant role in degrading the extracellular matrix in cancer development and metastasis. Overexpression of matrix metalloproteinases in tumour tissues relative to normal tissues has been exploited as a target for peptide-based therapeutics, to improve therapeutic index of currently used agents. The stability of MMP-activated prodrugs in normal tissue or organs is a significant challenge for their success in the clinic. In an in vitro study, the stability of twenty six prodrugs was studied in mouse liver, kidney, lung and tumour homogenates using HPLC and LC/MS. Selected agents were studied in vivo. Each prodrug has a characteristic amino acid sequence with dominant FITC N-terminal end cap. All prodrugs were conjugated to a colchicine derivative (ICT 2552) which is a vascular disrupting agent causing tumour vasculature shutdown and consequently, tumour necrosis. ICT 3146, ICT 3019, ICT 3120 and ICT 3115 prodrugs showed significant stability in normal tissues and considerable activation in certain tumour tissues compared to the lead compound ICT 2588. Also, the selectivity of promising prodrugs to the MMP family was confirmed by using leupeptin (serine, cysteine and threonine protease inhibitor), pepstatin A (aspartate protease inhibitor), phosphoramidon (nepralysin inhibitor), ilomastat (metalloproteinase inhibitor) and BML-P115 (matrix metalloproteinase inhibitor). Moreover, members of the MMP family responsible for cleaving the selected prodrugs were identified using recombinant MMP enzymes. Furthermore, a LC/MS-MS method was developed to specifically detect and quantify MMP-16 protein expression in H460 tumour. MMP- 16 was responsible for the cleavage of ICT 3146 and ICT 3115. Therefore, MMPactivated prodrugs could be a useful therapeutic approach to avoid off-site toxicities of currently used anti-tumour agents.