Hannover, Universiẗat, Diss., 1998.

Hannover, Universiẗat, Diss., 2003.

Hannover, Universiẗat, Diss., 2000.

Secondary metabolites play important roles in tree defense. Proanthocyanidins (PAs), one of the most common secondary metabolites, are widely distributed in trees and woody plants, and are abundant in poplar. In my research, molecular biology and biochemistry techniques were used to investigate the function of two important transcription factors, MYB115 and MYB134, in regulating the PA pathway in hybrid poplars. The importance of these transcription factors in regulating PA synthesis in leaves has recently emerged, but their roles in roots are not known. MYB134- and MYB115-overexpressing transgenic poplars showed a strong high-PA phenotype in leaves, but how these two regulators interact in vivo is still a mystery. This research aims to test the function of both MYBs in the regulation of PAs in poplar roots, and to explore the antimicrobial functions of root PAs. Both alleles of the MYB genes were sequenced in wild type poplars to design gRNAs for creating transgenic poplars with knocked-out (KO) MYB115 and MYB134 using the CRISPR Cas9 system. Both hairy root and whole plant transgenics with respective single- and double knock-outs were generated. Chemical and genetic characterization of both mutant types showed reduced PA content and down-regulated flavonoid genes in leaves. In poplar roots, only double-KOs showed a significant change in PA and salicinoid metabolism. These results indicated that the regulatory pathways for PA biosynthesis may differ in poplar leaves and roots. Significant PA concentrations remained in double-KO plants, suggesting other transcription factors for PA regulation are active. Because poplars accumulate large amounts of PAs in roots, potential functions of root tannins were also investigated. Antimicrobial activity of PAs was tested by disc inhibition assay in vitro and mycorrhizal co-culture sandwich assay in vivo. Pure PAs showed no inhibition towards the pathogenic fungi Armillaria ostoyae and A. sinapina but displayed slight inhibition to the mycorrhiza fungus Laccaria bicolor. These results provide preliminary insight into the functions of PAs in roots. / Graduate / 2023-04-24

Orientador: Marcelo Carnier Dornelas / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-09-05T12:21:39Z (GMT). No. of bitstreams: 1 Bello_CarolinaCassanoMonte_M.pdf: 4096413 bytes, checksum: 81120541bfee4c3700b982f05823abef (MD5) Previous issue date: 2013 / Resumo: A grande diversidade floral presente entre as espécies de Passiflora é o produto de diversas adaptações à grande variedade de polinizadores. A presença de um verticilo de filamentos denominado corona, entre os verticilos de pétalas e estames, é uma das principais características florais em Passiflora. Estes filamentos são de fundamental importância na interação com polinizadores e suas características (tamanho, forma, cor) estão sob forte pressão de seleção. A coloração dos filamentos da corona pode ter a mesma pigmentação do perianto, ou possuir cores contrastantes. Geralmente esta pigmentação é dada pela presença de antocianinas, garantindo um grande espectro de cores para as flores. Com frequência há uma distribuição diferencial dos pigmentos ao longo dos filamentos da corona. O estudo desta distribuição diferencial dos pigmentos permitiu a identificação de pelo menos 5 tipos de padrões de pigmentação da corona em Passiflora, sendo eles: "dégradée", bandeado e ponteado, bem como pigmentação uniforme e ausência aparente de pigmentação. Devido à grande importância da pigmentação da corona para o sucesso reprodutivo em Passiflora, propôs-se estudar a influência hormonal no processo de pigmentação desta estrutura. Pretendeu-se identificar o efeito da concentração de auxinas (por meio da aplicação de diferentes concentrações de ácido 2,4-diclorofenoxiacético, 2,4-D, e um inibidor do transporte polar de auxina, o ácido naftil-ftalâmico, NPA) e de giberelinas (por meio da aplicação de diferentes concentrações de ácido giberélico, GA3, e de um inibidor de sua síntese, o paclobutrazol, PACLO) na pigmentação de filamentos da corona em desenvolvimento das espécies de P. edulis, P. morifolia e P. suberosa, sendo os tratamentos hormonais realizados in vitro e in planta. Após as aplicações hormonais, os pigmentos florais foram analisados por espectrofotometria UV-vis. A aplicação de GA3 causou aumento da pigmentação floral e a aplicação de um inibidor de sua síntese, PACLO, causou a redução da pigmentação. A Aplicação de auxina (2,4-D) e um inibidor do seu transporte polar (NPA) tiveram efeitos similares no sentido da redução da pigmentação. Determinou-se o padrão de expressão, mediante RT-PCR e hibridização in situ, do gene MYB-R2R3/PACEPS7022E07 de P. suberosa, potencialmente envolvida na modulação da síntese de antocianinas, e demonstrou-se que o mesmo é expresso preferencialmente em órgãos florais / Abstract: The floral diversity present in the species of Passiflora is the product of several adaptations to the wide variety of pollinators. The presence of a whorl of filaments called corona, between the whorls of stamens and petals, is a major floral trait in Passiflora. These filaments are of fundamental importance in the interaction of pollinators and their characteristics (size, shape, color) are under strong selective pressure. The pigmentation of the corona filaments might have the same color of the perianth, or have contrasting colors. Generally the pigmentation is given by the presence of anthocyanins, ensuring a wide range of colors for the flowers. Often there is a differential distribution of pigment throughout the corona filament. Studying a variety of patterns of corona pigmentation, it was established that there are at least 5 types of pigmentation patterns in Passiflora: "dégradée", stripped and dotted, as well as uniformly pigmented and non-pigmented. Due to the great importance of the corona pigmentation for the reproductive success in Passiflora, we proposed to study the influence of hormones on the pigmentation process of this structure. In order to identify the effect of the application of auxins (through different doses of 2,4-dichlorophenoxyacetic acid, 2,4-D, and of the inhibitor of auxin polar transport, naphthyl ftalamic acid, NPA) and gibberellins (through the application of different doses of gibberellic acid, GA3, and its synthesis inhibitor, paclobutrazol, PACLO) in pigmentation of corona filament developing of species P. edulis, P. morifolia and P. suberosa, and hormonal treatments performed in vitro, in cultured corona filaments, and in planta spraying floral buds. For the verification of the effect of hormone applications, the flower pigments were analyzed by spectrophotometer UV-vis where only GA3 caused increased floral pigmentation contrary to PACLO, which caused the reduction of pigmentation. The auxin 2,4-D and an inhibitor of polar auxin transport, NPA had the same effect, reducing the floral pigmentation. We determined the pattern of expression (by RT-PCR and in situ hybridization) of the R2R3-MYB/PACEPS7022E07 gene of P. suberosa, potentially involved in the regulation of anthocyanin synthesis, demonstrating that it is expressed preferentially in floral organs / Mestrado / Biologia Vegetal / Mestra em Biologia Vegetal

c-Myb/MYB is an established and key player in hematopoietic malignancies but more recently a strong case for c-Myb as an oncogene in breast cancer has emerged. c-Myb and its transcriptional target genes have direct bearing on tumour initiation and progression and thus this has opened new opportunities to the development of therapeutic approaches in a range of cancer types with the aim of treating cancer at its various stages. In this study, the requirement of c-Myb during mammary gland tumourigenesis is being examined. In addition a direct therapeutic approach to targeting c-Myb-driven gene grp78/GRP78 in the context of primary and metastatic breast cancer was assessed. / The first aim of this study is to examine the expression of c-Myb during normal mammary gland development. The expression of c-Myb is extensively characterised in a temporal and spatial fashion. Nuclear staining of c-Myb by immunohistochemisty was found to be most elaborately expressed in the ductal epithelium during early mammary gland development. Mouse mammary gland lacking c-myb showed disorganised ductal structure in virgin mice, but did not affect subsequent pregnancy and lactation. / To extend the view that c-Myb is involved in mammary tumourigenesis c-myb-transduced immortalised mammary epithelial cells and two mammary tumour prone transgenic mouse models were examined. NMuMG cells transduced with c-myb showed enhanced proliferation and reduced Annexin V staining consistent with the protection from apoptosis. This reduced apoptosis is consistent with, and perhaps contingent upon, the elevated expression observed for bcl-2 and grp78. The data assembled by expression studies raised the possibility that c-Myb is essential for the establishment of mammary gland tumor in both MMTV-Neu and MMTV-PyMT spontaneous mammary gland tumor models. Loss of c-Myb expression in these models significantly delayed and in most instances completely abolished the onset of mammary gland tumours in both models. Preliminary evidence also indicated that Stat3 phosphorylation may underpin the elevated c-Myb expression in mouse mammary tumour cells. / The focus of my thesis then shifted to examining ways to exploit elevated c-Myb target gene GRP78 expression on the cell surface of mammary tumour cells. To do this I employed a GRP78 binding pro-apoptotic chimera peptide that specifically binds to GRP78 where I examined its efficacy against primary and metastatic breast cancer models. My results demonstrated the anti-tumour activity of the GRP78-chimera peptide both in vitro and in vivo. More importantly, the peptide is also effective at prolonging disease-free survival in mice with established metastatic disease. / Evidence obtained within these studies suggests that c-Myb plays an important role in mammary gland development and tumourgenesis. Although it may be difficult to directly target c-Myb in malignant disease, alternative anti-tumoural therapy may be developed against c-Myb-regulated target genes that are also implicated in mammary tumours. Collectively my thesis studies have advanced our understanding of c-Myb in mammary cancer initiation, progression and as a direct or indirect therapeutic target.

Univ., Diss., 2009--Würzburg.

In the model cruciferous plant, Arabidopsis thaliana, tryptophan (Trp) is a focal point for growth and defense as it is used for the production of secondary metabolites including the growth hormone indole-3-acetic acid (IAA, or auxin), and two classes of defense compounds: indole glucosinolates (IGs) and camalexin. Trp metabolism in plants is of general importance to agriculture because animals (including humans) cannot synthesize Trp and must obtain it from their diet. Questions remain about the synthesis and regulation of Trp and how it relates to secondary metabolism in Arabidopsis. In this thesis it is shown that IGs are a sink for Trp metabolism because auxotrophic mutants deficient in Trp production are suppressed in combination with the IG-deficient cyp79B2 cyp79B3 mutant and enhanced in combination with IG overproducing mutant, atr1D. Because Trp auxotrophic mutants were found to produce IGs, the four predicted Arabidopsis Trp Synthase Beta genes (TSB1, TSB2, TSB3 and TSBt2) were examined for their role in Trp primary and secondary metabolism. It was determined that members of this gene family, while being redundant for enzyme activity, may have unique functions in channeling Trp to different secondary endpoints. tsb1 tsb2 plants display a healthier phenotype and produce lower IG levels than the single tsb1 mutants, in contrast to tsb1 tsbt2 plants, which have elevated IG production and an enhanced auxotrophic phenotype. tsb2 tsbt2 plants are indiscernible from WT. Gene expression in Trp biosynthetic pathway steps, IG biosynthesis genes, and regulatory TFs is dysregulated in these mutants. In a second part of this thesis, transcriptional regulation of IG synthesis was examined with respect to tissue specificity and stress. In collaboration with Judith Bender's laboratory at Brown University, the function of a subfamily of three Myb transcription factors that have been implicated in regulating IG biosynthesis genes was studied. Using combinations of Myb knockout mutants and GUS reporter plants, tissue specific roles for MYB34 and MYB51 in root and shoot tissues, respectively, were found. In addition, roles were discovered for MYB34 in mediating anti-herbivory signals, and for both MYB51 and MYB122 in regulating defense against microbial pathogens.

Thesis (Ph. D.)--University of Virginia, 2001. / Includes bibliographical references (leaves 182-215). Also available online through Digital Dissertations.

Exposure to benzene, a ubiquitous environmental pollutant, has been linked to leukemogenesis, although the mechanism of benzene initiated carcinogenesis remains unclear. It has been proposed that benzene can be bioactivated to toxic metabolites such as 1,4 benzoquinone (BQ), which can alter signalling pathways and affect chromosomal integrity. BQ has been shown to increase the activity of c-Myb, which is an important transcription factor involved in hematopoiesis, cell proliferation, and cell differentiation. The c-Myb protein also increases topoisomerase IIα (topo IIα) promoter activity specifically in cell lines with hematopoietic origin. Topo II is a critical nuclear enzyme that removes torsional strain by cleaving, untangling and religating double-stranded DNA. Since topo II mediates DNA strand breaks, aberrant topo II activity or increased protein levels may increase the formation of DNA strand breaks, leaving the cell susceptible to mutational events. I hypothesize that BQ increases c-Myb activity, which in turn increases topo IIα promoter activity resulting in increased DNA strand breaks. Using luciferase reporter assays in K-562 cells (human chronic myeloid leukemic cells) I confirmed that BQ exposure (25 and 37 µM) caused an increase in c-Myb activity after 24 hours. Contradictory to previous findings, overexpression of exogenous c-Myb or a polypeptide consisting of c-Myb’s DNA binding domain (DBD), which competitively inhibits the binding of endogenous c-Myb to DNA, did not affect topo IIα promoter activity. However, BQ exposure (37 µM for 24 hours) caused a significant increase in topo IIα promoter activity, which could be blocked by the overexpression of the DBD polypeptide. Western immunoblotting analysis did not show any significant increases in topo IIα protein levels in cells exposed to 37 µM BQ for 24 hours. Overall, this study suggests that BQ exposure increases topo IIα promoter activity through the c-Myb signalling pathway and furthers our understanding of BQ-mediated toxicity. / Thesis (Master, Pharmacology & Toxicology) -- Queen's University, 2008-01-02 14:09:00.011