Allopolyploids are formed by interspecific hybridization and whole genome duplication, with the resulting organism contains multiple distinct subgenomes in one nucleus. Subgenomic interactions result in massive genetic and epigenetic reconstruction, contributing to variable phenotypic traits noted in newly formed allopolyploids. To better understand these mechanisms in the context of molecular biology, evolution, and plant breeding, plant biologists study the model organism Brassica napus (farmed as canola or oilseed rape). With white-flowering and yellow-flowering progenitors, flower color phenotype of B. napus presents an opportunity to examine subgenomic interactions. CAROTENOID CLEAVAGE DIOXYGENASE 4 (CCD4) is known to play a major role in determining flower color phenotype of carotenoid-synthesizing angiosperms. Here, we investigate the genetic and epigenetic role of CCD4 orthologs and their role in flower color phenotype of B. napus.

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Three 2-oxoglutarate-dependent dioxygenases required for flavonoid biosynthesis were characterized in Arabidopsis thaliana. Genes encoding flavanone 3-hydroxylase (F3H), flavonol synthase (FLS), and leucoanthocyanidin dioxygenase (LDOX) were cloned and sequenced. The predicted proteins encoded by each of these Arabidopsis genes shared high homology with all F3H, FLS, or LDOX sequences available in Genbank. Low-stringency DNA blot analysis indicated that F3H and LDOX are encoded by a single gene in Arabidopsis, while FLS may be encoded by two or three genes. RNA blot analysis was performed to determine the expression patterns of these three genes relative to previously-cloned genes encoding flavonoid biosynthetic enzymes. Light-induction experiments and analysis of regulatory mutants showed that the CHS, CHI, F3H, and FLS1 are coordinately regulated in Arabidopsis seedlings, encode enzymes acting near the beginning of the pathway, and are therefore referred to as "early" genes. The same experiments showed that DFR and LDOX are regulated distinctly from "early" genes, share similar expression patterns in response to light, and are not expressed in the ttg mutant. DFR and LDOX are therefore referred to as "late" genes due to the timing of expression in response to light and the fact that they encode enzymes acting late in flavonoid biosynthesis. To determine whether any of the previously-identified transparent testa mutants were defective in F3H, FLS, or LDOX, the chromosomal locations of these genes in the Arabidopsis genome were determined. The positions of these genes suggested that no previously-identified tt mutant was defective in the cloned FLS or LDOX structural genes, while tt6 was potentially the F3H locus. The coding region of F3H was amplified by PCR from tt6 genomic DNA and sequenced, and several point mutations were found in the coding region of this allele, three of which are predicted to result in amino acid substitutions. Polyclonal antibodies were also developed using four different purified, recombinant flavonoid enzymes as antigens. These antibodies were used to determine the pattern of accumulation of flavonoid enzymes in developing seedlings. Immunoblot analysis was also performed to determine whether mutations in genes encoding specific flavonoid enzymes or an enzyme in pathways that compete for or provide substrate for flavonoid biosynthesis (mutants defective in tryptophan or ferulic acid biosynthesis) affect the levels of flavonoid enzymes. These analyses showed that mutant seedlings which lacked specific flavonoid or tryptophan biosynthetic enzymes accumulated higher steady-state levels of other enzymes in the pathway. These results suggest that the accumulation of specific flavonoid intermediates or indole can lead directly or indirectly to higher levels of flavonoid enzymes. / Ph. D.

Yes / Alopecia areata (AA) is an autoimmune hair loss disease with infiltration of proinflammatory cells into hair follicles. Current therapeutic regimens are unsatisfactory mainly because of the potential for side effects and/or limited efficacy. Here we report that cultured, transduced fibroblasts, which express the immunomodulatory molecule indoleamine 2,3-dioxygenase (IDO), can be applied to prevent hair loss in an experimental AA model. A single intraperitoneal (IP) injection of IDO-expressing primary dermal fibroblasts was given to C3H/HeJ mice at the time of AA induction. While 60–70% of mice that received either control fibroblasts or vehicle injections developed extensive AA, none of the IDO-expressing fibroblast-treated mice showed new hair loss up to 20 weeks post injection. IDO cell therapy significantly reduced infiltration of CD4+ and CD8+ T cells into hair follicles and resulted in decreased expression of TNF-α, IFN-γ and IL-17 in the skin. Skin draining lymph nodes of IDO fibroblast-treated mice were significantly smaller, with more CD4+ CD25+ FoxP3+ regulatory T cells and fewer Th17 cells than those of control fibroblast and vehicle-injected mice. These findings indicate that IP injected IDO-expressing dermal fibroblasts can control inflammation and thereby prevent AA hair loss. / Canadian Institutes of Health Researches (Funding Reference Number: 134214 and 136945).

The pharmacologically important alkaloids morphine and codeine are found in latex of opium poppy (Papaver somniferum). Latex is harbored in laticifers, a specialized vascular cell-type. Isolation and characterization of latex-specific genes may provide a useful tool to metabolically engineer increased alkaloid production. Previous research in the Nessler laboratory identified genes that exhibit latex-specific gene expression. Latex-specific genes were an 2-oxoglutarate-dioxygense (DIOX), involved in hydroxylation, desaturation and epoxidation reactions, and two of the major latex proteins, MLP146 and MLP149. MLP-like proteins function in fruit ripening in various species that do not have the laticifer cell type. The latex-specific promoters (LSPs) for the three genes were sequenced. The 2.5 kb DIOX promoter was fused to the reporter gene Β-glucuronidase (GUS) to characterize its expression pattern. To assess the functional sites within the DIOX promoter, deletions were made 1.5 kb and 0.14 kb upstream of the ATG start codon, fused to GUS, and transformed into opium poppy, Arabidopsis thaliana, and tobacco (Nicotiana tabacum). The 2.5 kb DIOX:GUS and 1.5 kb EcoRIDIOX:GUS reporter gene constructs showed vascular specific expression in opium poppy, Arabidopsis, and tobacco. The 0.14 kb SpeIDIOX promoter deletion construct showed no activity in opium poppy, and limited expression in the shoot apical meristem and root hypocotyl axis in Arabidopsis. These results indicate that the minimum active DIOX promoter is greater than 0.14 kb. Over 1 kb of the LSPs were sequenced and analyzed for regulatory elements using the Plant cis-acting regulatory DNA elements database, PLACE (http://www.dna.affrc.go.jp/PLACE). Knowledge of the cis-elements and regulatory regions of LSPs would serve as a tool for metabolic engineering of poppy alkaloids. Sixty-five elements were conserved among 2 of the 3 LSPs. Among the cis-elements identified, some are associated with basic functions such as: light regulation, carbon metabolism and plant defense. Other elements include: WRKY elements that are binding sites of transcription factors known for signaling plant defense genes, a vascular cis-element, and a fruit specific element. The presence of plant defense and vascular cis-elements in the LSPs, correlate with the concept that latex is a protective defense mechanism found in the vascular system. The latex-specific promoters isolated and cis-elements identified in this research are potential tools for driving increased alkaloid production in opium poppy. / Master of Science

京都大学 / 新制・課程博士 / 博士(農学) / 甲第25333号 / 農博第2599号 / 新制||農||1106(附属図書館) / 京都大学大学院農学研究科応用生命科学専攻 / (主査)教授 小川 順, 教授 矢﨑 一史, 教授 栗原 達夫 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

Neste estudo avaliamos o papel do metabolismo do triptofano (Trp) na homeostasia, na vaginose bacteriana e nas lesões cervicais associadas ao HPV. A importância do metabolismo do Trp se deve a sua ação na proliferação de microrganismos e de células do sistema imune. O consumo de triptofano tem sido identificado como uma forma de controlar o crescimento bacteriano limitando a infecção. Por outro lado, a oxidação de Trp produz quinurenina (QUIN), que tem papel chave na tolerância imunológica. A formação de QUIN se dá através das enzimas indoleamina 2,3-dioxigenase (IDO) e triptofano 2,3- dioxigenase (TDO). A mais estudada delas no âmbito das infecções/ imuno escape é a enzima IDO. Mais recentemente, tem-se dado ênfase ao papel da TDO no câncer. Nesta dissertação, o interesse foi avaliar a expressão da IDO no epitélio cervicovaginal de mulheres com vaginose bacteriana e de IDO e TDO em amostras cervicais de mulheres com diferentes graus de lesão cervical associada ao HPV. Foram incluídas 165 mulheres atendidas no CAISM/UNICAMP, as quais foram divididas em dois grupos: grupo caso composto por mulheres com lesão de baixo ou alto grau e carcinoma invasor (n=42) e grupo controle composto por mulheres com citologia oncológica normal, independente de apresentar infecção genital (n=123). IDO foi avaliada por imunocitoquímica em citologia em base líquida e IDO e TDO em biópsias cervicais. Mulheres com vaginose bacteriana apresentaram expressão aumentada de IDO em células escamosas em comparação às mulheres sem vaginose bacteriana (OR=7.41; IC 95%= 2.50 a 21.4; p <0.0001). No epitélio vaginal normal com ou sem infecção por HPV houve uma expressão leve de IDO em células escamosas. Na presença de lesões ou carcinoma, houve um aumento no número de células escamosas displásicas e de leucócitos IDO-positivos; aumento de IDO também pôde ser observada em culturas de pele organotípicas transduzidas com as oncoproteínas E6/ E7 do HPV16. Nas lesões cervicais, assim como visto para a IDO, a TDO esteve expressa em leucócitos, especialmente os infiltrados na região estromal e na parede dos vasos sanguíneos. A expressão basal de IDO no epitélio cervical normal e sua regulação positiva na infecção por HPV e lesões associadas sugerem a participação do metabolismo do Trp nos mecanismos imunossupressores envolvidos na doença. Embora o papel do IDO já tenha sido abordada anteriormente, até onde sabemos esta é a primeira evidência da expressão de TDO no epitélio vaginal, na neoplasia intraepitelial cervical e carcinoma de células escamosas. Ainda, em leucócitos, especialmente aqueles com morfologia típica de polimorfonucleares, parecem ser importantes fontes de IDO na cérvix uterina. / In this study we evaluated the role of tryptophan (Trp) metabolism in cervix homeostasis, bacterial vaginosis and HPV-associated lesions. The importance of Trp metabolism is due to its action on microorganisms and immune cells. Tryptophan consumption has been identified as a way to controlling bacterial growth limiting infection. On the other hand, the oxidation of Trp produces kynurenine (Kyn) which plays a key role in immunological tolerance. The formation of Kyn occurs through the enzymes indoleamine 2,3-dioxygenase (IDO) and tryptophan 2,3-dioxygenase (TDO). IDO is the most studied of them within the context of infections / immune escape. More recently, TDO has also been considered in studies of cancer progression. In this thesis, we were interested in cervicovaginal epithelium IDO expression in women with bacterial vaginosis and of IDO and TDO in cervical samples of women with different degrees of cervical lesion associated with HPV. A total of 165 women attended at CAISM/UNICAMP were divided into two groups: a case group composed of women with low or high grade lesions and invasive carcinoma (n = 42) and a control group composed of women with normal cytology, independent to present genital infection (n =123). IDO was evaluated by immunocytochemistry in liquid-based cytology and IDO and TDO in cervical biopsies. Women with bacterial vaginosis had increased IDO expression in squamous cells compared to women without bacterial vaginosis (OR = 7.41, 95% CI = 2.50- 21.74; p<0.0001). In normal vaginal epithelium with or without HPV infection there was a mild IDO expression in squamous cells. In the presence of cervical intraepithelial lesions or squamous cell carcinoma, there was an increase in the number of IDO-positive dysplastic squamous cells and leukocytes; increase in IDO can also be observed in organotypic skin cultures transduced with HPV-16 E6/E7 oncoproteins. In cervical lesions, as observed for IDO, TDO was expressed in leukocytes, especially infiltrates in the stromal region and in the wall of blood vessels. The basal expression of IDO in the normal cervical epithelium and its positive regulation in HPV infection and associated lesions suggests the participation of Trp metabolism in the immunosuppressive mechanisms involved in the disease. Although some previous data have already considered the role of IDO, as far as we know this is the first evidence of the participation of TDO in the vaginal epithelium, cervical intraepithelial neoplasia and squamous cell carcinoma. In addition, in leukocytes, especially those with a typical polymorphonuclear morphology, appear to be important sources of IDO in the uterine cervix.

Eimeria falciformis ist ein Apicomplexa-Parasit, welcher das Blinddarmepithel der Maus befällt. Aufgrund des monoxenen Lebenszyklus in einem exzellent-erforschten Wirt, bietet sich E. falciformis als Modellorganismus an, um Wirts-Parasit-Interaktionen zu untersuchen. Im Rahmen dieser Arbeit wurden mit Hilfe von Genexpressionsanalysen bei E. falciformis-infizierten Zellen und Mäusen Wirtsfaktoren identifiziert, welche für die in vitro bzw. in vivo Entwicklung des Parasiten vonnöten sind. Der Transkriptionsfaktor c-FOS (FBJ osteosarcoma oncogene) zeigte eine erhöhte Expression bei der Infektion einer Epithelzelllinie mit E. falciformis. C-FOS ist ein Bestandteil des AP-1 (activator protein 1) Komplexes, welcher die Transkription zahlreicher Gene unterschiedlichster Funktion steuert. Unsere Ergebnisse zeigen, dass die Entwicklung von E. falciformis in Zellen, welche den Transkriptionsfaktor nicht besitzen (c-FOS knockout Zellen) beeinträchtigt war. Diese Beobachtung betont eine mögliche Ausbeutung des Transkriptionsfaktors des Wirtes durch den Parasiten. In E. falciformis-infizierten Mäusen war die Expression des Enzyms Indoleamin 2,3-Dioxygenase (IDO1) bemerkenswert induziert. IDO1 katalysiert die erste und geschwindigkeits-bestimmende Reaktion des Tryptophan-Abbaus innerhalb des Kynurenin-Stoffwechselweges. Wir zeigen in dieser Studie, dass in den E. falciformis-infizierten Epithelzellen IDO1 IFN-gamma abhängig induziert wird. Das Wachstum des Parasiten war zudem beeinträchtigt in IDO1-/- Mäusen sowie in Mäusen, in welchen zwei weiterer Enzyme des Kynurenin-Stoffwechselweges pharmakologisch inhibiert wurden. Bemerkenswerterweise konnte das Parasitenwachstum in IDO1-/- Mäusen durch Gabe von Xanthurensäure, ein Nebenprodukt des Tryptophan-Abbaus, auf Wildtyp-Niveau angehoben werden. Diese Daten demonstrieren, dass sich der intrazelluläre Parasit E. falciformis die wirtseigenen Verteidigungsmechanismen (IFN-gamma, IDO1) für seine eigene Entwicklung zu Nutze macht. / Eimeria falciformis is a highly host- and tissue-specific parasite of murine caecum epithelium. Its monoxenous life cycle in a well-investigated host makes it an excellent model to examine parasite-host interactions. To identify the host determinants of the parasite infection, this work involved the comparative in vitro and in vivo analyses of mouse gene modulation by E. falciformis. The in vitro microarray analyses identified the transcription factor FBJ osteosarcoma oncogene (c-FOS) as highly induced during E. falciformis infection. C-FOS is part of the activator protein 1 (AP-1) complex, which controls the transcription of genes involved in various biological processes. We show that infection of c-FOS-deficient mouse cells results in an impaired development of E. falciformis, highlighting an exploitation of the host transcription factor by an apicomplexan parasite. Our ex vivo gene expression analyses using mouse caecum cells revealed a substantial modulation of the host transcriptome. The indoleamine 2,3-dioxygenase 1 (IDO1), the first and rate-limiting enzyme of tryptophan catabolism in the kynurenine pathway, was one of the most up-regulated epithelial transcripts. Induction of IDO1 supposedly depletes tryptophan in host cells, which is proposed to inhibit the in vitro growth of pathogens auxotrophic for this essential amino acid. We show that E. falciformis induces IDO1 in the epithelial cells in an IFN-gamma-dependent manner. The absence or inhibition of IDO1 and two downstream enzymes of the pathway in the mouse impairs parasite growth. Noticeably, the parasite development was entirely rescued by xanthurenic acid, a by-product of tryptophan catabolism. These data demonstrate contrasting roles of IFN-gamma signaling and a conceptual subversion of the host defense (IFN-gamma, IDO1) by an intracellular pathogen for progression of its natural life cycle.

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