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Pleiotropy refers to the shared effects of a gene or genes on multiple phenotypes, a major reason for genetic correlation between phenotypes. For example, for osteoporosis, bone mineral densities at different skeletal sites may share common genetic factors; thus, examining the shared effects of genes may enable more effective fracture treatments. To date, methods are not available for estimating and testing the pleiotropic effects of single nucleotide polymorphisms (SNPs) in genetic association studies. In this dissertation, we explore two types of methods to evaluate the SNP-specific pleiotropic effect based on multivariate techniques. First, we propose two approaches based on variance components (VC) analysis for family-based studies, which quantify and test the pleiotropic effect by examining the contribution of specific genetic marker(s) to polygenic correlation or covariance of traits. Second, we propose a multivariate linear regression approach for population-based studies with samples of families or unrelated subjects. This method partitions the specific effect of the marker(s) from phenotypic covariance. We evaluate the performance of our proposed methods in simulation studies, compare them to existing multivariate analysis methods and illustrate their application using real data to assess candidate SNPs for osteoporosis-related phenotypes in the Framingham Osteoporosis Study. In contrast to existing methods, our newly proposed approaches allow the quantification of pleiotropic effects. The bootstrap resampling percentile method is used to construct confidence intervals for statistical hypothesis testing. Simulation results suggest that the VC-based approaches are affected by the polygenic correlation level. The covariance analysis approach outperforms the VC-based approaches, with unbiased estimates and better power, which remain consistent regardless of the polygenic correlation. In addition, the covariance analysis approach is simple to implement and can be applied to both family data and genetically unrelated data. Using simulation, we also show that existing methods, such as MANOVA, can have high rejection rates when a SNP has a large effect on a single trait, which prevent us from using them for pleiotropic effect analysis. In summary, this dissertation introduces promising new approaches in multiple phenotypic models for SNP-specific pleiotropic effect.

Leptin, a 16kDa protein, is secreted primarily by adipose tissue. Its effects are pleiotropic, with known roles in body mass regulation, haematopoiesis, immune function and reproduction. Studies of the leptin system in females were undertaken to complement existing animal and human studies. Serum leptin levels were found to increase in the luteal phase of the menstrual cycle raising the possibility of a role for leptin at the time of implantation. RT-PCR studies showed that leptin receptors were expressed in the human endometrium throughout the menstrual cycle. In these preliminary studies, these receptors appeared to be functional in response to leptin; increasing proliferation and decreasing TNF-a production in cultured endometrial cells. This implies that leptin has a local effect at the level of the endometrium. Many cytokines are bound to proteins in serum; a proportion of this binding can be attributed to a soluble cytokine receptor. Soluble receptors and binding proteins have a variety of roles, acting as scavengers, carriers, agonists and antagonists. In order to investigate this phenomenon in the leptin system, a radio-receptor assay was developed to measure leptin-binding activity (LBA). The leptin receptor has an extracellular domain that is common to all isoforms and therefore, LBA may also reflect the binding parameters of cell surface receptors. Serum leptin levels of LBA were found to be low at birth, high in early childhood, to fall steadily through puberty and remain at the post-pubertal levels throughout adult life. This suggests that leptin and LBA has an important role in the initiation of puberty. There was no significant variation in LBA during the menstrual cycle. Several single nucleotide polymorphisms (SNP) exist in the leptin receptor gene. Studies of two of these SNPs, GLN223ARG and LYS656ASN, present in the extracellular domain of the receptor were undertaken to assess if genetic changes are associated with differences in phenotype or effect ligand binding. Homozygosity of the G allele of GLN223ARG is associated with lower fat mass, BMI and leptin levels in postmenopausal Caucasian females. This polymorphism changes the binding characteristics of the receptor, with a higher LBA being associated with homozygosity of the G allele. This suggests that the actual binding of leptin to its receptor may be an important factor in the regulation of body weight and adiposity.

Human health is increasingly threatened by the emergence of multiply drug resistant malignant organisms. Yet, our understanding of the numerous ways by which such resistance arises is modest. Here, we present evidence of a bet hedging strategy in the budding yeast, Saccharomyces cerevisiae, to counter the effects of cytotoxic drugs through the action of Pdr5, an ATP-binding cassette transporter. We have employed flow cytometry and fluorescent activated cell sorting to probe the expression levels of a GFP-tagged version of PDR5 in individual cells. The results obtained from these experiments demonstrate that each yeast population is variable in the levels of Pdr5 production, and a small subpopulation of cells produces this efflux pump at much higher quantities than the population average. Consequently, cells with high and low levels of Pdr5 grow differentially in presence and absence of cycloheximide, a cytotoxic drug. These properties are highly suggestive of a bet hedging strategy mediated by Pdr5 levels. We further link this bet hedging strategy to the transcriptional regulatory network of PDR5 consisting of two major transcription factors, Pdr1 and Pdr3. Our analysis suggests that a self-activating feedback loop acting on Pdr3 plays an important role in generation of the aforementioned subpopulation. Furthermore, our results point to a large difference in the activity of these two regulators wherein Pdr3 is notably stronger than Pdr1. The disparity in their activity could indicate a mechanism for generation of the observed proportions of subpopulations with regards to the level of Pdr5.

Τα ευεργετικά αποτελέσματα των αναστολέων της αναγωγάσης του HMG-CoA (3-υδροξυ-3-μεθυλ-γλουταρυλ-συνένζυμο Α) έχουν αποδοθεί όχι μόνο στη μείωση των επιπέδων χοληστερόλης αλλά και στις πλειοτροπικές τους δράσεις και ιδιαίτερα στις αντιοξειδωτικές τους δράσεις. Ο Nrf2 (NF-E2-related factor 2) είναι ένας μεταγραφικός παράγοντας που ενορχηστρώνει τη μεταγραφική απάντηση των κυττάρων στο οξειδωτικό στρες και σε ηλεκτροφιλικά ξενοβιοτικά με την έκφραση αντιοξειδωτικών-κυτταροπροστατευτικών γονιδίων. Στην παρούσα μελέτη, χρησιμοποιήθηκαν επίμυες Wistar, πρωτογενείς καλλιέργειες ηπατοκυττάρων, ST-2 κύτταρα και πρωτογενείς εμβρυϊκοί ινοβλάστες από μυς C57BL6J αγρίου τύπου (WT) ή με απαλοιφή του Nrf2 (KO-Nrf2) για τη μελέτη του ρόλου του Nrf2 στη διαμεσολάβηση των αντιοξειδωτικών δράσεων των στατινών. Η σιμβαστατίνη ενεργοποίησε τον Nrf2, δηλαδή οδήγησε στη μετακίνησή του στον πυρήνα στο ήπαρ επίμυων και σε πρωτογενή καλλιέργεια ηπατοκυττάρων. Η ανωτέρω δράση ήταν εξαρτώμενη από το μεβαλονικό και ανεξάρτητη από τη χοληστερόλη. Σε πυρηνικά πρωτεϊνικά εκχυλίσματα από ήπαρ επίμυων που έλαβαν σιμβαστατίνη, η μεταγραφική ενεργότητα του Nrf2 αυξήθηκε σημαντικά και το mRNA δύο γνωστών στόχων του Nrf2, ΗΟ-1 (Heme Oxygenase 1) και GPX-2 (Glutathione Peroxidase 2) αυξήθηκε. Σε ST-2 κύτταρα μόνιμα διαμολυσμένα με πλασμίδιο που φέρει αλληλουχία DNA στην οποία προσκολλάται ο Nrf2 (ARE-Antioxidant Response Element) (ARE-ST2 κύτταρα), η σιμβαστατίνη αύξησε τη μεταγραφική ενεργότητα του Nrf2 με τρόπο εξαρτώμενο από το μεβαλονικό και ανεξάρτητο από τη χοληστερόλη. Επίσης, χρησιμοποιώντας πρωτογενείς καλλιέργειες εμβρυϊκών ινοβλαστών από μυς WT και ΚΟ-Nrf2 και μετρώντας τα επίπεδα των αντιδραστικών ειδών οξυγόνου (ROS) μετά από επώαση με οξειδάση της γλυκόζης έπειτα από επίδραση ή μη με σιμβαστατίνη, διαπιστώθηκε ότι η σιμβαστατίνη μειώνει τα επίπεδα των παραχθέντων ROS στους WT ινοβλάστες και όχι σε μεγάλο βαθμό στους KO-Nrf2 ινοβλάστες Τέλος, με τη χρήση αναστολέων του μονοπατιού της PI3K/Akt σε ARE-ST2 κύτταρα στα οποία είχαμε επιδράσει με σιμβαστατίνη, διαπιστώθηκε ότι αίρεται σε μεγάλο βαθμό η ενεργοποίηση του Nrf2 από τη σιμβαστατίνη. Στην παρούσα μελέτη παρουσιάζεται για πρώτη φορά α) η ενεργοποίηση του Keap1/Nrf2 σηματοδοτικού μονοπατιού σε in vivo και in vitro μοντέλα από τη σιμβαστατίνη με τρόπο εξαρτώμενο από το μεβαλονικό και ανεξάρτητο από τη χοληστερόλη και β) ότι η σιμβαστατίνη μειώνει τα παραγόμενα αντιδραστικά είδη οξυγόνου (ROS) μέσω της ενεργοποίησης του Nrf2 διαμέσου του μοριακού μονοπατιού της PI3K/Akt. H ενεργοποίηση αυτή του Nrf2 από τη σιμβαστατίνη εκτός του ότι προσφέρει αποτελεσματική προστασία του κυττάρου από τις δυσμενείς επιπτώσεις του οξειδωτικού στρες, εξηγεί και μέρος των πλειοτροπικών δράσεων των στατινών. / The beneficial effects of HMG-CoA (3-hydroxy-3-methyl-glutaryl-CoA) reductase inhibitors (statins) have been attributed not only to their cholesterol lowering effect but also to their pleiotropic actions and especially to their anti-oxidant activity. Nrf2 (NF-E2-related factor 2) is a transcription factor that orchestrates the transcriptional response of cells to oxidative stressors and electrophilic xenobiotics. In the present study, Wistar rats, primary hepatocytes, ST-2 cells and primary mouse embryonic fibroblasts from wild type or Nrf2 knock out C57BL6J mice were used to explore the potential role of Nrf2 in mediating the antioxidant effects of statins. Simvastatin triggered nuclear translocation of Nrf2 in rat liver and in primary rat hepatocytes in a mevalonate-dependent and cholesterol-independent way. In liver nuclear extracts from simvastatin-treated rats, the DNA-binding activity of Nrf2 was significantly increased and the mRNA of two known targets of Nrf2, HO-1 (Heme Oxygenase 1) and GPX-2 (Glutathione Peroxidase 2) was induced. In ST-2 cells (ARE-ST2) stably transfected with constructs bearing Nrf2-binding site (antioxidant responsive element), simvastatin enhanced Nrf2-mediated transcriptional activity in a mevalonate-dependent and cholesterol-independent fashion. Moreover, in WT and KO-Nrf2 primary mouse embryonic fibroblasts, after incubation with simvastatin, the addition of glucose-oxidase resulted in much greater levels of ROS (Reactive Oxygen Species) in simvastatin treated KO-Nrf2 fibroblasts compared to their WT counterparts, which means that simvastatin eliminates ROS in a Nrf2 dependent way. Finally, the use of PI3K/Akt pathway inhibitors in simvastatin treated ARE-ST2 cells leads to the attenuation of the simvastatin-induced enhanced Nrf2 transcriptional activity. In the present study, it is shown for the first time that a) simvastatin activates the Keap1/Nrf2 signaling pathway in in vivo and in vitro models in a mevalonate- dependent, cholesterol-independent fashion and b) that simvastatin lowers reactive oxygen species level by Nrf2 activation via PI3K/Akt pathway. The activation of Nrf2 by simvastatin besides providing effective protection of the cell from the deleterious effects of oxidative stress, it explains in part the pleiotropic actions of statins.

Alcohol addiction may increase the risk of obesity due to shared genetic components. The Copine V (CPNE5) gene is involved in Ca2+ binding and may play an important role in the development of the central nervous system. This study tested the genetic associations of 77 single-nucleotide polymorphisms (SNPs) within the CPNE5 gene with alcohol dependence (AD) and obesity using a Caucasian sample - The Study of Addiction - Genetics and Environment (SAGE) sample (1066 AD cases and 1278 non-AD controls, 422 obese cases and 1395 non-obese controls). The Marshfield sample (1442 obese cases and 2122 non-obese controls) was used for replication of obesity. Multiple logistic regression analysis was performed using the PLINK software. In the SAGE sample, we identified 10 SNPs associated with AD and 17 SNPs associated with obesity (p < 0.05). Interestingly, 6 SNPs (rs9986517, rs9470387, rs3213534, rs10456444, rs3752482, and rs9470386) were associated with both AD (OR = 0.77, 0.77, 0.83, 0.84, 0.79 and 1.14, respectively; p = 9.72 × 10-5, 1.1 × 10-4, 4.09 × 10-3, 5.26 × 10-3, 1.59 × 10-2, and 3.81 × 10-2, respectively) and obesity (OR = 0.77, 0.77, 0.78, 0.77, 0.68 and 1.18, respectively; p = 2.74 × 10-3, 2.69 × 10-3, 2.45 × 10-3, 1.01 × 10-3, 5.18 × 10-3 and 3.85 × 10-2, respectively). In the Marshfield sample, rs3752480 was associated with obesity (p = 0.0379). In addition, four SNPs (rs9986517, rs10456444, rs7763347 and rs4714010) showed associations with obesity in the meta-analysis using both samples (p = 0.00493, 0.0274, 0.00346, and 0.0141, respectively). These findings provide the first evidence of common genetic variants in the CPNE5 gene influencing both the AD and obesity; and will serve as a resource for replication in other populations.

Oxidative stress is a documented factor in the pathogenesis of inflammation and cancer. Vitamin E with its antioxidant properties holds promise for use in clinical practice. There are two main forms of vitamin E, tocopherols and tocotrienols. Palm oil contains almost 70% of tocotrienols. Tocotrienols exerts its antiproliferative activity against malignant cells but not on normal cells. Tocotrienols play an important role in counteracting cellular inflammatory response secondary to oxidative stress, thus exerting an anticancer property. Tocotrienols mediate function of NF-kappa B, STAT3 (signal transduction and activators), and COX-2. In addition to its role as an antioxidant and anti-inflammatory agent, tocotrienols also mediate multiple cell cycle pathways. More work needs to be done on animal models and in genetic models of pancreatic cancer to gather more data to eventually consider phase III clinical trial in human subjects.

One of the more fascinating Darwinian puzzles in sexual selection is that of copulatory wounding. For those taxa displaying this behavior, and that does not engage in traumatic insemination, the function of wound infliction during copulation remains unknown. In this study, the intention was to evaluate the purpose of copulatory wounding in Drosophila melanogaster and the consequences it might have for male and female fecundity. The present study measured the size and number of wounds that males from different stocks of Darwinian fitness imposed on females, as well as the copulation duration and the resulting fecundity. The number of wounds and the total wound size showed no apparent correlation to fecundity, but a significant difference in variation of the number of wounds inflicted on females was observed between males from two different lines of fitness. Wounded females were also considerably more common than non-wounded females, as were wounds that were paired compared to non-paired copulatory wounds. The results suggests that wounding might be an adaptive male trait and a copulatory courtship interpretable to females, implying a possible case of cryptic female choice. Since most wounds were paired, this implies that a bilaterally symmetrical organ is causing these wounds. Also, wounding was not as important for fecundity as previously thought. / Ett av de mer fascinerande Darwinistiska pusslen inom sexuell selektion är skadande under kopulationen. För de taxa som uppvisar detta beteende, och inte engagerar sig i traumatisk insemination, så är funktionen av kopulationsskador okänd. I denna studie så var avsikten att utvärdera syftet av kopulationsskador hos Drosophila melanogaster och de konsekvenser beteendet kan ha för det resulterande antalet avkomma. Denna studie uppmätte storleken och antalet skador som hanar av olika Darwinistisk fitness vållade honor, samt kopulationstiden och antalet avkomma. Antalet skador och den totala storleken av skador visade ingen signifikant korrelation till antalet avkommor, däremot uppvisades signifikanta variationsskillnader mellan hanar av två olika fitnessnivåer. Skadade honor visade sig också vara betydligt vanligare än icke skadade honor och parade skador var även vanligare än icke parade skador. Resultaten indikerade att kopulationsskador kan vara en adaptiv egenskap hos hanar och en form av uppvaktning som honorna kan tyda, vilket kan antyda ett möjligt fall av ”kryptiskt honligt val”. Eftersom de flesta skador var parade, så antyder detta att ett bilateralt symmetriskt organ orsakar skadorna. Kopulationsskador visade sig heller inte vara så viktiga som man tidigare antagit.

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第18326号 / 農博第2051号 / 新制||農||1022(附属図書館) / 学位論文||H26||N4833(農学部図書室) / 31184 / 京都大学大学院農学研究科応用生命科学専攻 / (主査)教授 植田 充美, 教授 喜多 恵子, 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

A epistasia tem sido considerada como um componente importante da herança de caracteres quantitativos, estando presente nos componentes de variância e covariância genética. No entanto, por ser desconsiderada na maioria dos modelos genético-estatísticos, seu impacto nos programas de melhoramento ainda é pouco conhecido. Além disso, como os programas de melhoramento de milho, em especial no Brasil, têm realizado seleção sob condições de estresse hídrico, é importante analisar o efeito desse estresse sobre a epistasia. Assim, este trabalho teve como objetivo a análise genética de caracteres quantitativos em milho, incluindo epistasia e epistasia pleiotrópica, em ambientes contrastantes para estresse hídrico. Foi utilizado o delineamento triple test cross modificado com testadores, em que cem progênies F2:3 foram retrocruzadas com suas linhagens genitoras L-08-05F e L-38-05D e sua geração F1. Posteriormente, as 300 progênies de retrocruzamento foram cruzadas com duas linhagens testadoras, L-02-03D e L-04-05F. Os testecrosses foram avaliados em dez ambientes no município de Piracicaba/SP, sendo cinco ambientes sem estresse hídrico e cinco com estresse hídrico, utilizando o delineamento &alpha;-látice em esquema fatorial com duas repetições por ambiente. Os caracteres analisados foram produção de grãos (PG), acamamento e quebramento de plantas (ACQ), prolificidade (PROL), altura da planta (AP) e da espiga (AE), posição relativa da espiga (PRE), florescimento masculino (FM) e feminino (FF) e intervalo entre florescimentos (IF). A presença de epistasia foi detectada para todos os caracteres. No entanto, os grupos de ambientes, sem e com estresse hídrico, e os diferentes testadores afetaram a detecção de epistasia para todos os caracteres, com exceção de PG, PROL e AP em que a epistasia foi detectada em todas as análises de variâncias conjuntas. A interação epistasia x grupos de ambientes foi significativa apenas para os caracteres PG e PROL dos testecrosses provenientes do testador L-02-03D (TC1) e AP e AE dos testecrosses provenientes do testador L-04-05F (TC2), enquanto que, a interação epistasia x testadores foi significativa para todos os caracteres. Os efeitos epistáticos de testecrosses referentes às plantas F2 significativos foram bidirecionais, com exceção do caráter PROL do TC2 no grupo de ambientes com estresse hídrico, em que os efeitos foram unidirecionais e positivos. Além disso, para cada testador e grupo de ambientes foram detectados efeitos epistáticos significativos para um grupo específico de testecrosses, ocorrendo variações na magnitude e/ou sinais dos testecrosses coincidentes. A presença de epistasia pleiotrópica foi detectada entre diversos pares de caracteres e, além disso, foi observada a formação de agrupamentos de caracteres relacionados, indicando que a epistasia pleiotrópica é modular, porém sofre influência do ambiente, presença ou ausência de estresse hídrico, e dos testadores. Portanto, na população analisada, a epistasia é um componente importante tanto da variância genética de caracteres quantitativos como da covariância genética entre caracteres, indicando que não considerá-la nas análises pode resultar em interpretações que não refletem a real complexidade do controle genético destes caracteres. / Epistasis has been considered an important part of quantitative traits inheritance and it is present in the components of genetic variance and covariance. However, its impact on breeding programs is still little-known because it is disregarded in most statistical genetic models. In addition, maize breeding programs, especially in Brazil, have been practicing selection under water stress conditions, so it is important to analyze the effects of these environments on epistasis. In this context, the objective of this study was to perform a genetic analysis of quantitative traits in maize, including epistasis and pleiotropic epistasis, in contrasting environments for water stress. The modified triple test cross design with testers was used, in which 100 F2:3 progenies were backcrossed to their parental inbred lines, L-08-05F and L-38-05D, and their F1 generation. Thereafter, the 300 backcrossed progenies were testcrossed to the inbred lines testers L-02-03D and L-04-05F. The testcrosses were evaluated in Piracicaba, SP, Brazil, in ten environments, which five environments presented water stress and the other five not, in an &alpha;-lattice design on a factorial scheme with two replications by environment. The analyzed traits were grain yield (GY), root and stalk lodging (PL), ear per plant (EPP), plant height (PH), ear height (EH), ear placement (EP), days to anthesis (DA), days to silking (DS) and anthesis-silking interval (ASI). Epistasis was detected for all traits. However, the environmental groups and the different testers affected epistasis detection for all traits, except for GY, EPP and PH, in which epistasis was detected in all joint analysis of variance. The interaction epistasis x environments groups was significant only for GY and EPP of testcrosses from the tester L-02-03D (TC1) and PH and EH of testcrosses from the tester L-04-05F (TC2), whereas the interaction epistasis x testers was significant for all traits. The significant epistatic effects of testcrosses referring to F2 plants were bidirectional, except for EPP of TC2 in the water stress environmental group, where the effects were unidirectional and positives. Besides that, for each tester and environmental group, significant epistatic effects for a specific group of testcrosses were detected, occurring variation in magnitude and/or sign of coincident testcrosses. Pleiotropic epistasis was detected between many pairs of traits and it was observed formation of related traits clusters, suggesting that pleiotropic epistasis is modular, but the modules suffered influence of environments and testers. Therefore, in the population analyzed, epistasis is an important component of genetic variance of quantitative traits and also of genetic covariance between traits, showing that disregarding epistasis in analyses may result in interpretations that do not reflect the real complexity of the genetic control of these traits.