O objetivo do experimento I foi avaliar a interação entre dois ganhos médios diários [GMD - fator nutricional (ganho elevado GA = 0,700 e ganho baixo GB = 0,300 Kg/dia)] e a diferença esperada da progênie (DEP para idade ao primeiro parto - fator genético) na puberdade de novilhas Nelore. Foram utilizadas 58 novilhas desmamadas com 8 meses de idade, filhas de 4 touros [2 precoces (P) e 2 tardios (T)], formando um esquema fatorial 2 x 2, no qual o fator 1 foi o GMD e o fator 2 foi a DEP do touro, constituindo assim quatro tratamentos: Touro precoce com GMD elevado (PGA), touro precoce com GMD baixo (PGB), touro tardio com GMD elevado (TGA), touro tardio com GMD baixo (TGB). Houve efeito dos tratamentos na porcentagem de novilhas púberes durante o experimento (PGA e PGB = 100%; TGA = 83% até 27 meses de idade; TGB = 38% até 3 anos de idade). As novilhas do PGA apresentaram a menor (P = 0,0001) idade na puberdade (18 ± 1 meses). Contudo, a nutrição atrasou a puberdade em cerca de 10 meses nas novilhas filhas de touros precoces e tardios (PGA = 18 ± 1 vs. PGB = 29 ± 1 meses; TGA = 24 ± 1 vs. TGB = 34 ± 1 meses). Assim podemos concluir que a DEP para IPP foi o fator necessário para o adiantamento da idade a puberdade. No entanto, o GMD foi um fator limitante para o aparecimento da puberdade. O objetivo do experimento II foi avaliar o efeito do imprinting metabólico dos 3 aos 7 meses de idade na puberdade em novilhas cruzadas (Angus x Nelore). Os tratamentos foram arranjados em esquema fatorial 2 x 2, no qual o fator 1 foi GMD elevado e médio dos 3 aos 7 meses de idade (fase 1) e o fator 2 foi GMD elevado e médio dos 7 meses de idade até a puberdade (fase 2). Formando os seguintes tratamentos: CAA Novilhas cruzadas submetidas ao GMD elevado nas fases 1 e 2; CAM - GMD elevado na fase 1 e GMD médio na fase 2; CMA- GMD médio na fase1 e GMD elevado na fase 2; CMM GMD médio nas fases 1 e 2. O GMD médio foi atingido pela restrição do consumo de matéria seca (CMS) no GMD médio. As novilhas dos tratamentos CAA, CAM e CMA apresentaram puberdade na mesma idade (cerca de 12 meses). As novilhas cruzadas submetidas ao GMD elevado na fase 2 foram mais pesadas (P = 0,0379) na puberdade do que novilhas submetidas ao baixo GMD. O GMD elevado dos 3 aos 7 meses de idade não impactou na puberdade das novilhas cruzadas. O objetivo e a metodologia do experimento III foram semelhantes ao experimento II, no entanto foram utilizadas novilhas Nelore (N) ao invés de cruzadas e o experimento terminou aos 19 meses de idade. O tratamento NAA (84%) induziu maior (P = 0,0145) proporção de novilhas púberes do que o NMM (23%), mas não houve diferença na taxa de puberdade entre os tratamentos NAM (60%) e NMA (50%). Além disso, o comportamento das curvas de puberdade dos tratamentos NAM e NMA foram semelhantes, sendo assim não houve efeito de imprinting metabólico nas novilhas Nelore. O GMD elevado dos 3 aos 19 meses idade aumentou a porcentagem de novilhas Nelore púberes aos 19 meses de idade / The objective of experiment I was to evaluate the interaction between two average daily gain (ADG nutritional factor (high gain HG = 0,700 and low gain LG = 0,300 kg/day)] and expected progeny differences (EPD to age at first calving; genetic factor) on puberty attainment of Nellore heifers. Fifth eight heifers weaned at 8 months of age and daughters of 4 sires [2 precocious (P) and 2 later (L)]. A factorial design 2 x 2 was used, where the factor 1 was the ADG and the factor 2 was the EPD of sire, resulting in four treatments: Precocious sire with HG (PHG), precocious sire with LG (PLG), later sire with HG (LHG) and later sire with LG (LLG). There was effect of treatment in the percentage of pubertal heifers (PHG and PLG = 100%; LHG = 83% until 27 month of age; LLG = 38% until 3 years old). Heifers from the PHG treatment were the youngest (P = 0,0001) (18 months of age). However, the nutritional factor delayed puberty achievement by approximately 10 months the age at puberty in precocious and later heifers (PHG = 18 ± 1 vs. PLG = 29 ± 1 months; LHG = 24 ± 1 vs. LLG = 34 ± 1 months of age). The EPD to age at first calving was the main factor affecting the age at puberty. However, the ADG was limiting to puberty onset. The objective of experiment II was to evaluate the effect of metabolic imprinting from 3 to 7 months of age on puberty attainment of crossbreed heifers (Angus x Nellore). The treatments were arranged in 2 x 2 factorial design, where the factor 1 was high (H) and medium (M) ADG from 3 to 7 months of age (phase 1) and the factor 2 was high and medium ADG from 7 months of age to puberty onset (phase 2), resulting in the following treatments: CHH crossbreed heifers submitted to high ADG in both phases of experiment; CHM high ADG in phase 1 and medium ADG in phase 2; CMH medium ADG in phase 1 and high ADG in phase 2; CMM Medium ADG in both phases. The medium ADG were targeted by restricting of dry mater intake (DMI) in medium ADG. Crossbreed heifers of treatments CHH, CHM, CMH showed the same age at puberty, about 12 months. Heifers submitted to high ADG on phase 2 were heavier (P = 0,0379) at puberty than heifers submitted to low ADG. The high ADG from 3 to 7 months did not affect puberty onset in crossbreed heifers. The objective and methodology used in experiment III was the same of used in experiment II. However, Nellore heifers (N) were used instead of crossbreed heifers and the experimental period ended at 19 months of age. The NHH treatment (84%) induced higher (P = 0,0145) percentage of pubertal heifers than NMM (23%), but there was not difference in puberty rate between NHM (60%) and NMH (50%) treatments. Furthermore, the comportment of curves of puberty of treatments NHM and NMH were similar, thus there was not effect of metabolic imprinting in Nellore heifers. The high ADG from 3 to 19 months of age increased the percentage of pubertal Nellore heifers at 19 months of age

Epigenética é o ramo da biologia que estuda as características herdáveis não associadas a alterações na seqüência de nucleotídeos do DNA. Um dos principais processos epigenético estudados é a metilação do DNA, a qual está associada a diversos mecanismos de regulação gênica, entre eles o imprinting (marcação) genômico. Esse tipo de regulação caracteriza-se pela expressão parental específica dos loci associados e à metilação diferencial em regiões regulatórias conhecidas como centros de imprinting (ICs). Alterações desse mecanismo estão relacionadas com síndromes de hipo e hipercrescimento em humanos e animais domésticos, desenvolvimento de tumores, doenças associadas com alterações de comportamento e já foram detectadas em indivíduos concebidos por técnicas de reprodução assistida e em células-tronco embrionárias derivadas de diferentes espécies. Essas duas últimas evidenciam que genes marcados são particularmente lábeis ao estresse induzido por manipulação celular in vitro. As possíveis causas dessas epimutações não estão completamente esclarecidas. Os bovinos parecem ser um melhor modelo comparativo no estudo dessas alterações, evitando a utilização de embriões humanos. No entanto, existem poucas seqüências descritas de genes marcados nessa espécie. No presente estudo, duas regiões diferencialmente metiladas (H19DMR e KvDMR1) foram caracterizadas em bovinos em termos de elementos conservados (EC), enriquecimento de elementos repetitivos (ERs) e padrões de metilação. A análise de ECs e ERs foi realizada utilizandose os programas VISTA e RepeatMasker, respectivamente. Os padrões de metilação para ambas as DMRs foram analisados utilizando-se o ensaio de COBRA (do inglês COmbined Bisulfite Restriction Analysis) em DNA de sangue periférico e espermatozóides em amostras de animais concebidos in vivo. Também foi pesquisada a possível ocorrência de perda de imprinting em uma amostra de quatro animais clonados. A análise dos resultados indicou que os padrões de imprinting observados nas DMRs bovinas estudadas são semelhantes aos descritos para regiões homólogas em outras espécies de mamíferos. As características genômicas mostraram uma maior similaridade nas regiões analisadas entre bovinos e humanos do que entre humanos e camundongos. Não foram encontradas diferenças entre o padrão de imprinting de animais gerados naturalmente ou por transferência nuclear. Os resultados desse trabalho poderão auxiliar em futuras pesquisas de genes marcados em bovinos, além de contribuir para o melhoramento na utilização dessa espécie como modelo de comparação para desenvolvimento humano. / Epigenetics is the branch of biology which studies heritable changes in genome function that occur without a change in nucleotide sequence within the DNA. One of the most studied epigenetic process is the DNA methylation, which is associated with several gene regulation mechanisms such as genomic imprinting. This type of regulation is characterized by parental specific gene expression and differential methylation of the associated loci in regulatory sequences named imprinting centers (ICs). Alterations of this mechanism has been related to hypo and hypergrowth syndromes in humans and domestic animals, tumor development, behavior disorders, and it has also been associated with epimutations in individuals conceived by assisted reproduction (AR) techniques and stem cells derived from different species. These last two evidences are indicatives of the imprinted genes lability to in vitro cell manipulation. The possible causes of these epimutations are not completely clear. Cattle seem to be a better comparative model in the study of this epigenetic alterations, and it can avoid the use of human embryos. However, there is few description of imprinted gene sequences this species. In the present work, two differently methylated regions (H19DMR and KvDMR1) were characterized in terms of conserved elements (CEs), enrichment of repetitive elements (RE) and methylation patterns. The CEs and REs analysis was carried out using the VISTA and RepeatMasker softwares, respectively. The methylation patterns for both DMRs were analyzed by COBRA (COmbined Bisulfite Restriction Analysis) assay in DNA from peripherical blood and sperm samples of in vivo conceived animals. It also was investigated the loss of imprinting in samples of four cloned animals. The results indicated that the imprinting patterns of the studied bovine DMRs are similar to the other homologue regions in mammals. The genomic features demonstrated a bigger similarity of the analyzed regions between cattle and humans than between humans and mice. Differences between the imprinting patterns of in vivo conceived versus cloned animals were not found. The results of this work can help future studies of imprinted genes in cattle, and, in addition, can contribute for the improvements of this animal model as a comparative to the human development.

O imprinting genômico, processo regulado epigeneticamente segundo o qual os genes se expressam de acordo com sua origem parental, está envolvido no crescimento e desenvolvimento placentário. Na região 11p15.5 encontram-se vários genes regulados por duas regiões controladoras de imprinting (ICR1 e ICR2), onde se encontram as regiões diferencialmente metiladas H19DMR e KvDMR1. Acredita-se que o padrão de imprinting seja dinamicamente regulado durante o desenvolvimento da placenta. Em humanos, há poucas informações sobre imprinting genômico e desenvolvimento placentário, principalmente para estágios precoces do desenvolvimento devido às dificuldades técnicas de obtenção dessas placentas. A descrição de mosaicismo do padrão de metilação restrito a placenta ou entre a placenta e o feto evidencia um perfil epigenético único deste órgão. A 5-hidroximetilação, a qual não tem um papel de silenciamento gênico, pode ser confundida com a metilação do DNA nas análises moleculares. O objetivo principal do presente estudo foi o de verificar a influência da idade gestacional (IG) no perfil de metilação do DNA das ICRs 1 e 2 em vilosidade coriônica, bem como a existência de mosaicismo do perfil de metilação intra-placentário. Neste trabalho também foi investigada a presença de hidroximetilação na KvDMR1. Foram coletadas amostras de tecido placentário, sendo 25 de vilosidades coriônicas (VC) (15 de 3° trimestre gestacional e 10 do 1° trimestre) e nove de cordão umbilical (UC) de 1° trimestre (pareadas com a VC). Quatro placentas de 3° trimestre foram analisadas em separado para o estudo de mosaicismo. O perfil de metilação do DNA das regiões foi verificado por PCR Específica para a Metilação (MS-PCR), Análise Combinada de Bissulfito e Restrição Enzimática (COBRA) e Método de Digestão Enzimática Sensível à Metilação Associada à PCR em Tempo Real (DESM-RT), além do ensaio para hidroximetilação na KvDMR1. Com os ensaios qualitativos (MS-PCR e COBRA) foi observado um perfil de metilação monoalélico, sendo que na H19DMR foi identificada a presença de CpGs diferentemente metilados. Para a H19DMR foram observadas médias de 0,43 de metilação em VC e 0,31 em UC de 1° trimestre, e de 0,41 em VC de 3° trimestre. Para a KvDMR1, foram encontradas médias de 0,47 em VC e 0,57 em UC de 1° trimestre, e de 0,41 em VC de 3° trimestre. A presença de hidroximetilação na KvDMR1 foi excluída. Não foram observadas diferenças significativas entre as médias das diferentes IGs ou entre tecidos pelos testes t e F para ambas as regiões. Não foi observada correlação positiva no perfil de metilação para H19DMR e KvDMR1 entre os tecidos. Em relação ao mosaicismo, não houve diferenças significativas no perfil de metilação entre os diferentes cotilédones amostrados numa mesma placenta. Os resultados demonstram uma discordância entre tecido embrionário (UC) e extraembrionário (VC). Apesar de não serem observadas alterações significantes nos perfis de metilação da H19DMR e KvDMR1 em diferentes IGs, as informações apresentadas são importantes para as pesquisas sobre a dinâmica do fenômeno de imprinting genômico ao longo da gestação, para os estudos de mosaicismo intraplacentário bem como o perfil epigenético da placenta em relação a outros tecidos. / Genomic imprinting, an epigenetically regulated process by which genes are expressed accordingly to their parental origin, is involved in placental growth and development. In 11p15.5 region, there are many genes regulated by two Imprinting Control Regions (ICR1 and ICR2), in which are found two Differentially Methylated Regions, H19DMR and KvDMR1, respectively. Imprinting patterns seem to be adjusted during placenta development. In humans, there is little information on genomic imprinting and placental development, especially for early stages of development due to technical difficulties in obtaining these placentas. The description of mosaicism in methylation pattern restricted to placenta or between placenta and fetus shows a unique epigenetic profile of this organ. The 5-hidroxymethylation, which has no role in gene silencing, can be confused with DNA methylation in molecular analysis. The main aim of our study was to verify the influence of gestational age (GA) in DNA methylation profile of ICRs 1 and 2 in chorionic villi, as well as the existence of intra-placental methylation profile mosaicism. The presence of hydroximethylation in the KvDMR1 was also investigated. Samples were collected from placentas, 25 from chorionic villi (CV) (15 of the 3rd gestational trimester and 10 of the 1st trimester) and nine from umbilical cord (UC) in 1st trimester (paired with the CV samples). Four 3rd trimester placentas were separately analyzed for mosaicism. DNA methylation profile was verified by Methylation Specific PCR (MS-PCR), and Combined Bisulfite Restriction Analysis (COBRA) and Methylation-Sensitive Enzyme Digestion Method associated with Real-Time PCR (DESM-RT), in addition to hydroximethylation test in the KvDMR1 region. With qualitative assays (MS-PCR and COBRA), it was observed a monoallelic methylation pattern, and, only for the H19DMR, differently methylated CpGs were observed. For the H19DMR, we observed methylation means of 0.43 in CV and 0.31 in UC of 1st trimester, and 0.41 in CV of 3rd trimester. For KvDMR1, we observed means of 0.47 in CV and 0.57 in UC of 1st trimester, and 0.41 in CV of 3rd trimester. No hydroximethylation in the KvDMR1 was observed. There were no significant differences between the means of different GAs or between tissues by F and t tests for both regions. No positive correlation was found on methylation profile for H19DMR and KvDMR1 between tissues. In relation to mosaicism, there were no significant differences in methylation profile between different cotyledons sampled in the same placenta. The results showed a discrepancy between embryonic (UC) and extra-embryonic (CV) tissues. Although it was not observed significant changes in methylation profiles of H19DMR and KvDMR1 in different GAs, the presented results are important to research on dynamics of genomic imprinting phenomenon during pregnancy, studies of intra-placental mosaicism and placenta epigenetic profile in relation to other tissues.

Submitted by Nadir Basilio (nadirsb@uninove.br) on 2017-06-14T20:05:20Z No. of bitstreams: 1 Wilson Agnaldo Horvath.pdf: 1349211 bytes, checksum: ff3284d5e9f032d141582eab3ca0fb81 (MD5) / Made available in DSpace on 2017-06-14T20:05:20Z (GMT). No. of bitstreams: 1 Wilson Agnaldo Horvath.pdf: 1349211 bytes, checksum: ff3284d5e9f032d141582eab3ca0fb81 (MD5) Previous issue date: 2017-04-07 / This research presents an analysis of the trajectory of four professors with a Master´s degree from the Brazilian popular strata that teach at two private universities in the city of São Paulo. The objective of the study was to understand how these subjects managed to re-significate the oppressive elements present in their life histories, especially in childhood and youth, and transcend a cruel and merciless reality, becoming university professors. The methodological procedure used in the interviews was the oral history and narratives were assessed by two main concepts present in the Complex Thought written by Edgar Morin, the Cultural Imprinting and Action Ecology. The results confirm culture as constitutive and intrinsic part of human nature. The individual is marked by the social and cultural context in which he lives. The subjects of research were crossed by modernity, capitalism and its ills, by the slave-like and violent past of Brazilian historiography and patriarchalism. Nonetheless, they have managed, nevertheless, to advance and grow personally and professionally. It is concluded that, although there is a strong conditioning of the cultural elements, over determination can be subverted, since there is always the opening for chance, for the imponderable, for the new, allowing the subjects to resize their lives and transpose situations from disrepute, dishonor and debasement towards success and achievement of goals and purposes. / Esta pesquisa presenta un análisis de la trayectoria de cuatro profesores con título de Maestría, venidos de las capas populares brasileñas que imparten clases en dos universidades privadas en la ciudad de São Paulo. El objetivo del trabajo fue comprender cómo estos sujetos lograron dar nuevo significado a los elementos tiranos presentes en sus historias de vida, principalmente en la niñez y juventud, y transcender una realidad cruel e implacable, volviéndose profesores universitarios. El procedimiento metodológico utilizado en las entrevistas fue la historia oral y las narrativas fueron apreciadas por dos conceptos principales presentes en el Pensamiento Complejo elaborado por Edgar Morin, lo de Imprinting Cultural y de Ecología de la Acción. Los resultados confirman la cultura como parte constituyente e intrínseca a la naturaleza humana. El individuo es aplastado por el contexto social y cultural donde vive. Los sujetos de esa pesquisa fueron atravesados por la modernidad, por el capitalismo y sus males, por el pasado de esclavitud y violento de la historiografía brasileña y por el patriarcalismo. Sin embargo, lograron, a pesar de esto, avanzar y crecer personal y profesionalmente. Se concluye que, aunque haya un fuerte condicionamiento de los elementos culturales, el determinismo histórico puede ser subvertido, pues siempre hay apertura hacia el acaso, el improbable, en dirección de lo novedoso, posibilitando a los sujetos la reorganización de sus vidas y la superación de situaciones de desconfianza, deshonra y humillación rumbo al éxito y a la conquista de metas y propósitos. / Esta pesquisa apresenta uma análise da trajetória de quatro professores com título de Mestre, oriundos das camadas populares brasileira que lecionam em duas universidades particulares da cidade de São Paulo. O objetivo do trabalho foi entender como estes sujeitos conseguiram ressignificar os elementos opressores presentes em suas histórias de vida, principalmente na infância e juventude, e transcender uma realidade cruel e impiedosa, tornando-se professores universitários. O procedimento metodológico utilizado nas entrevistas foi a história oral e as narrativas foram apreciadas por dois conceitos principais presentes no Pensamento Complexo elaborado por Edgar Morin, o de Imprinting Cultural e de Ecologia da Ação. Os resultados confirmam a cultura como parte constitutiva e intrínseca da natureza humana. O indivíduo é marcado pelo contexto social e cultural em que vive. Os sujeitos de pesquisa foram atravessados pela modernidade, pelo capitalismo e suas mazelas, pelo passado escravocrata e violento da historiografia brasileira e pelo patriarcalismo. Todavia, conseguiram, apesar disto, avançar e crescer pessoal e profissionalmente. Conclui-se que, embora haja um forte condicionamento dos elementos culturais, a sobredeterminação pode ser subvertida, pois há sempre a abertura para o acaso, para o imponderável, para o novo, possibilitando aos sujeitos o redimensionamento de suas vidas e a transposição de situações de descrédito, desonra e aviltamento rumo ao sucesso e à conquista de metas e propósitos.

published_or_final_version / abstract / Statistics and Actuarial Science / Doctoral / Doctor of Philosophy

Molecularly imprinted polymers (MIPs) are a novel, promising family of porous materials with potential applications ranging from separations, chemical sensing and catalysis to drug delivery and artificial immunoassays. The unique feature of these materials is their biomimetic molecular recognition functionality. Molecular recognition is the biological phenomenon of specific, selective and strong association between a substrate and a ligand. In man made MIPs this functionality is implemented via templated synthesis protocol. MIPs are synthesized in the presence of additional template molecules which form complexes with functional monomers in the pre‐polymerization mixture. After polymerization, the template is removed, leaving cavities in the structure which are complementary in shape and interaction patterns to the template molecules. These cavities act as mimics of biological receptors and are able to recognize and rebind template molecules. Although the imprinting concept is simple in principle, synthesis of MIPs with precisely controlled characteristics and performance remains a challenging task. Composition, polymerization conditions, template removal process and application conditions all affect the properties of MIPs. The material is affected at different scales, but crucially at the microscopic level, the number, fidelity and accessibility of binding sites are dependent on all the factors mentioned. The full potential of these materials can only be achieved if researchers can control and optimize the properties of MIPs through detailed understanding of adsorption and molecular recognition processes in these materials. The objective of this work is to, using computer simulations and statistical mechanics; develop a fundamental description of MIP formation and function, and to link morphological features of the model materials to their molecular recognition capabilities. In general, molecular simulations employed in this study should allow easier and more efficient exploration of a vast number of factors influencing the behaviour of MIPs. At the heart of the approach developed in this thesis is a computational strategy that imitates all the stages of MIP formation and function. First, the model simulates the pre‐polymerization mixture, allowing the formation of template‐functional monomer complexes. (This stage is implemented via canonical Monte Carlo simulation). Complexes can have different structures, depending on the chemical nature of template and functional monomer; therefore complexes can have a range of association constants. The distribution of template‐functional monomer complexes also translates into a distribution of binding sites of different specificity after template removal. In the second stage of the process, adsorption simulations (grand canonical Monte Carlo) are performed for a variety of model MIPs prepared to assess the role of various processing conditions such as composition, density and binding sites degeneration. This strategy was first applied to a simplified description of MIP species in order to identify the minimal model capable of molecular recognition and thus shed the light on the very nature of this phenomenon. In the developed model, the molecular species are constructed from hard spheres, featuring small interaction sites on their surfaces. The bond between two interaction sites has the strength and topological features of a typical hydrogen bond. The model exhibits molecular recognition, being able to preferentially adsorb template molecules. The associations between template and functional monomers were analyzed and classified to describe the distribution of binding sites and their heterogeneity. Using this model, several experimental trends typically observed in MIP studies could be explained, such as maximum in the selectivity as a function of monomer concentration. Using this model, we were also able to explore hypothetical, alternative protocols for MIP synthesis in order to improve their performance. These include the use of alternative templates and the post‐synthetic surface modifications of MIPs. The general strategy to modelling MIP, employed in this thesis, was then applied to a more detailed description of MIPs with realistic force field potentials for all the species involved. This more elaborate model is simulated with a combination of molecular dynamics (MD) and Monte Carlo techniques. This detailed model provided a wealth of information on various types of complexes observed in the pre‐polymerization mixture. Specifically, it revealed the relative weight of different interactions in the complex and their role in the binding energy of adsorbates. These simulations also provided the comparison of the relative contribution of different types of interactions (van der Waals, Coulombic) involved in a molecular recognition process. We believe the insights gained in this work will contribute to the development of rational MIP design strategies. In the discussion of the results of the thesis we speculate on how these models can be further developed in order to generate quantitative predictions and what type of systems it would be interesting and important to investigate in the future.

Nespas is the non-coding antisense transcript of the imprinted Gnas cluster; it is expressed from the paternal allele and is located on mouse distal chromosome 2. In this thesis new transcripts of >10 kb and 0.8 kb have been identified. The 0.8 kb transcript is a spliced variant that is retained in the nucleus and its 3’ end lies approximately 30 kb from the start site. Transcription from the Nespas promoter does not proceed beyond this point. A collection of previously known splice variants have also been detected and are exported to the cytoplasm. Nespas is expressed in the embryo during the second half of gestation and peaks at 13.5 dpc. Nespas is imprinted in the placenta at 11.5, 15.5 and 17.5 dpc. The Nespastm4Jop allele, to truncate the Nespas transcript 10.5 kb from the start site, has been transmitted through the germline and a breeding colony established. Preliminary analysis shows Nespas has a regulatory function. A second targeting construct to truncate Nespas 12.5 kb from the start site has been designed and assembled to investigate whether the 3’ end of the Nespas transcript that is transcribed upstream of the Nesp promoter is required for Nespas-mediated silencing of Nesp.

Genomic imprinting results in monoallelic parent-of-origin gene expression. Therian mammals show conclusive evidence for imprinting, while the evidence in Aves is conflicting. It’s unclear if Aves have the proteins necessary for establishment and maintenance of imprinting loci. Every examined avian orthologue to mammalian imprinted genes shows biallelic expression providing evidence for a lack of imprinting in Aves. While the known parent-of-origin quantitative trait loci in chicken do not overlap with differentiated methylated regions, further analysis with a larger sample size is required. No transcript in the chicken transcriptome at incubation day 4.5 shows parent-of-origin expression, providing strong evidence for a lack of imprinting at this stage of development. Investigating expression of the chicken transcriptome at additional developmental time points, and the transcriptome of other Aves would provide decisive evidence on the presence or lack of imprinting in Aves. Based on current knowledge, Aves lack imprinting as observed in mammals.

Existing studies have demonstrated that choosing a partner is strongly determined by physical and personal characteristics of a parent of the opposite sex. This influence is affected by the quality of the relationship with the parent during one's childhood. There are many studies focused on choosing a partner in relation to self-similarity. They show that many characteristics are shared between partners. The partner self-similarity has a positive impact on one's satisfaction in and stability of a relationship. Previous research also shows consistency in choosing a partner, meaning there exists a resemblance among ex-partners. The main objective of this thesis is to make a contribution to understanding the mechanisms of choosing a partner based on similarity (colour of eyes and hair) with the parent of the opposite sex and/or based on self-similarity. Next aim is to explore consistency in choosing a partner according to phenotype characteristics (eye and hair colour). The online survey involved overall 1 266 participants, 942 women and 324 men. The survey provided following results. Women choose self-similar partners in terms of eye colour; they also choose their partners depending on the eye colour of their father. Those women with hair colour similar to their partner's show higher relationship...

Imprinted genes are a subset of loci, positioned on autosomes and the X-chromosome, which are expressed monoallelically in a parent-of-origin specific manner. The influence of such genes on the regulation of embryonic growth and postnatal energy homeostasis is well established. The parental conflict hypothesis predicts that, in utero, paternally-expressed genes will promote maternal resource acquisition and thus growth, whereas maternally-expressed genes will oppose this action, restricting resource investment in a single brood in the interests of the lifetime reproductive success of the mother. Grb10 is an imprinted gene which encodes the cytoplasmic adaptor protein Growth factor receptor bound protein 10. In the majority of tissues, Grb10 is expressed from the maternally-derived chromosome. Consistent with conflict theory, transgenic mice inheriting a disrupted Grb10 allele through the maternal line (Grb10Δ2-4m/+) exhibit embryonic overgrowth, although the mechanisms and signalling pathways responsible for this effect are unclear. Grb10Δ2-4m/+ mice also demonstrate enhanced insulin signalling and improved whole body glucose clearance, consistent with the established role of imprinted genes in the regulation of postnatal metabolism. An integrated LacZ gene-trap in the Grb10Δ2-4 allele failed to fully recapitulate endogenous Grb10 expression, notably within the central nervous system. To address this issue, a second transgenic mouse line, Grb10KO, was created. This allele produced strong LacZ reporter expression in the central nervous system, but only when transmitted through the paternal line (Grb10KO+/p), establishing Grb10 as the only known imprinted gene with a reciprocal imprinting profile between the central nervous system and peripheral tissues. Grb10KO+/p mice exhibit a social dominance phenotype, suggesting distinct roles for maternally- and paternally-expressed Grb10, consistent with their respective sites of expression. The current study characterised the Grb10KO allele at the genetic level, and in doing so, revealed a phenotypic difference between Grb10KOm/p and Grb10Δ2-4m/p mice for which a possible explanation was provided. Importantly, with this knowledge, the current study elucidated the genetic and molecular basis for inconsistencies in reporter expression between the two transgenic lines, identifying a novel tissue-specific enhancer element at the locus. In addition to the central nervous system, this enhancer appeared to be active in the mammary epithelium, identifying a novel site of Grb10 expression, which was pregnancy-dependent and specifically from the maternally-inherited chromosome. Characterisation of the functional significance of expression in this tissue revealed that maternally-expressed Grb10 mediates a supply/demand system between lactating mother and suckling pup, acting as a supply promoter and demand suppressor. This role is inconsistent with conflict theory, but suggests the maintenance of the Grb10 imprint in the mammary epithelium might be associated with improved coadaptiveness between mother and offspring. Intriguingly, in utero, Grb10 is both a demand and supply suppressor. When considered together, these findings suggest a wider role for maternally-expressed Grb10 in the homeostatic control of growth and achievement of optimal fitness.