Wie in anderen Zellen sind auch bei Säugerspermien spezifische Lipide und Proteine der Zellmembran aufgrund ihrer heterogenen lateralen Verteilung in speziellen Domänen angereichert, die in unterschiedlichen räumlichen und zeitlichen Dimensionen existieren und der Zelle funktionale Variabilität ermöglichen. Aufgrund der fehlenden aktiven Proteinbiosynthese bietet dies den Spermien eine Möglichkeit, auf unterschiedliche Anforderungen zu reagieren. In der vorliegenden Arbeit wurden daher sogenannte detergenzresistente Membrandomänen (DRMs) aus Eberspermien unterschiedlicher Reifestadien präpariert und untersucht. Dabei stieg bereits in den Dichtegradienten mit zunehmender Reife die Dichte, bei der die opaleszenten Banden auftraten. Eine Analyse dieser mittels 31P-NMR zeigte mit zunehmender Reife eine Anreicherung an Glycerophosphatidylethanolamin und Phosphatidylinositol bei den Glycerophospholipiden, der Gehalt an Sphingomyelin hingegen nahm während der Nebenhodenreifung und auch nach der Ejakulation ab. Diese Veränderungen könnten auf eine Destabilisierung von Membrandomänen hindeuten, um eine Zusammenlagerung zu größeren Domänenclustern zu erleichtern, möglicherweise in Vorbereitung auf Kapazitation und Akrosomenreaktion. Zunächst werden die destabilisierten Membrandomänen jedoch durch die Anlagerung von Seminalplasmaproteinen geschützt, was vermutlich für das verringerte Lipid- zu Proteinverhältnis der DRMs bei Ejakulatspermien sorgt. Aufgrund der generellen Kälteempfindlichkeit von Eberspermien findet ihre Lagerung üblicherweise bei 16°C statt. Dies ist aus mikrobiologischer Sicht nachteilig gegenüber einer kälteren Lagerungstemperatur. Eine Untersuchung der Spermien von 64 Ebern zeigte jedoch bei 10% der Ejakulate eine individuum-spezifische Resistenz gegenüber der Lagerung bei 4°C. Die DRMs der kälteresistenten Spermien hatten einen erhöhten Anteil an langkettigen, mehrfach ungesättigten Fettsäuren, wie 31P-NMR und MALDI-TOF MS Analysen zeigten. / The lateral distribution of lipids and proteins in the plasma membrane is heterogeneous. Therefore specific lipids and proteins in membranes of mammalian spermatozoa are enriched in special domains of varying size and different time scales enabling the cell’s membrane functional variability. Being transcriptional inactive this is especially relevant for spermatozoa in responding to multiple challenges on their way to fertilization. Therefore so called detergent resistant membrane domains (DRMs) from boar spermatozoa of different developmental stages were investigated. Already in the sucrose density gradients differences were visible, so the opalescent bands of more maturated sperm had a higher density. An analysis of these bands by 31P-NMR showed an enrichment of glycerophosphatidylethanolamine and phosphatidylinositol during maturation and a decrease of sphingomyelin during maturation in the epididymis and even after ejaculation. This suggests destabilization of DRMs and hence of putative membrane domains. This could enable clustering to bigger membrane domain platforms in preparation for capacitation and acrosome reaction. First, however, seminal fluid proteins cover the spermatozoa protecting the membrane with the destabilized membrane domains. This could have led to the detected decrease of the lipid to protein ratio in DRMs of ejaculated sperm. Boar spermatozoa are sensitive to storage at cold temperatures and are therefore usually stored at 16°C, which is especially disadvantageous with regard to growing of bacteria. A screening of sperm from 64 boars showed a ratio of 10% individuals with cold resistant sperm which could be stored at 4°C without quality loss. The DRMs of cold resistant sperm had a higher proportion of longchained, polyunsaturated fatty acids, as shown by analysis with 31P-NMR und MALDI-TOF MS.

Nicht-codierende RNAs sind ein bedeutender Bestandteil genregulatorischer Prozesse. Ihre Fehlregulierung wird mit zellulärer Dysfunktion und der Entstehung von Krankheiten in Zusammenhang gebracht. Ziel dieser Arbeit war die Entwicklung verschiedener Testsysteme zur Untersuchung nicht codierender RNAs mit dem Schwerpunkt microRNA (miRNA), precursor miRNA (pre-miRNA) und circular RNA (circRNA). Für eine Zyklisierung und Funktionalisierung von circRNA mittels Cu-katalysierter Click-Chemie zur Identifizierung zellulärer Interaktionspartner und zugehöriger Wirkmechanismen wurden die Termini linearer RNA-Template modifiziert. Mit Hilfe enzymatischer Techniken wie Transkription und Ligation konnte in vitro die Inkorporation Azid- und Alkin-funktionalisierter Nukleotid-Bausteine am 5‘- und 3‘-Terminus gezeigt werden. Zur Untersuchung der miRNA-Reifung in cellulo wurde die pre-miRNA-134 unter Verwendung chemo-enzymatischer Methoden mit einem Fluorophor/Quencher-Paar an den Termini ausgestattet. Durch intrazelluläre Reifung der gelabelten pre-miRNA mit einhergehender Fluoreszenzfreisetzung sollte die Visualisierung und damit die Lokalisierung des miRNA-Reifungsortes innerhalb von Neuronen realisiert werden. Zudem gelang die Entwicklung eines auf branched rolling-circle amplification (BRCA) basierenden Argonaute2(Ago2)-vermittelten Spaltungsassays. Ein Enzymkomplex aus rekombinantem, humanem Ago2 und der miRNA miR 122, genannt minimal RISC, wurde dabei zur Substrat-Spaltung eingesetzt. Zur Etablierung des BRCA-basierenden Ago2-vermittelten Spaltungsassays als Screening-Tool für die Identifizierung potentieller Inhibitoren der mRNA-Spaltung wurden exemplarisch sechs Testsubstanzen aus der Gruppe der Aminoglykoside untersucht. Der BRCA-basierende Ago2-vermittelte Spaltungsassay stellte eine einfache und zuverlässige Detektionsmethode dar, der die Untersuchung einer größeren Probenzahl mit geringem Aufwand und ohne Verwendung von fluorogen gelabeltem Substrat ermöglichte. / Non-coding RNAs are an important factor in gene regulation in which their deregulation is associated with cellular dysfunction and disease. Here, the development of different test systems for the investigation of non-coding RNAs, namely microRNA (miRNA), precursor miRNA (pre-miRNA), and circular RNA (circRNA), was on focus. In order to circularize and functionalize circRNA with the purpose of identifying cellular interaction partners and possible mechanisms of action, 5‘- and 3‘-terminal modifications were added to a linear RNA template. This was accomplished by using azide- and alkyne-functionalized nucleotides which were incorporated by enzymatic approaches like transcription and ligation to be followed by Cu-catalyzed click chemistry for circularization. For investigating miRNA maturation in neuronal cells, pre-miR-134 was modified by chemo-enzymatic approach with fluorophore and quencher at its 5‘ and 3‘ ends, respectively. Intracellular maturation of labeled pre-miRNA would produce a fluorescent signal upon cleavage, thus enabling visualization and localization of miRNA maturation in neuronal cells. Furthermore, the development of Ago2-mediated mRNA cleavage assay based on branched rolling-circle amplification (BRCA) was accomplished. A complex of recombinant human Ago2 and miRNA miR-122, called minimal RISC, was used for substrate cleavage. To establish this assay as adequate screening method for identifying potential inhibitors of mRNA cleavage, a group of six aminoglycosides was tested. The BRCA-based Ago2-mediated cleavage assay showed to be a simple and reliable detection method and screening tool for small molecule binders with little effort and without fluorescent labeling of substrate.

Das Proteasom spielt eine zentrale Rolle beim Proteinabbau und der Antigen-Generierung für die adaptive Immunantwort. Vertebraten exprimieren zwei Typen des proteolytischen 20S-Kernkomplexes: das konstitutive c20S (mit den aktiven Untereinheiten beta 1, 2, 5) und das Immunoproteasom i20S (mit den Immunountereinheiten LMP2, MECL-1, LMP7). Die i20S-Expression wird durch Interferon_gamma (IFNg) induziert, was die Antigen-Präsentation auf MHC Klasse I und die Immunantwort gegen infizierte bzw. maligne entartete Zellen durch cytotoxische T-Zellen steigert. Proteasomen werden über komplexe, bisher unvollständig verstandene Biogenese-Prozesse formiert. Die initialen Schritte der humanen 20S-Formation wurden in dieser Arbeit untersucht und eine Methode zur Isolation früher Assemblierungsintermediate (EPIs) etabliert. Die 20S-Biogenese bedarf der Assistenz von Hilfsfaktoren wie dem Proteasom-Maturierungsprotein POMP. Diese Komponente von Precursorkomplexen stellt das erste Substrat gereifter c20S dar. In dieser Arbeit konnte erstmalig gezeigt werden, dass POMP ebenfalls die i20S-Formation vermittelt und sich die Biogenese von c20S und i20S hinsichtlich der Maturierungskinetik unterscheidet. POMP wird durch IFNg induziert und interagiert mit der Immunountereinheit LMP7. Dieses molekulare Zusammenspiel bewirkt eine schnellere Maturierung von i20S- im Vergleich zu c20S- Precursorkomplexen, wodurch POMP einem schnelleren Abbau unterliegt. Die forcierte i20S-Biogenese ist eine intrinsische Eigenschaft und unabhängig von weiteren, IFNg-induzierten Faktoren. Nur die LMP7_E2-Variante vermittelt die schnelle Degradation von POMP, während das nicht funktionelle LMP7_E1 mit einer anderen Prosequenz nicht in i20S-Vorläufer inkorporiert wird. Somit führt die alleinige LMP7_E1-Expression in IFNg-stimulierten Carcinom-Zellen zu einer i20S-Defizienz, was eine mögliche Immunevasions-Strategie darstellt. Weiterhin besitzen beide 20S-Typen unterschiedliche Halbwertszeiten: i20S sind, unabhängig von weiteren IFNg-induzierten Proteinen, signifikant instabiler als c20S. Somit werden i20S sowohl schneller formiert als auch zügiger wieder abgebaut als c20S, womit sie typische Eigenschaften cytokin-regulierter Proteine aufweisen. Die i20S-Formation ist also eine transiente Antwort und stellt ein effizientes Instrument zur schnellen Reaktion auf immunologische Herausforderungen wie z.B. eine Infektion dar. Nach einer wirksamen Immunantwort erlaubt die geringere i20S-Stabilität eine schnelle Rückkehr zur standardmäßigen c20S-Expression. / The proteasome plays a crucial role in protein degradation and antigen generation for the adaptive immune response. Vertebrates express two types of the proteolytic 20S core complex: the constitutive proteasome c20S (with the active subunits beta 1, 2 and 5) and the immunoproteasome i20S (with the immunosubunits LMP2, MECL-1 and LMP7). Interferon_gamma (IFNg) induces the i20S expression, that supports a more efficient MHC class I antigen presentation and an effective immune response against infected or malignant cells by cytotoxic T-cells. Proteasomes are formed by a complex and not well understood biogenesis program. The initial steps in the human 20S formation have been analyzed in this thesis and a method for the isolation of ´early proteasome assembly intermediates´ (EPIs) has been established. The 20S biogenesis requires the assistance of accessory factors like the proteasome maturation protein POMP. This component of precursor complexes becomes the first substrate of the matured c20S. The described experiments demonstrate for the first time that POMP mediates the i20S formation and that biogenesis of c20S and i20S differ in their maturation kinetics. POMP is induced by IFNg and interacts with the immunosubunit LMP7. This molecular interplay provokes a faster maturation of i20S compared to c20S precursor complexes, whereby POMP becomes subject to a faster degradation. The accelerated i20S biogenesis is an intrinsic characteristic and independent of additional IFNg-induced factors. Exclusively the LMP7_E2 variant causes the rapid degradation of POMP, whereas the non-functional LMP7_E1 bearing another prosequence is not incorporated into i20S precursor complexes. Thus, LMP7_E1 expression in IFNg-stimulated carcinoma cells leads to a i20S deficiency pointing out a possible immune evasion strategy. In addition, both 20S types display different half-life values: i20S are, independent of other IFNg-induced proteins, significantly less stable than c20S. Thus, i20S are not only faster assembled, but also more quickly decomposed compared to c20S, showing typical attributes of proteins regulated by cytokines. Consequently, i20S formation is a transient response and represents an efficient instrument for a rapid adjustment to varying immunological challenges like an infection. Once the immune response has been effective, the lower stability of i20S permits an expeditious return to the standard c20S expression.

Die gegenwärtige Vorstellung von Genexpression in Plastiden leitet sich von der Analyse weniger, individueller Gene ab und ist deshalb noch relativ lückenhaft. In dieser Arbeit sollte daher differenzierende RNA Sequenzierung- eine neue Methode, die zwischen prozessierten und Primärtranskripten unterscheiden kann, verwendet werden, um ein vollständigeres Bild des Transkriptionsprozesses und der RNA Prozessierung von Hordeum vulgare L. (Gerste) Chloroplasten zu erhalten. Plastidengene in höheren Pflanzen können sowohl von einer plastidenkodierten, bakterienähnlichen RNA-Polymerase (PEP), als auch von einer kernkodierten, phagenähnlichen RNA-Polymerase (NEP), die beide unterschiedliche Promotoren erkennen, abgelesen werden. In dieser Arbeit wurde die Verteilung von Transkriptionsstartstellen innerhalb des Plastidengenoms von grünen (reife Chloroplasten; Transkriptionsaktivität von PEP und NEP) und weißen Plastiden (Transkriptionsaktivität von NEP) der Gerstenmutantenlinie albostrians analysiert. Dies führte zu neuen Erkenntnissen bezüglich polymerasenspezifischer Genexpression in Plastiden. Auf Grundlage neuerer Arbeiten wird angenommen, daß nicht kodierende RNAs (ncRNAs) in Chloroplasten vorkommen. Die bisher verwendeten Methoden waren jedoch nicht geeignet, ncRNAs als Primärtranskripte zu identifizieren, die zumindest in Prokaryoten die häufigste Klasse von ncRNAs darstellen. In dieser Arbeit konnte durch dRNA-seq gezeigt werden, daß auch in Plastiden zahlreiche ncRNAs als Primärtranskripte generiert werden. Die wichtigsten Schritte im Prozess der mRNA Reifung in Plastiden sind 5´und 3´ Endformation und intercistronische Prozessierung. Vor Kurzem wurde gezeigt, daß ein PPR (Pentatricopeptide repeat) Protein zur Bildung der Ende von einigen prozessierten Plastiden mRNAs beiträgt, indem es als Hindernis für Exonukleasen wirkt. Mit dieser Arbeit konnte gezeigt werden, daß dies ein genereller Mechanismus zur Bildung prozessierter mRNA-Enden in Chloroplasten ist. / The current view on plastid gene expression is mainly based on the analysis of a few individual genes, and thus it is lacking in comprehensiveness. Here, a novel differential RNA-seq approach, designed to discriminate between primary and processed transcripts, was used to obtain a deeper insight into the plastid transcription and RNA maturation of mature barley (Hordeum vulgare L.) chloroplasts. Transcription in plastids of higher plants is dependent on two different transcription machineries, a plastid-encoded bacterial-type RNA polymerase (PEP) and a nuclear-encoded phage-type RNA polymerase (NEP), which recognize distinct types of promoters. This study provided a thorough investigation into the distribution of transcription start sites within the plastid genome of green (mature chloroplasts; transcription by both PEP and NEP) and white (PEP-deficient plastids; transcription by NEP) plastids of the barley line albostrians. This analysis led to new insights on polymerase specific gene expression in plastids. Recent studies have suggested that non-coding RNAs (ncRNAs) are common in chloroplasts. However, they did not directly detect ncRNAs generated via transcription, the so far most abundant class of known regulatory ncRNAs in bacteria. Here, dRNA-seq analysis of the transcriptome of barley chloroplasts demonstrated the existence of numerous ncRNA generated via transcription of free-standing genes. Major events in plastid mRNA maturation include 5’ and 3’ processed end formation and intercistronic processing. Recently, a PPR (pentatricopeptide repeat) protein was shown to participate in the generation of several plastid mRNA processed ends by serving as a barrier to exonucleases. This study provided evidence for the global impact of this mechanism on processed termini formation in chloroplasts.

Pétrole et gaz proviennent de la décomposition de la matière organique dans la croûte terrestre. En s’enfouissant, les résidus organiques se décomposent en un solide poreux et carboné, appelé kérogène et en un fluide composé d’hydrocarbures et de petites molécules telles que de l’eau. Le processus de formation du kérogène n’est pas totalement élucidé et une modélisation aiderait à une meilleure compréhension à la fois de sa structure et de sa composition et serait utile à l’industrie pétrolière.Dans le présent travail, nous adoptons une approche thermodynamique ayant pour but, à l’aide de simulations numériques, de d’étudier la décomposition de précurseurs de kérogène d’un type donné –ici le type III- dans les conditions d’un réservoir géologique. La méthode dite de Replica Exchange Molecular Dynamics (REMD) est appliquée pour étudier la décomposition de cristaux de cellulose et de lignine. Le potentiel d’interaction ReaxFF et le code LAMMPS sont utilisés. La REMD est une façon de surmonter de larges barrières d’énergie libre, en améliorant l’échantillonnage de configurations d’une dynamique moléculaire conventionnelle à température constante, en utilisant des états générés à températures supérieures.En fin de simulation, les systèmes ont atteint un état d’équilibre entre deux phases : une phase riche en carbone, composée d’amas de macromolécules, que nous appelons « solide » et d’une phase riche en oxygène et en hydrogène, composée de petites molécules, que nous dénommons « fluide ». L’évolution des parties solides de nos systèmes coïncide avec celle d’échantillons naturels de kérogènes de type III. / In deep underground, organic residues decompose into a carbonaceous porous solid, called kerogen and a fluid usually composed of hydrocarbons and other small molecules such as water, carbon monoxide. The formation process of the kerogen remains poorly understood. Modeling its geological maturation could widen the understanding of both structure and composition of kerogen, and could be useful to oil and gas industry.In this work we adopt a purely thermodynamic approach in which we aim, through molecular simulations, at determining the thermodynamic equilibrium corresponding to the decomposition of given organic precursors of a specific type of kerogen –namely type III- under reservoir conditions. Starting from cellulose and lignin crystal structures we use replica exchange molecular dynamics (REMD) simulations, using the reactive force field ReaxFF and the open-source code LAMMPS. The REMD method is a way ofovercoming large free energy barriers, by enhancing the configurational sampling of a conventional constant temperature MD using states from higher temperatures.At the end of the simulations, we have reached for both systems, a stage where they can clearly be cast into two phases: a carbon-rich phase made of large molecular clusters that we call here the "solid" phase, and a oxygen and hydrogen rich phase made of small molecules that we call "fluid" phase.The evolution of solid parts for both systems and the natural evolution of a type III kerogen clearly match. Evolution of our systems follows the one of natural samples, as well as the one of a type III kerogen submitted to an experimental confined pyrolysis.

German text / Gutes Altern gelingt selbst bei den zumeist günstigen äusseren Voraussetzungen in der Schweiz nicht allen Menschen. Die Frage stellt sich, unter welchen Bedingungen sowohl echtes Wohlbefinden im Alltag als auch Lebenssinn und -perspektive erreicht und erhalten werden können. Gelingt dies bekennenden Christen besser als nicht religiösen Menschen? Im Hinblick auf diese Fragestellung untersuchte die vorliegende Arbeit die bisherige gerontologische Forschung hauptsächlich in der Schweiz. Es wurde versucht, die Begriffe Wohlbefinden, Lebenssinn in Verbindung mit Religionsgerontologie anhand einzelner Exponenten zu definieren und das Zusammenspiel der Faktoren aufzuzeigen. In einer qualitativen empirischen Studie wurden gemäss der Grounded Theory zwölf Personen zwischen 50 und 80 Jahren (drittes Alter) mit zehn verschiedenen religiös/spirituellen Hintergründen mit Hilfe eines halbstrukturierten Leitfadens interviewt. Die Hauptthemen waren Wohlbefinden, Lebenssinn, Glaube/Spiritualität und Sterben/Tod. Der Zusammenhang zwischen alltäglichem Wohlbefinden und Lebenssinn bzw. -perspektive erwies sich als stark. Menschen mit einer persönlichen Gottesbeziehung schienen über mehr Ressourcen zu verfügen, nicht nur, um die eigene Endlichkeit zu akzeptieren oder schwierige Ereignisse im Leben zu meistern, sondern auch, um ihren letzten Lebenssinn in Gott zu erkennen, also ausserhalb von sich selbst. So waren sie besser befähigt, in ihrem Lebenslauf Erreichtes wieder abzugeben. Dieser Lebensstil benötigt Einübung, er kann jedoch auch gezielt gefördert werden. Aufgrund der Erkenntnisse konnten eine Reihe von Aufgaben formuliert werden, die den älteren Menschen Anweisung geben, wie sie diesen Lebensstil konkret in ihrem Alltag umsetzen könnten. Zudem scheint christliche Gemeinde ein geeigneter Rahmen darzustellen, wenn sie für das dritte Alter interdisziplinäre Lernangebote aus den Bereichen Medizin, Psychologie, Soziologie und Theologie wie auch die Möglichkeit persönlicher Seelsorge bereitstellt. Der ältere Mensch wird dadurch frei, sich generativ für andere einzusetzen und für sie da zu sein, innerhalb der christlichen Gemeinde oder auch ausserhalb als Teil der Gesellschaft. Somit wird dem neutestamentlichen Gebot der Nächstenliebe nachgekommen, Solidarität anderen gegenüber wird gelebt. Stärkeres Sinnempfinden und höheres Wohlbefinden sind geschenkte Nebenprodukte, die zu einem erfüllten Leben beitragen. / In Switzerland ageing is not necessarily unproblematic, even in the most favorable conditions. The question to answer is on what conditions in everday life a high level of wellbeing as well as meaning and perspective of life can be attained and preserved. Do professing Christians succeed better than non-religious people? The present study examined the results of current gerontological resarch in this respect, primarily in Switzerland. It attempted to define the terms of wellbeing, meaning of life in connection with religious gerontology with specific criteria and to show up the interaction of their several components. In a qualitative empirical study, according to the Grounded Theory, twelve persons between fifty and eighty years of age with ten different religious/spiritual backgrounds were interviewed with the aid of a semistructured interview guide. The main topics were wellbeing, meaning of life, faith/spirituality and dying/death. The relationship between everyday wellbeing and meaning of life and life perspective proved to be strong. People with a personal relationship with God seemed to have more resources to accept not only their own mortality or to cope with negative events in life, but to recognize their ultimate meaning of life to be in God, which is outside themselves. In consequence they were better able to let go any of their own achievements. This lifestyle requires practise and promotion. On account of the results a number of tasks were formulated to guide the older people in their daily life in practicing this lifestyle. The Christian community seems to be a suitable framework to provide interdisciplinary learning opportunities for the third age in the fields of medicine, psychology, sociology and theology as well as the opportunity of personal counseling. In this way, the never-ending process of maturation of individual elderly people should be encouraged. They are free, according to their talents, to get themselves involved with other people, to have time for them, that is within the Christian community or outside, in society. Thus, the New Testament commandment to love one another can be complied with, and solidarity with others will truly be fact. A stronger sense of meaning and a higher grade of wellbeing will be welcome by-products, which will contribute to a fulfilled life. / Practical Theology / D. Th. (Practical Theology)

INTRODUÇÃO: A puberdade reuni as mudanças físicas da adolescência como o crescimento pondoestatural e a evolução da maturação sexual. Um marco importante da puberdade no sexo feminino é a menarca. Para sua ocorrência é necessária a interação de múltiplos fatores, destacando-se por sua importância a condição nutricional, da qual faz parte um teor de gordura corporal suficiente para deflagrar não só a primeira menstruação, como também a manutenção dos ciclos menstruais regulares. OBJETIVOS: Este estudo avaliou a associação entre indicadores de estado nutricional (peso, estatura, circunferência braquial e índice de massa corpórea) e de maturidade (idade e maturação sexual) para ocorrência da menarca e avaliou a percepção da imagem corporal em relação a presença ou não deste evento. MÉTODOS: Trata-se de um estudo transversal, retrospectivo, observacional, de 98 adolescentes femininas, entre 10 e 19 anos, do subdistrito do Butantã, São Paulo, Brasil, que comparou dois grupos: as que apresentaram menarca até três meses antes da pesquisa (27) e as que ainda não haviam menstruado (71). Os dados são provenientes de um banco de dados, coletados na década de 80, mediante questionários padrão, em pesquisa domiciliar e fizeram parte de estudos anteriores. A variável resposta foi a menarca e as explanatórias foram peso, estatura, circunferência braquial e índice de massa corpórea, idade, desenvolvimento mamário, impressão da estatura e impressão da condição nutricional. O processamento e análise dos dados foi realizado no Departamento Materno- Infantil da Faculdade de Saúde Pública da Universidade de São Paulo, digitados em planilha Excel® e analisados pelo programa EpiInfo versão 3.5.3 CDC e WHO Anthro versão 3.2.2 OMS. As variáveis foram descritas através de suas distribuições, frequências, médias, desvios-padrão e medianas. Testes paramétricos e não paramétricos foram usados na dependência da distribuição normal ou não das variáveis. Os indicadores que apresentaram significância estatística nos modelos univariados e apresentaram baixa correlação entre si foram testados em modelo de regressão logística para controle das variáveis de confusão. A análise da percepção da autoimagem foi realizada pela correlação entre impressão da estatura e condição nutricional com classificação do referencial gráfico. RESULTADOS: Peso, estatura, circunferência braquial, índice de massa corpórea sempre foram superiores para adolescentes com menarca em relação às sem menarca (diferença estatisticamente significativa). A primeira menstruação ocorreu mais frequentemente no estágio de desenvolvimento M4 de Tanner, com média idade de 13,26 anos. Todas as variáveis estudadas se mostraram associadas à ocorrência da menarca nas análises univariadas. No modelo multivariado, o índice de massa corpórea se revelou o principal desencadeante da menarca. Mostraram-se também associados à primeira menstruação: o desenvolvimento mamário e a idade. Foi observada uma correlação positiva entre a percepção da imagem corporal e a classificação gráfica nas meninas com e sem menarca. CONCLUSÃO: O estado nutricional, traduzido pelo índice de massa corpórea, destacou-se como principal fator desencadeante para a ocorrência da menarca, mesmo frente aos indicadores neuroendócrinos e a idade, confirmando a relevância da força do ambiente sobre a herança. Não houve diferença da percepção da imagem corporal para meninas com e sem menarca / INTRODUCTION: Puberty gathers the physical changes of adolescence such as the growth spurt and evolution of sexual maturation. An important biologic mark of puberty in females is menarche. For its occurrence an interaction of multiple factors are required, mainly the nutritional status whose body fat, depending on its amount, might trigger not only the first period, but also the maintenance of regular menstrual cycles. OBJECTIVES: This study evaluated the association between indicators of nutritional status (weight, height, arm circumference and body mass index) and maturity (age and sexual maturity) for the occurrence of menarche and it assessed the perception of body image compared with the presence or absence of this event. METHODS: It is a cross-sectional, retrospective, observational study of 98 female adolescents between 10 and 19 years old, in the sub-district of Butantan, Sao Paulo, Brazil, which compared two groups: those that had menarche up to three months before the survey (27) and those that had not menstruated (71). The data come from a database, collected in 80, through standard questionnaires on household survey. These data have already been part of previous studies. The variable response was the menarche, and the explanatory variables were weight, height, arm circumference, body mass index, age and breast development, height perception and nutritional status perception. The processing and analysis of data were performed in the Department of Maternal and Child School of Public Health, University of São Paulo, typed in Excel ® spreadsheet and analyzed by using EpiInfo version 3.5.3 CDC and WHO Anthro version 3.2.2 WHO. The variables were described according to their distributions, frequencies, means, standard deviations and medians. Parametric and non-parametric tests were used depending on the normal distribution of the variables or not. The indicators that showed statistical significance in the univariate models and showed low correlation between them were tested in a logistic regression model for the control of the confusion variables. The analysis of the perception of selfimage was performed by correlation between height and nutritional status perception with reference graph. RESULTS: Weight, height, arm circumference, body mass index were always higher for the adolescents who had menarche than for those who did not (statistically significant difference). The first period occurred more often in adolescents in stage of breast development M4, at the mean age of 13.26. In univariate analysis, all variables were associated with the occurrence of menarche. In the final model of logistic regression the body mass index was the main trigger of menarche. The other variables associated with the menarche were: breast development and age. It was a positive correlation between the perception of body image and graphical classification in girls with and without menarche. CONCLUSIONS: The nutritional status, reflected by body mass index, stood out as the main triggering factor for the occurrence of menarche, even against neuroendocrine indicators and age, confirming the relevance of the strength of the environment on the inheritance. There was no difference in the perception of body image for girls with and without menarche

Chez les plantes à fleurs, l’ovaire est l’organe reproducteur femelle et il interagit de façon importante avec les gamètes mâles durant la croissance, le guidage, la réception et la rupture du tube pollinique ainsi que la fusion des gamètes. Le processus débute lorsque de nombreux gènes de l’ovule sont activés à longue distance lors de la réception du pollen sur le stigmate. Afin d’explorer les signaux provenant de l’ovule ayant un impact important sur les interactions pollen–pistil, particulièrement les molécules sécrétées impliquées dans la signalisation espècespécifique, l’expression génique des ovules sous forme d’ARNm ainsi et la sécrétion protéique ont été étudiées chez Solanum chacoense, une espèce diploïde de pomme de terre sauvage. S. chacoense a subi beaucoup d’hybridation interspécifique avec d’autres espèces sympathiques de solanacées, facilitant ainsi grandement l’étude des interactions pollen–ovule de façon espècespécifique ainsi que leur évolution. Dans ce projet, des ovules provenant de trois conditions différentes ont été comparés: des ovules matures de type sauvage, des ovules légèrement immatures, récoltés deux jours avant l’anthèse et des ovules provenant du mutant frk1 pour lesquels le sac embryonnaire est absent. Un séquençage d’ARN à haut débit a d’abord été effectué sur les ovules de type sauvage de S. chacoense afin de générer un assemblage de référence comprenant 33852 séquences codantes. D’autres séquençages ont été effectués sur les trois conditions d’ovules et sur les feuilles afin de faire une analyse d’expression différentielle des gènes. En comparaison avec les ovules de type sauvage, 818 gènes sont réprimés dans les ovules du mutant frk1. Un sous-groupe de 284 gènes, étaient également sous-exprimés dans les ovules légèrement immatures, suggérant un rôle spécifique dans les stades tardifs de la maturation du sac embryonnaire (stade de développent FG6 à FG7) ainsi que du guidage du tube pollinique, puisque ni les ovules du mutant frk1 ni ceux légèrement immatures ne sont capables d’attirer les tubes polliniques lors d’essais de croissance semi in vivo. De plus, 21% de ces gènes sont des peptides riches en cystéines (CRPs). En utilisant un transcriptome assemblé de novo provenant de deux proches parents de S. chacoense, S. gandarillasii et S. tarijense, une analyse d’orthologie a été effectuée sur ces CRPs, révélant une grande variabilité et une évolution rapide chez les solanacées. De nouveaux motifs de cystéine uniques à cette famille ont également été découverts. En comparant avec des études similaires chez Arabidopsis, le sac embryonnaire de S. chacoense montre un transcriptome fortement divergent, particulièrement en en ce qui a trait à la catégorisation fonctionnelle des gènes et de la similarité entre les gènes orthologues. De plus,même si la glycosylation n’est pas requise lors du guidage mycropylaire du tube pollinique chez Arabidopsis, Torenia ou le maïs, des extraits d’ovules glycosylés de S. chacoense sont capables d’augmenter la capacité de guidage de 18%. Cette étude est donc la première à montrer une corrélation entre glycosylation et le guidage du tube pollinique par l’ovule. En complément à l’approche transcriptomique, une approche protéomique portant sur les protéine sécrétées par l’ovule (le secrétome) a été utilisée afin d’identifier des protéines impliquées dans l’interaction entre ovule et tube pollinique. Des exsudats d’ovules matures (capables d’attirer le tube pollinique) et d’ovules immatures (incapables d’attirer le tube pollinique) ont été récoltés en utilisant une nouvelle méthode d’extraction par gravité permettant de réduire efficacement les contaminants cytosoliques à moins de 1% de l’échantillon. Un total de 305 protéines sécrétées par les ovules (OSPs) ont été identifiées par spectrométrie de masse, parmi lesquelles 58% étaient spécifiques aux ovules lorsque comparées avec des données de protéines sécrétées par des tissus végétatifs. De plus, la sécrétion de 128 OSPs est augmentée dans les ovules matures par rapport aux ovules immatures. Ces 128 protéines sont donc considérées en tant que candidates potentiellement impliquées dans la maturation tardive de l’ovule et dans le guidage du tube pollinique. Cette étude a également montré que la maturation du sac embryonnaire du stade FG6 au stade FG7 influence le niveau de sécrétion de 44% du sécrétome total de l’ovule. De façon surprenante, la grande majorité (83%) de ces protéines n’est pas régulée au niveau de l’ARN, soulignant ainsi l’importance de cette approche dans l’étude du guidage du tube pollinique comme complément essentiel aux études transcriptomiques. Parmi tous les signaux sécrétés par l’ovule et reliés au guidage, obtenus à partir des approches transcriptomiques et protéomiques décrites ci-haut, nous avons spécifiquement évalué l’implication des CRPs dans le guidage du tube pollinique par l’ovule chez S. chacoense, vu l’implication de ce type de protéine dans les interactions pollen-pistil et le guidage du tube pollinique chez d’autres espèces. Au total, 28 CRPs étaient présentes dans les ovules capables d’attirer le tube pollinique tout en étant absentes dans les ovules incapables de l’attirer, et ce, soit au niveau de l’ARNm et/ou au niveau du sécrétome. De celles-ci, 17 CRPs ont été exprimées dans un système bactérien et purifiées en quantité suffisante pour tester le guidage. Alors que des exsudats d’ovules ont été utilisés avec succès pour attirer par chimiotactisme le tube pollinique, les candidats exprimés dans les bactéries n’ont quant à eux pas été capables d’attirer les tubes polliniques. Comme l’utilisation de systèmes d’expression hétérologue eucaryote peut permettre un meilleur repliement et une plus grande activité des protéines, les candidats restants seront de nouveau exprimés, cette fois dans un système de levure ainsi que dans un système végétal pour produire les peptides sécrétés. Ceux-ci seront ensuite utilisés lors d’essais fonctionnels pour évaluer leur capacité à guider les tubes polliniques et ainsi isoler les attractants chimiques responsable du guidage du tube pollinique chez les solanacées comme S. chacoense. / In flowering plants, the ovary is the female reproductive organ that interacts extensively with the male gametophyte during pollen tube (PT) growth, guidance, reception, discharge and gamete fusion. The process begins when numerous ovule-expressed genes are activated when pollen lands on the stigma. To explore the ovular signals that have a great impact on successful pollen–pistil interactions, especially the secreted molecules that mediate species-specific signalling events, ovule mRNA expression and protein secretion profiles were studied in Solanum chacoense, a wild diploid potato species. Solanum chacoense has undergone extensive interspecific hybridization with sympatric solanaceous species that greatly facilitates the study of species-specific pollen–ovule interactions and evolution. In this project, three ovule conditions were studied: wild-type mature ovules, slightly immature ovules at two days before anthesis (2DBA), and frk1 mutant ovules that lack an embryo sac (ES). RNA-seq was performed on S. chacoense ovules to provide a scaffold assembly comprising 33852 CDS-containing sequences, then to provide read counts for differential gene expression analyses on three ovule conditions as well as on leaf. Compared to wild-type ovules, 818 genes were downregulated in frk1 ovules. A subset of 284 genes was concurrently under-expressed in 2DBA ovules, suggestive of their specific involvement in late stages of ES maturation (female gametophyte (FG), FG6 to FG7 developmental stage), as well as in PT guidance processes, as neither frk1 nor 2DBA ovules attract semi in vivo-grown PTs. Of these 284, 21% encoded cysteine-rich peptides (CRPs). Using de novo assembled ovule transcriptomes of two close relatives, S. gandarillasii and S. tarijense, an orthology survey was conducted on these CRPs, revealing their highly polymorphic nature among species and rapid evolution. Interestingly, novel cysteine motifs unique to this family were also uncovered. As compared to parallel studies in Arabidopsis, S. chacoense was found to possess a highly divergent ES transcriptome, in terms of both functional categories and individual ortholog similarities. Although glycosylation is not required for micropylar guidance cues to attract PTs in Arabidopsis, Torenia or maize, glycosylated ovule extracts from S. chacoense showed enhanced PT guidance competency by 18%. This is the first time a positive regulation between glycosylation and ovular PT guidance has been observed. As a complement to the transcriptomic approach, a proteomic approach using secreted proteins from the ovule (secretome) was employed to identify proteins involved in pollen–pistil interactions. Ovule exudates were collected from mature ovules (PT attracting) and immature ovules at 2DBA (PT nonattracting), using a novel tissue free-gravity extraction method (tf-GEM), which efficiently reduced the cytosolic contamination to less than 1%. Through mass spectrometry analyses, a total of 305 ovule-secreted proteins (OSPs) were identified, of which 58% were considered ovule-specific when compared to secretome studies conducted in other plant tissues. The secretion of 128 OSPs was upregulated in mature ovules vs. immature ovules. These OSPs were considered as candidate proteins involved in late ovule maturation and PT guidance. This study demonstrated that the ES maturation from FG6 to FG7 stages influenced the secretion status of 44% of ovule secretome. Surprisingly, the majority (83%) of these proteins were not regulated at the RNA level, vindicating this novel approach in the study of PT guidance as a robust complement to transcriptomic studies. Among all identified guidance-related ovular signals from the transcriptomic and proteomic approaches described above, we focused on the evaluation of the involvement of CRPs in ovular PT guidance of S. chacoense, due to the implication of various CRPs in pollen–pistil interactions and, especially, in PT guidance. A total of 28 CRPs were present in PT attracting ovules while being low or absent in nonattracting ovules, at the mRNA and/or protein secretion levels. Of these, 17 CRPs were expressed in bacteria and purified in sufficient amount for PT guidance assays. However, while ovule exudates were shown to induce PT chemotropism in the bead assay, refolded candidates did not show guidance competency. Since the use of eukaryotic protein expression systems might lead to better refolding and higher protein activity, the remaining candidates will be expressed in both yeast and plant-based expression systems and tested for their ability to attract PTs in a semi in-vivo assay, in order to lead us toward the isolation of PT guidance chemoattractants in solanaceous species like S. chacoense.

INTRODUÇÃO: A puberdade reuni as mudanças físicas da adolescência como o crescimento pondoestatural e a evolução da maturação sexual. Um marco importante da puberdade no sexo feminino é a menarca. Para sua ocorrência é necessária a interação de múltiplos fatores, destacando-se por sua importância a condição nutricional, da qual faz parte um teor de gordura corporal suficiente para deflagrar não só a primeira menstruação, como também a manutenção dos ciclos menstruais regulares. OBJETIVOS: Este estudo avaliou a associação entre indicadores de estado nutricional (peso, estatura, circunferência braquial e índice de massa corpórea) e de maturidade (idade e maturação sexual) para ocorrência da menarca e avaliou a percepção da imagem corporal em relação a presença ou não deste evento. MÉTODOS: Trata-se de um estudo transversal, retrospectivo, observacional, de 98 adolescentes femininas, entre 10 e 19 anos, do subdistrito do Butantã, São Paulo, Brasil, que comparou dois grupos: as que apresentaram menarca até três meses antes da pesquisa (27) e as que ainda não haviam menstruado (71). Os dados são provenientes de um banco de dados, coletados na década de 80, mediante questionários padrão, em pesquisa domiciliar e fizeram parte de estudos anteriores. A variável resposta foi a menarca e as explanatórias foram peso, estatura, circunferência braquial e índice de massa corpórea, idade, desenvolvimento mamário, impressão da estatura e impressão da condição nutricional. O processamento e análise dos dados foi realizado no Departamento Materno- Infantil da Faculdade de Saúde Pública da Universidade de São Paulo, digitados em planilha Excel® e analisados pelo programa EpiInfo versão 3.5.3 CDC e WHO Anthro versão 3.2.2 OMS. As variáveis foram descritas através de suas distribuições, frequências, médias, desvios-padrão e medianas. Testes paramétricos e não paramétricos foram usados na dependência da distribuição normal ou não das variáveis. Os indicadores que apresentaram significância estatística nos modelos univariados e apresentaram baixa correlação entre si foram testados em modelo de regressão logística para controle das variáveis de confusão. A análise da percepção da autoimagem foi realizada pela correlação entre impressão da estatura e condição nutricional com classificação do referencial gráfico. RESULTADOS: Peso, estatura, circunferência braquial, índice de massa corpórea sempre foram superiores para adolescentes com menarca em relação às sem menarca (diferença estatisticamente significativa). A primeira menstruação ocorreu mais frequentemente no estágio de desenvolvimento M4 de Tanner, com média idade de 13,26 anos. Todas as variáveis estudadas se mostraram associadas à ocorrência da menarca nas análises univariadas. No modelo multivariado, o índice de massa corpórea se revelou o principal desencadeante da menarca. Mostraram-se também associados à primeira menstruação: o desenvolvimento mamário e a idade. Foi observada uma correlação positiva entre a percepção da imagem corporal e a classificação gráfica nas meninas com e sem menarca. CONCLUSÃO: O estado nutricional, traduzido pelo índice de massa corpórea, destacou-se como principal fator desencadeante para a ocorrência da menarca, mesmo frente aos indicadores neuroendócrinos e a idade, confirmando a relevância da força do ambiente sobre a herança. Não houve diferença da percepção da imagem corporal para meninas com e sem menarca / INTRODUCTION: Puberty gathers the physical changes of adolescence such as the growth spurt and evolution of sexual maturation. An important biologic mark of puberty in females is menarche. For its occurrence an interaction of multiple factors are required, mainly the nutritional status whose body fat, depending on its amount, might trigger not only the first period, but also the maintenance of regular menstrual cycles. OBJECTIVES: This study evaluated the association between indicators of nutritional status (weight, height, arm circumference and body mass index) and maturity (age and sexual maturity) for the occurrence of menarche and it assessed the perception of body image compared with the presence or absence of this event. METHODS: It is a cross-sectional, retrospective, observational study of 98 female adolescents between 10 and 19 years old, in the sub-district of Butantan, Sao Paulo, Brazil, which compared two groups: those that had menarche up to three months before the survey (27) and those that had not menstruated (71). The data come from a database, collected in 80, through standard questionnaires on household survey. These data have already been part of previous studies. The variable response was the menarche, and the explanatory variables were weight, height, arm circumference, body mass index, age and breast development, height perception and nutritional status perception. The processing and analysis of data were performed in the Department of Maternal and Child School of Public Health, University of São Paulo, typed in Excel ® spreadsheet and analyzed by using EpiInfo version 3.5.3 CDC and WHO Anthro version 3.2.2 WHO. The variables were described according to their distributions, frequencies, means, standard deviations and medians. Parametric and non-parametric tests were used depending on the normal distribution of the variables or not. The indicators that showed statistical significance in the univariate models and showed low correlation between them were tested in a logistic regression model for the control of the confusion variables. The analysis of the perception of selfimage was performed by correlation between height and nutritional status perception with reference graph. RESULTS: Weight, height, arm circumference, body mass index were always higher for the adolescents who had menarche than for those who did not (statistically significant difference). The first period occurred more often in adolescents in stage of breast development M4, at the mean age of 13.26. In univariate analysis, all variables were associated with the occurrence of menarche. In the final model of logistic regression the body mass index was the main trigger of menarche. The other variables associated with the menarche were: breast development and age. It was a positive correlation between the perception of body image and graphical classification in girls with and without menarche. CONCLUSIONS: The nutritional status, reflected by body mass index, stood out as the main triggering factor for the occurrence of menarche, even against neuroendocrine indicators and age, confirming the relevance of the strength of the environment on the inheritance. There was no difference in the perception of body image for girls with and without menarche

This thesis examined body dissatisfaction and body change behaviors among adolescent girls and boys from a biopsychosocial framework. The contribution of biological, psychological and sociocultural factors were examined in relation to body dissatisfaction, weight loss, weight gain and increased muscle tone behaviors among early adolescent girls and boys. In particular, pubertal maturation, body mass index (BMI), perception of body shape and size and psychological factors, such as depression, anxiety, ineffectiveness, self-esteem and perfectionism, were examined as possible factors that may precipitate or maintain body dissatisfaction and engagement in body change strategies. The sociocultural factors evaluated were the quality of family and peer relationships, as well as the influence of family and peers in predicting the adoption of specific body change strategies. The specific mechanisms by which these influences were transmitted were also examined. These included perceived discussion, encouragement and modelling of various body change strategies, as well as perceived teasing about body shape and size. A number of separate cross-sectional and longitudinal studies were conducted to examine the above relationships and identify the factors that contribute to weight loss, weight gain and increased muscle tone behaviors in adolescents. Study 1 examined the psychometric properties and principal components structure of the Bulimia Test Revised (BULIT-R; Thelen, Farmer, Wonderlich, & Smith, 1991) to assess its applicability to adolescent samples. Study 2 investigated the nature of body dissatisfaction and weight loss behaviors among 603 adolescents (306 girls and 297 boys) using a standardised questionnaire. This preliminary study was conducted to ascertain whether variables previously found to be relevant to adolescent girls, could also be related to the development of body dissatisfaction and weight loss behaviors among adolescent boys. Studies 3 and 4 described the development and validation of a body modification scale that measured weight loss, weight gain and increased muscle tone behaviors. Studies 5 and 6 were designed to modify an Excessive Exercise Scale developed by Long, Smith, Midgley, and Cassidy (1993) into a shorter form, and validate this scale with an adolescent sample. Study 7 investigated the factors that contribute to weight loss, weight gain and increased muscle among adolescent girls and boys both cross-sectionally and longitudinally (over one year). Structural equation modelling was used to examine associations among self-reported body dissatisfaction, body change strategies and a range of biological, psychological and sociocultural variables both cross-sectionally and longitudinally. Overall, the results suggested that both girls and boys experience body dissatisfaction and engage in a number of different body change strategies in order to achieve an ideal size. A number of gender similarities and differences were identified in the expression of body dissatisfaction and the adoption of body change strategies for both girls and boys. Girls were more likely than boys to report body dissatisfaction and engage in weight loss behaviors, while boys were more likely than girls to engage in weight gain and increased muscle tone behaviors. Generally, the same factors were found to contribute to weight loss, and more specifically, bulimic symptomatology, ad weight gain in both adolescent girls and boys. While a combination of biological, psychological and sociocultural factors contributed to bulimic symptomatology, only biological and psychological factors were found to contribute to weight gain in adolescents. The most notable gender differences were found in the model of increased muscle tone. Sociocultural and biological factors contributed to increased muscle tone behaviors in girls, while sociocultural and psychological factors were implicated in these behaviors in adolescent boys. With the exception of the model of increased muscle tone for boys, body dissatisfaction was a consistent factor in the adoption of body change behaviors. Consistent with previous investigations, the present thesis provides empirical support for the need to examine the etiology and maintenance of such concerns and behaviors from a multifaceted perspective.