Thèses sur le sujet « Physiology and psycology analyses »

Conflicts in published research raised a series of questions on the precision of the measurements used to differentiate between vertical jumps performed with and without pre-stretch. Procedures outlined by previous researchers (eg. Komi and Bosco, 1978a; Bedi et al., 1987) were repeated and extended. Force plate data were collected for a series of squat, counter movement and rebound jumps. Individual subjects responded differently and no evidence could be found for an optimal rebound dropping height. Modal analysis of the force plate highlighted the need for improving its mounting. A frame was designed to raise the resonant frequency of the plate and static and dynamic calibrations revealed point of force application errors. 16 mm cinefilm was selected in preference to video for the subsequent inverse dynamics analysis of rebound jumping. French physiologist, Marey, observed that people appeared to jump higher following a rebound than a counter movement. A 'Marey' style jumping exercise was used to examine different takeoff and landing strategies. Variations in kinematic data filtering, body segment inertia parameters and quasi-static analysis techniques on the resultant moment moments were investigated. No differences in maximum jump height were found between counter movement and rebound jump takeoffs. This apparent contraction to the findings in previous research was accounted for by variations in the subjects' stretch heights at takeoff. A general proximal to distal sequencing of muscle moment peaking was observed in both takeoff actions, but moments peaked later in rebound takeoffs than when following counter movements. Larger peak moments occurred during landings preceding coming to rest than during the landing phase of the rebound jump. Quasi-statically determined muscle moments about the ankles and knees matched closely with the inverse dynamics values, but joint and overall support moments were consistently over estimated. Conflicts with selected published research findings were shown to arise from a lack of measurement precision. Takeoff velocities were greater following counter movements, but were insufficient to differentiate between jumping techniques. Rebounding was found to increase leg extension. Improvements in automatic measurement procedures combined with an enhanced understanding of musculo-skeletal modelling were seen as a way of improving future knowledge of neuromuscular coordination and power production in jumping.

P-proteins are structurally distinct proteins present in the sieve element-companion cell complexes of phloem tissue. Genomic clones encoding the two major P-proteins, the phloem filament protein (PP1) and the phloem lectin (PP2), were isolated and characterized. To understand mechanisms that control phloem-specific expression of these two genes, approximately 1 kb of the 5' flanking region from PP1 and PP2 genomic clones were fused with the GUS reporter gene and introduced into tobacco plants using Agrobacterium tumefaciens-mediated gene transfer. For both promoters, histochemical staining detected GUS activity specifically in the phloem tissue that was most easily detected in stems followed by midrib, secondary veins, roots, and leaf lamina of transgenic tobacco plants. GUS activity directed by the PP2 promoter was approximately 23 times greater than GUS activity directed by the PPI promoter. A nested set of 5' deletions between nucleotides -1014 and +32 were constructed to localize cis-elements that specify the patterns of PP2 gene expression in the phloem. Deletions within this region revealed that nucleotides -228 to +32 relative to the transcription initiation site contained sufficient information to direct phloem-specific gene expression, while positive regulators of promoter activity appeared to be located upstream of nucleotide -621. Mutation of a conserved 13-bp sequence, TTAAAAGAAGATA, found in the minimal PP2 promoter did not affect reporter gene expression. Sucrose responsive elements were identified in the PP2 promoter that could contribute to increased promoter activity in response to sugar. Finally we initiated studies to construct a phloem-specific promoter that could be induced by wound released compounds such as ethylene. Although not conclusive, our results suggest that it is possible to enhance phloem-specific expression in response to ethylene.

The growing knowledge of cell biology and evidence for the role of β-catenin signalling network in homeostasis and carcinogenesis encourages further investigation into the regulatory network of nuclear β-catenin signalling complex. While the role of canonical Wnt signalling in the development of both normal tissue and malignant tumours is well documented, the molecular basis of these functionally distinct nuclear transcriptional programs is poorly understood. Many proteins are associated with cytoplasmic β-catenin for regulation of Wnt/β-catenin pathway activities. However, in the nucleus, the LEF/TCF family of transcription factors, which have DNA binding properties, remains the sole focus as unambiguous partners of β-catenin. In addition to LEF/TCFs, interaction of β-catenin with several other transcriptional co-activators and/or co-repressors is required for gene regulation. This regulation may also be influenced by alterations of β-catenin protein such phosphorylation of β-catenin which dramatically alters its trafficking and function. Delineation and functional description of nuclear cofactors that interact with unphosphorylated (i.e. nuclear) β-catenin will further unravel the mechanisms of β-catenin-mediated nuclear transcription, and may also identify whether distinct patterns of transcriptional cofactors are engaged in normal development versus tumour progression. Human FLYWCH1, a conserved member of the mammalian C2H2 zinc finger proteins, was identified as one of the phosphorylation-independent Catenin- Interacting-Proteins (CIPs) in a recent screening performed in Dr Nateri's laboratory using a modified yeast-2-hybrid RRS. FLYWCH1 is a previously uncharacterized protein with no known function in mammals. Herein, we have shown that; i) in human cells, FLYWCH1 physically interacts with β-catenin and represses its transcriptional activity, ii) it regulates the expression of some if not all downstream target genes, iii) in the intestine, Flywch1 marks the crypt-based columnar-cells (CBCs), which function as stem cells, but does not mark any of the differentiated cells in normal villi, iv) FLYWCH1 expression is strongly down-regulated in CRC cell lines but its expression is up-regulated and restricted to a subpopulation of tumour cells in both human and ApcMin/+ mouse. Our data also showed that v) FLYWCH1 controls CRC cell morphology and inhibits cell migration through up-regulation of E-cadherin which may not be related to ZEB2-mediated EMT. Collectively, our data suggest that FLYWCH1 is a novel nuclear β-catenin interacting protein that inhibits cell motility by antagonizing the activity of Wnt/β-catenin signalling pathway. As changes in cell motility is a key step toward invasion and metastasis, FLYWCH1, therefore, may function as a metastasis-suppressing factor which could potentially be of use in the therapeutic field of colon cancer to control cancer spread.

The mouse pelage hair consists of three types of hair coined primary (guard), secondary (awls and auchenes) and tertiary (zigzag) hair. They display distinct morphologies and are induced consecutively during hair morphogenesis. Previously two identified regulatory mouse mutants, Yellow submarine (Ysb) and Light coat and circling (Lcc) which the chromosomal rearrangements have disrupted the cis-acting regulatory elements of Sox2; resulting in the loss of Sox2 expression in the inner ear. The mutants displayed lighter hair coat color due to a reduction in the proportion of secondary hair and increased proportion of tertiary hair. Sox18 null mutants display darker coat colour and reduced proportion of zigzag hair. To dissect the underlying mechanisms of the phenotypes in hair type specification in 〖Sox2〗^Ysb and 〖Sox2 〗^Lcc mutants and the role of Sox2 and Sox18 in regulating the process; the expression of Sox2 in the hair follicle and the change in the density of hair types in mutants were analyzed. I have identified the expression pattern of Sox2 in the dermal papilla (DP) of the hair follicle and verified its down-regulation in 〖Sox2〗^Ysband 〖Sox2 〗^Lcc mutants. The DP at the base of hair follicle is the signaling center for the regulation of hair development. Sox2 is specifically expressed in the DP of primary and secondary but not in tertiary hair while Sox18 is expressed in the DP of all hair types. Analysis of Sox2 mutants showed that the number of secondary hair was normal at induction but was reduced and accompanied by an increase in tertiary hair in adult mice. The number of tertiary hair was reduced in Sox18 null mutants. To gain insight into the molecular basis of hair type specification and potential targets of Sox2 in the regulation, gene expression profile in DP cells of 〖Sox2 〗^(EGFP/+)and 〖Sox2 〗^(EGFP/Ysb) mice was examined; the data suggests that genes in the Wnt and BMP signalling pathway were down-regulated in Sox2 mutants; while Runx3 and Corin may act downstream of Sox2 in regulating hair type specification and pigmentation. Hair follicles enter cycles of growth and regression throughout life during the hair cycle. Sox2 was only expressed in the growth phase while Sox18 was persistently expressed throughout the hair cycle. I further asked if Sox2 and Sox18 regulate post-natal hair development by analysing the expression pattern of Sox2 and Sox18 in wildtype mice and mutants throughout the hair cycle and the progression of hair growth in the mutants. The growth phase of the first hair cycle was extended in Sox2 mutants while the hair cycle in Sox18 null mutants was normal. Cell proliferation was compromised during hair regeneration leading to a delay in hair regeneration in Sox2 mutants. Sox2 and Sox18 showed overlapping expression in the DP and both regulate hair type specification. To test if Sox2 and Sox18 synergistically regulate hair development, the 〖Sox2〗^(Ysb/Ysb);〖Sox18〗^(-/-) mutants have been generated. Hair morphogenesis and differentiation were impaired; while the number of tertiary hair was increased with reduced number of secondary hair, which phenocopied that of Sox2 mutants. In conclusion, the results suggest that Sox2 and Sox18 functions synergistically on the regulation of hair growth and differentiation.
published_or_final_version
Biochemistry
Doctoral
Doctor of Philosophy

Geobacillus thermoglucosidasius has been identified as an organism capable of producing bioethanol from lignocellulosic biomass based on its ability to ferment both hexose and pentose sugars. Engineering of the wild-type strain DL33 (wt) has produced a single knock out strain DL44 (Δldh) and a double knock out strain DL66 (ΔldhΔpfl↑pdh), both of which have increased capacity for bioethanol production. The nutritional requirements of the strains under anaerobic conditions are yet to be fully understood. In this study, a systems approach to understanding the metabolism of the wild-type and engineered strains has been taken in order to further understand the changes in metabolism resulting from the mutations introduced. For the first time 13C-metabolic flux analysis has been applied to the comparative study of the wild-type and engineered strains using global isotopomer balancing. This has revealed flux through the anaplerotic reactions has reversed from being in the direction of pyruvate/phosphoenolpyruvate in the wild-type, to being in the direction of oxaloacetate/malate in the engineered strains. Alterations in TCA cycle flux between the strains were also seen. Furthermore alanine was found to be produced as a fermentation product in each strain. Analysis of the genome sequence has revealed an unusual oxidative branch of the pentose phosphate pathway, missing 6-phosphogluconolactonase but with genes encoding the rest of the pathway still present, suggesting that flux through this pathway may still proceed, dependent on the themolability of glucono-1,5-lactone-6-phosphate. It has been found that RNA extracted from G. thermoglucosidasius is prone to rapid degradation which may affect the outcome of analysis of the transcriptome by RNA-seq. Nonetheless, it has been possible to apply RNA-seq to the wild-type organism grown aerobically and use this to identify transcripts for the major pathways of central carbon metabolism and the most highly expressed transcripts of the culture.

Vasculogenesis is a complex process by which endothelial stem and progenitor cells undergo de novo vessel formation. Quantitative assessment of vasculogenesis is a central readout of endothelial progenitor cell functionality. However, current assays lack kinetic measurements. To address this issue, new approaches were developed to quantitatively assess in vitro endothelial colony forming cell (ECFC) network formation in real time. Eight parameters of network structure were quantified using novel Kinetic Analysis of Vasculogenesis (KAV) software. KAV assessment of structure complexity identified two phases of network formation. This observation guided the development of additional vasculogenic readouts, including a tissue cytometry approach to quantify the frequency and localization of dividing ECFCs within cell networks. Additionally, FIJI TrackMate was used to quantify ECFC displacement and speed at the single cell level during network formation. These novel approaches were then applied to determine how intrauterine exposure to maternal type 2 diabetes mellitus (T2DM) impairs fetal ECFC vasculogenesis, and whether increased Transgelin 1 (TAGLN) expression in ECFCs from pregnancies complicated by gestational diabetes (GDM) was sufficient to impair vasculogenesis. Fetal ECFCs exposed to maternal T2DM formed fewer initial network structures, which were not stable over time. Correlation analyses identified that ECFC samples with greater division in branches formed fewer closed network structures and that reductions in ECFC movement decreased structural connectivity. To identify specific cellular mechanisms and signaling pathways altered in ECFCs following intrauterine GDM exposure, these new techniques were also applied in TAGLN expression studies. Similarly, ECFCs from GDM pregnancies and ECFCs overexpressing TAGLN exhibited impaired vasculogenesis and decreased migration. Both ECFCs from GDM pregnancies as well as ECFCs over-expressing TAGLN exhibited increased phosphorylation of myosin light chain. Reduction of myosin light chain phosphorylation via Rho kinase inhibition increased ECFC migration; therefore, increased TAGLN was sufficient to impair ECFC vasculogenic function. Overall, identification of these novel phenotypes provides evidence for the molecular mechanisms contributing to aberrant ECFC vasculogenesis. Determining how intrauterine exposure to maternal T2DM and GDM alters fetal ECFC function will enable greater understanding of the chronic vascular pathologies observed in children from pregnancies complicated by diabetes mellitus.

The electrophysiological and morphological properties of small networks of mammalian neurons were investigated with mouse spinal cord monolayer cultures of 2 mm diameter grown on multimicroelectrode plates (MMEPs). Such cultures were viewed microscopically and their activity simultaneously recorded from 2 of any 36 fixed recording sites. The specific aims achieved were: development of techniques for production of functional MMEPs and maintenance of mini-cultures, characterization of the spontaneous activity of mini-cultures, application of inhibitory and disinhibitory agents, development of staining methods for cultured neurons and initial light microscopic analysis with correlation of electrophysiological and morphological characteristics.

Pubertal development is an outcome of genes\' interaction producing molecular signals that rule physiological mechanisms. Understanding the genetic changes surrounding the peripubertal period in Nellore heifers could unveil certain differences in age at puberty because they reach sexual maturity much later than European heifers. The adipose tissue regulates energy metabolism and is an endocrine organ with active participation in reproductive processes .The present study aimed at understanding the functions of the adipose tissue around pubertal time in Nellore heifers. Our strategy was to search for differences in gene expression between prepubertal and pubertal animals. We quantified gene expression in the adipose tissue for Nellore heifers who reached puberty according to either the early or late pattern. This research was done on 30 Nellore heifers monitored weekly from 230 kg bodyweight until the onset of puberty through gynaecological ultrasound examination. The onset of puberty was defined by the presence of corpus luteum in the ovary and blood progesterone > 1 ng/ml. Adipose tissue was collected through subcutaneous biopsy; the first biopsy was done when heifers reached 230 kilos bodyweight and subsequently once a month until they entered puberty. Biopsies were collected of each heifer that reached puberty alongside its half-sister who had not reached puberty. We analysed samples of six early pubertal heifers and their six late pubertal sisters at two different stages: at the time of ovulation and approximately 50 days before ovulation. The RNA was extracted from the biopsies\' tissue and gene expression quantified through RNA sequencing. There were nine genes differentially expressed in pubertal heifers that have been found previously correlated with reproductive traits and/or processes in mammals on other studies. These genes were APOD, CYP17A1, DNMT3B, AKR1C4, TENM1/ODZ3, HPSE, SMPD3, FAM134B and CYP26B1, already found expressed in adipocytes, hypothalamus, ovaries and uterus, corroborating previous knowledge of a link among energy metabolism, reproductive organs and neural influence. By measuring the genetic changes and correlating them with known physiological mechanisms on the nervous system and reproductive organs during puberty, we detected genes expressed here that are related to modifications in the organism as a whole. Therefore the adipose tissue may be regarded as an instrument to indicate what is going on during reproductive processes. The results presented here may aid in a better understanding of genetic changes happening during peripubertal period.
O desenvolvimento púbere é o resultado da interação de genes que produzem sinais moleculares regulando os mecanismos fisiológicos. A compreensão das mudanças genéticas que permeiam o período peripuberal em novilhas Nelore (Bos indicus) poderia desvendar o porquê das diferenças em idade a puberdade, pois elas atingem a maturidade sexual mais tarde que o gado europeu (Bos taurus). O tecido adiposo regula o metabolismo de energia e é um órgão endócrino que tem participação ativa em processos reprodutivos. O presente estudo teve como objetivo entender as funções do tecido adiposo na época peripubertal em novilhas Nelore. Nossa estratégia foi procurar diferenças em expressão gênica entre animais pré-púberes e púberes. Nós quantificamos a expressão gênica no tecido adiposo de novilhas Nelores precoces ou tardias. A pesquisa foi feita em 30 novilhas Nelore, monitoradas semanalmente a partir do momento em que atingiram 230 kg de peso até a manifestação da puberdade por exame de ultrassom ginecológico. O momento da puberdade foi definido pela presença de corpo lúteo no ovário e concentração sanguínea de progesterona maior que > 1 ng/ml. O tecido adiposo foi coletado por biópsia subcutânea; a primeira biópsia foi feita quando as novilhas alcançaram 230 quilos de peso e subsequentemente uma vez por mês ate que elas entrassem em puberdade. As biópsias foram coletadas de cada novilha que atingiu a puberdade e de sua meia-irmã que não tinha atingido a puberdade. Nós analisamos amostras de seis novilhas púberes precoces e de seis novilhas púberes tardias em dois estágios diferentes: no momento da ovulação e aproximadamente 50 dias antes da ovulação. O RNA foi extraído do tecido das biopsias e a expressão genica quantificada através de sequenciamento de RNA. Encontramos nove genes diferencialmente expressos em novilhas púberes que foram previamente relacionados com características e/ou processos reprodutivos de mamíferos em outros estudos. Esses genes foram APOD, CYP17A1, DNMT3B, AKR1C4, TENM1/ODZ3, HPSE, SMPD3, FAM134B e CYP26B1, já encontrados expressos em adipócitos, hipotálamo, ovários e útero, comprovando conhecimento prévio da conexão existente entre metabolismo de energia, órgãos reprodutivos e influencia neural. Ao medir as mudanças genéticas e correlacioná-las com mecanismos fisiológicos conhecidos em sistema nervoso e órgãos reprodutivos durante a puberdade, nós conseguimos identificar genes expressos em adiposo que são relacionados a modificações no organismo como um todo. Sendo assim, o tecido adiposo pode ser considerado um instrumento para indicar o que está acontecendo durante os processos reprodutivos. .Os resultados apresentados aqui podem ajudar na melhor compreensão de mudanças genéticas durante o período peripuberal.

Borrelia burgdorferi, the causative agent of Lyme disease, exists in a defined enzootic cycle involving Ixodes scapularis ticks and various vertebrates. Humans can serve as an accidental host, if a tick colonized with B. burgdorferi happens to feed on a human. B. burgdorferi are also accidental pathogens: they do not make toxins, or destroy host tissue by other mechanisms. They merely transmit between vector and host to survive. In order to do this, they must effectively sense their current environment, and appropriately alter cellular processes. Understanding the regulatory mechanisms of how B. burgdorferi manages to do this has been a focus of the Stevenson lab for many years. Previous work identified SpoVG as a DNA-binding protein. Although a homologue of this protein had been implicated to serve a regulatory role in other bacteria, the Stevenson lab was the first to demonstrate a function for the protein, both for B. burgdorferi and two other bacteria. Studies contained in this body of work aim to provide insight into regulation of SpoVG by B. burgdorferi as well the impact that it has on gene regulation. By using genetic mutants, we determined that SpoVG is regulated at the levels of transcription and translation in culture by growth rate, temperature, and other regulatory factors. Additionally, we provide evidence that SpoVG regulates its own expression. Numerous genes are under control of SpoVG. Biochemical analyses revealed that SpoVG specifically interacts with DNAs and RNAs associated with genes found to be under its regulatory control. Finally, we provide evidence for SpoVG acting in concert with other known regulatory factors such as other DNA-binding proteins and the cyclic di-nucleotide second messengers cyclic-di-GMP and cyclic-di-AMP. All together, these studies provide insight into how B. burgdorferi broadly regulates cellular processes during different stages of the enzootic cycle. We hypothesize that SpoVG does this through globally manipulating the three-dimensional structure of the bacterial chromosome, and that exactly how SpoVG acts at any given point will be dependent on the other regulatory factors that are also present in the cell.

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This dissertation analyses the psychology concept on Nietzsche?s philosophy and its relation to the typology and hierarchy. First of all, it was verified that the meaning of the Nietzsche psychological research is based on the concept of power will which is considered by the German philosopher as an interpretation of the world and not a description. The power will has as mean characteristic to affirm the effectiveness as a come of strengths in fight for more power and depose of truth the metaphysics oppositions. These strengths, or impulses, are understood as power quanta that differ each other and they relate with all of other quanta in order to intensify. Therefore, the power intensity is what differ the strengths. This way, we understand the fight of the strengths as a domination process, which produce impulses complex or hierarchies. The development of these hierarchical configurations of strengths comes from the dynamic establishment of commander and commanded, that is, from dominion relations that are expressed in ways of life, human types. Types, however, are not understood as essential manifestations of a being, but as strength relation?s symptoms and their hierarchical configurations. These symptoms are the object of the psychology proposed by Nietzsche, and not the human soul or conscience. The human types are analyzed by the German philosopher through two great and focused perspectives, one of ascendancy (powerful and heavily hierarchizated impulses) and another of life?s decadence (weak and anarchic impulses). From this general aspect of Nietzsche typology, is verified the importance of the types, considering that for the appearance of a type is necessary the existence of its differentiated, because there is a relation of domination between them. Thus, the job of the nietzschean psychologist is, with grounding in the interpretation of the world as power will, to evaluate the various ways of life and make them hierarchical. Therefore, Nietzsche psychological evaluation corresponds to differ and make hierarchical the humans types, understood as symptoms of strength relations, in affirmation, growth, strength and overcome ways of life and negation, decline, weakness and conservation ways of life. This way, it is concluded that Nietzsche psychology is an evaluation exercise of various human kinds, understood as morphology and theory of the power will improvement
Esta disserta??o analisa o conceito de psicologia na filosofia de Nietzsche e sua rela??o com a tipologia e a hierarquia. Inicialmente, verificamos que o sentido da pesquisa psicol?gica de Nietzsche ? alicer?ado sobre o conceito de vontade de pot?ncia, que ? considerado pelo fil?sofo alem?o uma interpreta??o e n?o uma descri??o do mundo. A vontade de pot?ncia tem como caracter?stica principal afirmar a efetividade enquanto um devir de for?as em luta por mais pot?ncia e destituir de verdade as oposi??es metaf?sicas. Estas for?as, ou impulsos, s?o compreendidas enquanto quanta de pot?ncia que se diferenciam e se relacionam com todos os outros quanta de forma a se intensificar. O que diferencia as for?as, portanto, ? a intensidade de pot?ncia. Assim, entendemos a luta das for?as enquanto um processo de domina??o, o que produz conjuntos de impulsos ou hierarquias. O desenvolvimento destas configura??es hier?rquicas de for?as se d? pelo estabelecimento din?mico de mandantes e mandados, ou seja, por rela??es de dom?nio, que se expressam em modos de vida, tipos humanos. Os tipos, entretanto, n?o s?o entendidos enquanto manifesta??es essenciais de um ser, mas sim enquanto sintomas das rela??es das for?as e suas conseq?entes configura??es hier?rquicas. Esses sintomas s?o o objeto da psicologia proposta por Nietzsche, e n?o a alma ou a consci?ncia humana. Os tipos humanos s?o analisados pelo fil?sofo alem?o mediante duas grandes e centrais perspectivas, uma de ascend?ncia (impulsos potentes e fortemente hierarquizados) e outra de decad?ncia da vida (impulsos fracos e an?rquicos). A partir deste aspecto geral da tipologia de Nietzsche, verificamos a imprescindibilidade dos tipos, sendo que para o surgimento de um tipo ? necess?ria a exist?ncia do seu diferenciado, pois h? rela??o de domina??o entre eles. Assim, a fun??o do psic?logo nietzschiano ?, com base na interpreta??o do mundo enquanto vontade de pot?ncia, avaliar os diversos modos de vida e hierarquiz?-los. A avalia??o psicol?gica de Nietzsche, portanto, corresponde a diferenciar e hierarquizar os tipos humanos, compreendidos enquanto sintomas das rela??es de for?as, em modos de vida de afirma??o, crescimento, for?a, supera??o, e em modos de vida de nega??o, de decl?nio, fraqueza e conserva??o. Conclu?mos, desse modo, que a psicologia de Nietzsche ? um exerc?cio de avalia??o dos diversos tipos humanos, entendida enquanto morfologia e teoria do desenvolvimento da vontade de pot?ncia

It is often presumed that evolutionary reduction is tantamount to deconstruction, or even destruction, because relaxed selective forces have been insufficient to maintain the organ in its original state. However, studies on reduction are often limited by a lack of diversity, both of related species exhibiting reduction and of the reduced form itself. There have also been very few studies on the reduction of compound eyes, despite the fact that their near ubiquity among arthropods alone makes them perhaps the most common type of eye. Bat flies (Streblidae and Nycteribiidae) are a group of dipterans that exhibit variable degrees of compound eye reduction, and therefore provide the opportunity to study reduction of this organ in a phylogenetic context. The first chapter of this work reports on behavioral experiments demonstrating that the eyes of one bat fly species, Trichobius frequens, are functional, and that they neither exhibit phototaxis typical of other dipteran species, nor move toward a light source. The second chapter uses molecular phylogenetics to identify a correlation between eye and wing morphology. The results also suggest that secondary to their eye reduction, bat flies (at least in the case of New World specie, including Trichobius spp.) have secondarily experienced a shift in the structure of their facets that is convergent with other insects whose eyes have been selected for increased sensitivity. In the final chapter, histological and optical analyses of T. frequens eyes are used to reveal significant structural changes to the microstructure of its ommatidia that increase sensitivity at the expense of acuity. Many of these changes are also convergent with similar adaptations that have been demonstrated to increase sensitivity in organisms that function in reduced light environments. The results of these analyses suggest that reduction in T. frequens eyes may have been part of an active remodeling process resulting from a shift in the relative importance of sensitivity and acuity. As this is a process of reduction not generally considered, the findings here turn our attention to alternative hypotheses that should be considered when studying evolutionary reduction of any organ.

The 33 kDa manganese stabilizing protein (MSP) has been proposed to provide ligands to stabilize Mn ions in the water lysis reaction of photosystem II of photosynthesis. In previous research site-directed mutagenesis had been performed on regions of the psbO gene encoding two aspartic acid residues of MSP which were thought to have the potential to form carboxyl bridges with Mn ions. The purpose of this research was to analyze these mutants. Plasmids pUC120-33 (#1,3,5,7,9,11,15) containing mutant psbO genes could not be isolated from E.coli because the expressed MSP was toxic to the cells. However, a psbO mutant gene carried in pPGV5-33 (#7) was isolated from E.coli and transformed into cyanobacterium Svnechococcus PCC 7942. Cyanobacterial cells carrying the MSP mutant showed a susceptibility to intensive light (100 footcandles) with a decrease of 30% in the growth rate within the first 100 hours after inoculation. This result suggested a possible function of the MSP in protecting the oxygen evolving complex from intensive light exposure. However, the mutant appeared to revert after this time probably due to homologous gene recombination with the wild type gene. In order to further analyze the function of mutants without recombination occurring, the construction of an MSP deletion was attempted using insertion of a kanamycin cartridge into the middle of the psbO gene. The inactivated psbO gene was transformed into E.coli and transformants were selected by kanamycin resistance. However, plasmid DNA carrying the interrupted genes could not be isolated, probably due to toxicity of the expression product in E.coli cells. Thus, future studies should be directed to reconstruction of a deletion mutant by direct transformation into cyanobacterial cells. Once a deletion mutant has been constructed analyses of the site-directed mutations could be performed in cyanobacteria.
Department of Biology

Previous studies have identified stem cell populations in articular cartilage using colony forming assays and mesenchymal stem cell (MSC) marker expression. The specificity of classical MSC markers for isolation of stem cells within articular cartilage is insufficient, with large and highly variable quantities being reported in the literature. This study has demonstrated, for the first time, a panel of stem cell markers specific for articular cartilage-derived stem cells (ACSC). ACSCs were isolated, quantified and cultured from healthy and OA joints. Stem cells were clonally-derived cell lines that proliferated beyond 50 population doublings whilst maintaining a phenotype, and demonstrated tri-lineage potential. We discovered that OA cartilage had a two-fold increase in stem cell number, consisting of two divergent stem cell sub-populations. These divergent populations varied in proliferative capacity with only 50% of stem cells from the OA joint capable of extended proliferation in vitro. Using transcriptomic next generation sequencing of culture-expanded chondrocytes and ACSCs we successfully identified differentially expressed genes and a panel of novel markers of cartilage-specific stem cells. Novel markers were validated using qPCR and protein labelling and, were specifically expressed in ACSCs, with no expression in the culture-expanded full-depth chondrocytes. Using immunofluorescence for novel stem cell markers we found articular cartilage-derived stem cells are localised within the transitional zone in normal cartilage and the superficial zone in OA cartilage. OA cartilage was found to contain a 2-fold increase in stem cells using immunofluorescence. Subsequently, we used the panel of novel markers and fluorescent active cell sorting to isolate a sub-population from full-depth cartilage with stem cell characteristics. These cells were plastic adherent, clonogenic, with proliferative capacity greater than 50PD and displayed tri-lineage potential, therefore meeting all criteria for classification as a MSC population. The use of specific markers to isolate ACSCs will allow for further characterisation of stem cells, including a more in-depth understanding of the mechanisms of proliferation, differentiation and degeneration within articular cartilage.

Die Länge der Vegetationsperiode (VP) spielt eine zentrale Rolle für die interannuelle Variation der Kohlenstoffspeicherung terrestrischer Ökosysteme. Die Analyse von Beobachtungsdaten hat gezeigt, dass sich die VP in den letzten Jahrzehnten in den nördlichen Breiten verlängert hat. Dieses Phänomen wurde oft im Zusammenhang mit der globalen Erwärmung diskutiert, da die Phänologie von der Temperatur beeinflusst wird.

Die Analyse der Pflanzenphänologie in Süddeutschland im 20. Jahrhundert zeigte:
- Die starke Verfrühung der Frühjahrsphasen in dem Jahrzehnt vor 1999 war kein singuläres Ereignis im 20. Jahrhundert. Schon in früheren Dekaden gab es ähnliche Trends. Es konnten Perioden mit unterschiedlichem Trendverhalten identifiziert werden.
- Es gab deutliche Unterschiede in den Trends von frühen und späten Frühjahrsphasen. Die frühen Frühjahrsphasen haben sich stetig verfrüht, mit deutlicher Verfrühung zwischen 1931 und 1948, moderater Verfrühung zwischen 1948 und 1984 und starker Verfrühung zwischen 1984 und 1999. Die späten Frühjahrsphasen hingegen, wechselten ihr Trendverhalten in diesen Perioden von einer Verfrühung zu einer deutlichen Verspätung wieder zu einer starken Verfrühung.

Dieser Unterschied in der Trendentwicklung zwischen frühen und späten Frühjahrsphasen konnte auch für ganz Deutschland in den Perioden 1951 bis 1984 und 1984 bis 1999 beobachtet werden.
Der bestimmende Einfluss der Temperatur auf die Frühjahrsphasen und ihr modifizierender Einfluss auf die Herbstphasen konnte bestätigt werden. Es zeigt sich jedoch, dass
- die Phänologie bestimmende Funktionen der Temperatur nicht mit einem globalen jährlichen CO2 Signal korreliert waren, welches als Index für die globale Erwärmung verwendet wurde
- ein Index für grossräumige regionale Zirkulationsmuster (NAO-Index) nur zu einem kleinen Teil die beobachtete phänologischen Variabilität erklären konnte.

Das beobachtete unterschiedliche Trendverhalten zwischen frühen und späten Frühjahrsphasen konnte auf die unterschiedliche Entwicklung von März- und Apriltemperaturen zurückgeführt werden. Während sich die Märztemperaturen im Laufe des 20. Jahrhunderts mit einer zunehmenden Variabilität in den letzten 50 Jahren stetig erhöht haben, haben sich die Apriltemperaturen zwischen dem Ende der 1940er und Mitte der 1980er merklich abgekühlt und dann wieder deutlich erwärmt.
Es wurde geschlussfolgert, dass die Verfrühungen in der Frühjahrsphänologie in den letzten Dekaden Teile multi-dekadischer Fluktuationen sind, welche sich nach Spezies und relevanter saisonaler Temperatur unterscheiden. Aufgrund dieser Fluktuationen konnte kein Zusammenhang mit einem globalen Erwärmungsignal gefunden werden.
Im Durchschnitt haben sich alle betrachteten Frühjahrsphasen zwischen 1951 und 1999 in Naturräumen in Deutschland zwischen 5 und 20 Tagen verfrüht. Ein starker Unterschied in der Verfrühung zwischen frühen und späten Frühjahrsphasen liegt an deren erwähntem unterschiedlichen Verhalten. Die Blattverfärbung hat sich zwischen 1951 und 1999 für alle Spezies verspätet, aber nach 1984 im Durchschnitt verfrüht. Die VP hat sich in Deutschland zwischen 1951 und 1999 um ca. 10 Tage verlängert.
Es ist hauptsächlich die Änderung in den Frühjahrphasen, die zu einer Änderung in der potentiell absorbierten Strahlung (PAS) führt. Darüber hinaus sind es die späten Frühjahrsphasen, die pro Tag Verfrühung stärker profitieren, da die zusätzlichen Tage länger undwärmer sind als dies für die frühen Phasen der Fall ist. Um die relative Änderung in PAS im Vergleich der Spezies abzuschätzen, müssen allerdings auch die Veränderungen in den Herbstphasen berücksichtigt werden.
Der deutliche Unterschied zwischen frühen und späten Frühjahrsphasen konnte durch die Anwendung einer neuen Methode zur Konstruktion von Zeitreihen herausgearbeitet werden. Der neue methodische Ansatz erlaubte die Ableitung verlässlicher 100-jähriger Zeitreihen und die Konstruktion von lokalen kombinierten Zeitreihen, welche die Datenverfügbarkeit für die Modellentwicklung erhöhten.
Ausser analysierten Protokollierungsfehlern wurden mikroklimatische, genetische und Beobachtereinflüsse als Quellen von Unsicherheit in phänologischen Daten identifiziert. Phänologischen Beobachtungen eines Ortes können schätzungsweise 24 Tage um das parametrische Mittel schwanken.Dies unterstützt die 30-Tage Regel für die Detektion von Ausreissern.
Neue Phänologiemodelle, die den Blattaustrieb aus täglichen Temperaturreihen simulieren, wurden entwickelt. Diese Modelle basieren auf einfachen Interaktionen zwischen aktivierenden und hemmenden Substanzen, welche die Entwicklungsstadien einer Pflanze bestimmen. Im Allgemeinen konnten die neuen Modelle die Beobachtungsdaten besser simulieren als die klassischen Modelle.

Weitere Hauptresultate waren:
- Der Bias der klassischen Modelle, d.h. Überschätzung von frühen und Unterschätzung von späten Beobachtungen, konnte reduziert, aber nicht vollständig eliminiert werden.
- Die besten Modellvarianten für verschiedene Spezies wiesen darauf hin, dass für die späten Frühjahrsphasen die Tageslänge eine wichtigere Rolle spielt als für die frühen Phasen.
- Die Vernalisation spielte gegenüber den Temperaturen kurz vor dem Blattaustrieb nur eine untergeordnete Rolle.
The length of the vegetation period (VP) plays a central role for the interannual variation of carbon fixation of terrestrial ecosystems. Observational data analysis has indicated that the length of the VP has increased in the last decades in the northern latitudes mainly due to an advancement of bud burst (BB). This phenomenon has been widely discussed in the context of Global Warming because phenology is correlated to temperatures.

Analyzing the patterns of spring phenology over the last century in Southern Germany provided two main findings:
- The strong advancement of spring phases especially in the decade before 1999 is not a singular event in the course of the 20th century. Similar trends were also observed in earlier decades. Distinct periods of varying trend behavior for important spring phases could be distinguished.
- Marked differences in trend behavior between the early and late spring phases were detected. Early spring phases changed as regards the magnitude of their negative trends from strong negative trends between 1931 and 1948 to moderate negative trends between 1948 and 1984 and back to strong negative trends between 1984 and 1999. Late spring phases showed a different behavior. Negative trends between 1931 and 1948 are followed by marked positive trends between 1948 and 1984 and then strong negative trends between 1984 and 1999.
This marked difference in trend development between early and late spring phases was also found all over Germany for the two periods 1951 to 1984 and 1984 to 1999.

The dominating influence of temperature on spring phenology and its modifying effect on autumn phenology was confirmed in this thesis. However,
- temperature functions determining spring phenology were not significantly correlated with a global annual CO2 signal which was taken as a proxy for a Global Warming pattern.
- an index for large scale regional circulation patterns (NAO index) could only to a small part explain the observed phenological variability in spring.

The observed different trend behavior of early and late spring phases is explained by the differing behavior of mean March and April temperatures. Mean March temperatures have increased on average over the 20th century accompanied by an increasing variation in the last 50 years. April temperatures, however, decreased between the end of the 1940s and the mid-1980s, followed by a marked warming after the mid-1980s.
It can be concluded that the advancement of spring phenology in recent decades are part of multi-decadal fluctuations over the 20th century that vary with the species and the relevant seasonal temperatures. Because of these fluctuations a correlation with an observed Global Warming signal could not be found.
On average all investigated spring phases advanced between 5 and 20 days between 1951 and 1999 for all Natural Regions in Germany. A marked difference be! tween late and early spring phases is due to the above mentioned differing behavior before and after the mid-1980s. Leaf coloring (LC) was delayed between 1951 and 1984 for all tree species. However, after 1984 LC was advanced. Length of the VP increased between 1951 and 1999 for all considered tree species by an average of ten days throughout Germany.
It is predominately the change in spring phases which contributes to a change in the potentially absorbed radiation. Additionally, it is the late spring species that are relatively more favored by an advanced BB because they can additionally exploit longer days and higher temperatures per day advancement. To assess the relative change in potentially absorbed radiation among species, changes in both spring and autumn phenology have to be considered as well as where these changes are located in the year.
For the detection of the marked difference between early and late spring phenology a new time series construction method was developed. This method allowed the derivation of reliable time series that spanned over 100 years and the construction of locally combined time series increasing the available data for model development.
Apart from analyzed protocolling errors, microclimatic site influences, genetic variation and the observers were identified as sources of uncertainty of phenological observational data. It was concluded that 99% of all phenological observations at a certain site will vary within approximately 24 days around the parametric mean. This supports to the proposed 30-day rule to detect outliers.
New phenology models that predict local BB from daily temperature time series were developed. These models were based on simple interactions between inhibitory and promotory agents that are assumed to control the developmental status of a plant. Apart from the fact that, in general, the new models fitted and predicted the observations better than classical models, the main modeling results were:
- The bias of the classical models, i.e. overestimation of early observations and underestimation of late observations, could be reduced but not completely removed.
- The different favored model structures for each species indicated that for the late spring phases photoperiod played a more dominant role than for early spring phases.
- Chilling only plays a subordinate role for spring BB compared to temperatures directly preceding BB.

The primary amino acid sequence of a protein governs its specific cellular functions. Since the cracking of the genetic code in the late 1950’s, it has been possible to predict the amino acid sequence of a given protein from the DNA sequence of a gene. Nevertheless, the ability to predict a protein’s function from its primary sequence remains a great challenge in biology. In order to address this problem, we combined recent advances in next generation sequencing technologies with systematic mutagenesis strategies to assess the function of thousands of protein variants in a single experiment. Using this strategy, my dissertation describes the effects of most possible single point mutants in the multifunctional Ubiquitin protein in yeast. The effects of these mutants on the essential activation of ubiquitin by the ubiquitin activating protein (E1, Uba1p) as well as their effects on overall yeast growth were measured. Ubiquitin mutants defective for E1 activation were found to correlate with growth defects, although in a non-linear fashion. Further examination of select point mutants indicated that E1 activation deficiencies predict downstream defects in Ubiquitin function, resulting in the observed growth phenotypes. These results indicate that there may be selective pressure for the activity of the E1enzyme to selectively activate ubiquitin protein variants that do not result in functional downstream defects. Additionally, I will describe the use of similar techniques to discover drug resistant mutants of the oncogenic protein BRAFV600E in human melanoma cell lines as an example of the widespread applicability of our strategy for addressing the relationship between protein function and biological fitness.

The primary amino acid sequence of a protein governs its specific cellular functions. Since the cracking of the genetic code in the late 1950’s, it has been possible to predict the amino acid sequence of a given protein from the DNA sequence of a gene. Nevertheless, the ability to predict a protein’s function from its primary sequence remains a great challenge in biology. In order to address this problem, we combined recent advances in next generation sequencing technologies with systematic mutagenesis strategies to assess the function of thousands of protein variants in a single experiment. Using this strategy, my dissertation describes the effects of most possible single point mutants in the multifunctional Ubiquitin protein in yeast. The effects of these mutants on the essential activation of ubiquitin by the ubiquitin activating protein (E1, Uba1p) as well as their effects on overall yeast growth were measured. Ubiquitin mutants defective for E1 activation were found to correlate with growth defects, although in a non-linear fashion. Further examination of select point mutants indicated that E1 activation deficiencies predict downstream defects in Ubiquitin function, resulting in the observed growth phenotypes. These results indicate that there may be selective pressure for the activity of the E1enzyme to selectively activate ubiquitin protein variants that do not result in functional downstream defects. Additionally, I will describe the use of similar techniques to discover drug resistant mutants of the oncogenic protein BRAFV600E in human melanoma cell lines as an example of the widespread applicability of our strategy for addressing the relationship between protein function and biological fitness.

Tandis que les bénéfices physiologiques de chaussures de référence dans le milieu de la course à pied d’endurance sont montrés dans la littérature scientifique, les effets spécifiques et contrôlés de certaines propriétés mécaniques des chaussures demeurent peu connus. L’objectif général de ce travail de thèse était d’étudier les effets du retour d’énergie des semelles intermédiaire des chaussures et de la raideur en flexion des chaussures sur la performance physiologique et biomécanique en course à pied d'endurance. Que ce soit à court-terme ou lors d’une course à pied prolongée, le coût énergétique métabolique (critère utilisé pour évaluer la performance en course à pied) n’était pas significativement modifié par les propriétés mécaniques testées en moyenne parmi le groupe complet de participants. En revanche, les réponses spécifiques aux participants, à la fois à court-terme et lors d’une course à pied prolongée, ont permis de mettre en évidence des combinaisons de réponses biomécaniques et de caractéristiques intrinsèques aux participants expliquant les variations du coût énergétique métabolique en fonction des propriétés mécaniques chaussantes. Une nouvelle stratégie a notamment été mise en évidence chez les participants bénéficiant de la raideur en flexion des chaussures qui se traduisait par une redistribution descendante des activations musculaires des articulations de la hanche et du genou vers l’articulation de la cheville avec la durée de course. Ce travail de thèse soulignait l’importance de considérer une offre de conception de chaussures adaptées à des groupes de coureurs aux réponses biomécaniques et/ou aux caractéristiques intrinsèques similaires
While physiological benefits of baseline running racing shoes are shown in the scientific literature, the specific and controlled effects of some shoe mechanical features remain not well known. The main purpose of this work was to study the effects of the midsole energy return and the shoe longitudinal bending stiffness on the physiological and biomechanical performance during endurance running. In both short-term and prolonged running duration, the metabolic energetic cost (criteria used to evaluate the endurance running performance) was not significantly altered by the tested mechanical features in average over the group of participants. The main finding was that the shoe mechanical features induced different effects on the metabolic energetic cost depending on the participants. Taking into account the participant-specific responses (in both short-term and during a prolonged running duration) enabled to highlight combinations of biomechanical responses and intrinsic participant characteristics explaining the variations of the metabolic energetic cost as a function of shoe mechanical features. A novel strategy has been highlighted in participants benefiting from the shoe longitudinal bending stiffness resulting in descendant redistribution of the muscular coordination from the hip and knee joints to the ankle joint with the running duration. This work showed the importance of considering a footwear design offer suitable to groups of runners with similar biomechanical responses and/or intrinsic characteristics

Previous studies: starting point. Aim: training optimization to grow from Talent to Peak Performance. SEARCH → Recruitment → Detection and discovery; DIAGNOSIS → Identification and validation → Goodwill; PROGNOSIS → Development, maintenance and control → Selection; Dosage and methodological; TREATMENT → Strengthening care. SUBJECTS FOR THE STUDIES: 16 athletes (gymnasts Team FGI / Olympic Club CONI). Average age: 20 years (± 4,17). DATA ALREADY KNOWN: genotype; history; athletic and physical parameters; performance indicators. MONITORED FOR: training plans and technical control. PERIOD OF INVESTAGATION: 2013 - 2015, still TEST EVENT qualifying final OG. THIS POJECT WORK WILL BE DEVELOPED INTO FOLLOWING FOUR OPERATIVE SECTIONS. I study: Biological Survey. Task: monitor also try to do it or how, any DNA methylation and histone modifications, which lead to persistent effects on the availability of DNA for transcription. Goal: induced a result of special agents such as nutrition, nutraceuticals, nutrigenomics/genetic and stress factors. II study: Training to peak performance. Task: implementation and monitoring of the new system of performance gymnastics workout through specific experimental protocols intake on the field. Goal: surveying methods, control, adjustment and validation shall be those provided by the FIG and therefore relevant to so-called "International Standard” for a peak performance in male artistic gymnastics. III study: Time related muscle adaptations. Task: evaluation of different dimensions of phenomena that characterize, from time to time, all the “adaptation to performance" caused by the administration of loads (chronic) and overloads (acute). Goal: definition of the specific time related of changes after high intensity training including the phenomena of repairs after ‘regular’ damages producing by strenous exercise (vigorous eccentric muscle contraction). IV study: Training load analysis. Task: analysis of the specific effects of the training loads on muscle morphology and functions with relationship to the technical performance Goal: to meet the three main factors for the conditioning of high level performance, namely the "Olympic Records".

During my PhD I studied two principal biological aspects employing Drosophila melanogaster. Therefore, this study is divided into Part I and II. Part I: Bruchpilot and Complexin interact to regulate synaptic vesicle tethering to the active zone cytomatrix At the presynaptic active zone (AZ) synaptic vesicles (SVs) are often physically linked to an electron-dense cytomatrix – a process referred to as “SV tethering”. This process serves to concentrate SVs in close proximity to their release sites before contacting the SNARE complex for subsequent fusion (Hallermann and Silver, 2013). In Drosophila, the AZ protein Bruchpilot (BRP) is part of the proteinous cytomatrix at which SVs accumulate (Kittel et al., 2006b; Wagh et al., 2006; Fouquet et al., 2009). Intriguingly, truncation of only 1% of the C-terminal region of BRP results in a severe defect in SV tethering to this AZ scaffold (hence named brpnude; Hallermann et al., 2010b). Consistent with these findings, cell-specific overexpression of a C-terminal BRP fragment, named mBRPC-tip (corresponds to 1% absent in brpnude; m = mobile) phenocopied the brpnude mutant in behavioral and functional experiments. These data indicate that mBRPC-tip suffices to saturate putative SV binding sites, which induced a functional tethering deficit at motoneuronal AZs. However, the molecular identity of the BRP complement to tether SVs to the presynaptic AZ scaffold remains unknown. Moreover, within larval motoneurons membrane-attached C-terminal portions of BRP were sufficient to tether SVs to sites outside of the AZ. Based on this finding a genetic screen was designed to identify BRP interactors in vivo. This screen identified Complexin (CPX), which is known to inhibit spontaneous SV fusion and to enhance stimulus evoked SV release (Huntwork and Littleton, 2007; Cho et al., 2010; Martin et al., 2011). However, so far CPX has not been associated with a function upstream of priming/docking and release of SVs. This work provides morphological and functional evidence, which suggests that CPX promotes recruitment of SVs to the AZ and thereby curtails synaptic short-term depression. Together, the presented findings indicate a functional interaction between BRP and CPX at Drosophila AZs. Part II: The Adhesion-GPCR Latrophilin/CIRL shapes mechanosensation The calcium independent receptor of α-latrotoxin (CIRL), also named Latrophilin, represents a prototypic Adhesion class G-protein coupled-receptor (aGPCR). Initially, Latrophilin was identified based on its capacity to bind the α-component of latrotoxin (α-LTX; Davletov et al., 1996; Krasnoperov et al., 1996), which triggers massive exocytotic activity from neurons of the peripheral nervous system (Scheer et al., 1984; Umbach et al., 1998; Orlova et al., 2000). As a result Latrophilin is considered to play a role in synaptic transmission. Later on, Latrophilins have been associated with other biological processes including tissue polarity (Langenhan et al., 2009), fertility (Prömel et al., 2012) and synaptogenesis (Silva et al., 2011). However, thus far its subcellular localization and the identity of endogenous ligands, two aspects crucial for the comprehension of Latrophilin’s in vivo function, remain enigmatic. Drosophila contains only one latrophilin homolog, named dCirl, whose function has not been investigated thus far. This study demonstrates abundant dCirl expression throughout the nervous system of Drosophila larvae. dCirlKO animals are viable and display no defects in development and neuronal differentiation. However, dCirl appears to influence the dimension of the postsynaptic sub-synaptic reticulum (SSR), which was accompanied by an increase in the postsynaptic Discs-large abundance (DLG). In contrast, morphological and functional properties of presynaptic motoneurons were not compromised by the removal of dCirl. Instead, dCirl is required for the perception of mechanical challenges (acoustic-, tactile- and proprioceptive stimuli) through specialized mechanosensory devices, chordotonal organs (Eberl, 1999). The data indicate that dCirl modulates the sensitivity of chordotonal neurons towards mechanical stimulation and thereby adjusts their input-output relation. Genetic interaction analyses suggest that adaption of the molecular mechanotransduction machinery by dCirl may underlie this process. Together, these results uncover an unexpected function of Latrophilin/dCIRL in mechanosensation and imply general modulatory roles of aGPCR in mechanoception
In dieser These wurden zwei grundlegende biologische Aspekte mittels Drosophila melanogaster untersucht, weshalb diese in zwei Teile gegliedert ist. TeiL I: Die Interaktion von Bruchpilot und Complexin vermittelt die Anbindung von synaptischen Vesikeln an die Zytomatrix der aktiven Zone Oft findet man an aktiven Zonen (AZ) von Präsynapsen elektronendichte Matrices, welche meist in physischem Kontakt mit synaptischen Vesikeln (SV) stehen. Dieser als „SV Tethering“ bezeichnete Prozess dient der Anreicherung SV in der unmittelbaren Nähe ihrer Freisetzungszonen, noch bevor diese mit dem SNARE Komplex interagieren, um mit der präsynapti-schen Plasmamembran zu fusionieren (Hallermann und Silver, 2013). In der Taufliege Drosophila melanogaster bildet das AZ Protein Bruchpilot (BRP) Protrusionen, um welche SV akkumulieren (Kittel et al., 2006b; Wagh et al., 2006; Fouquet et al., 2009). Interessan-terweise resultiert bereits eine minimale Verkürzung von BRP (1% der Gesamtlänge) am C-terminalen Ende in einem schwerwiegenden Anbindedefekt von SV, der mit einem Funkti-onsverlust dieser Synapsen einhergeht (brpnude; Hallermann et al., 2010b). Entsprechend diesem Vorbefund resultierte die gewebespezifische Überexpression eines C-terminalen BRP Fragments - mBRPC-tip (entspricht dem fehlenden Fragment der brpnude Mu-tante; m = mobil) - sowohl in Verhaltens- als auch funktionellen Analysen in einer Phänoko-pie der brpnude Mutante. Dies deutet daraufhin, dass mBRPC-tip vermeintliche vesikuläre Interaktionspartner blockiert und so die Anreicherung von SV an motoneuronalen AZ verhindert, was ähnlich wie in brpnude Mutanten zu einem funktionellen Tethering-Defekt führt. Die molekulare Identität eines BRP Partners zur Anreicherung von SV an der Zytomatrix der AZ wurde bisher nicht beschrieben. Weiterhin zeigt diese Arbeit, dass membrangebundene C-terminale BRP Anteile genügen, um SV an Positionen außerhalb von AZ zu binden. Basierend auf diesem Befund wurde ein gene-tischer in vivo Screen zur Identifikation von BRP Interaktoren entwickelt. Dieser Screen identifizierte Complexin (CPX), ein Protein, dessen hemmende beziehungsweise fördernde Wirkung auf die spontane und reizinduzierte Vesikelfusion bekannt ist (Huntwork und Littleton, 2007; Cho et al., 2010; Martin et al., 2011). CPX wurde bisher nicht mit einer Funktion ober-halb von Vesikelpriming und -fusion in Verbindung gebracht. Diese Studie dokumentiert strukturelle und funktionelle Hinweise, die darauf hindeuten, dass CPX mit BRP interagiert, um Vesikelakkumulation an AZ zu fördern und dadurch synaptischer Kurzzeit-Depression entgegen zu wirken. Teil II: Adhäsions-GPCR Latrophilin/CIRL moduliert die Wahrnehmung mechanischer Reize Der Kalzium-unabhängige Rezeptor für α-Latrotoxin (CIRL), oder Latrophilin, ist ein prototypischer Rezeptor der Adhäsions G-Protein gekoppelten Klasse (aGPCR). Identifiziert wurde Latrophilin ursprünglich aufgrund seiner Fähigkeit die α-Komponente von Latrotoxin (α-LTX) zu binden (Davletov et al., 1996; Krasnoperov et al., 1996), welches seine Wirkung am peripheren Nervensystem entfaltet und dort übermäßige Transmitterausschüttung an neuronalen Endigungen induziert (Scheer et al., 1984; Umbach et al., 1998; Orlova et al., 2000). Basierend auf diesem Effekt wurde Latrophilin eine Rolle bei der synaptischen Transmission zugesprochen. Später wurden Latrophiline mit weiteren biologischen Prozessen in Zusammenhang gebracht, darunter Gewebepolarität (Langenhan et al., 2009), Fertilität (Prömel et al., 2012) und Synaptogenese (Silva et al., 2011). Allerdings blieb sowohl die subzelluläre Lokalisation als auch die Identität endogener Liganden, zwei Schlüsselaspekte im Verständnis der in vivo Funktion von Latrophilinen bisher rätselhaft. Drosophila besitzt lediglich ein latrophilin Homolog, dCirl, dessen Funktion bisher nicht untersucht wurde. Diese Arbeit zeigt, dass dCirl in weiten Teilen des larvalen Nervensystems von Drosophila exprimiert ist. dCirl knock-out Mutanten sind lebensfähig und weisen keine Störungen in der Entwicklung und neuronalen Differenzierung auf. Allerdings schien dCirl Einfluss auf die Ausdehnung des postsynaptischen subsynaptischen Retikulums (SSR) zu nehmen, was mit einer erhöhten Menge an Discs-large (DLG) assoziiert war. Die morphologischen und funktionellen Eigenschaften präsynaptischer Motoneurone der Fliegenlarve hingegen, waren durch den Verlust von dCirl funktionell weitestgehend unbeeinträchtigt. Vielmehr ist dCirl notwendig für die Wahrnehmung mechanischer Reize (akustische-, taktile und propriozeptive) durch spezialisierte Vorrichtungen - Chordotonalorgane (Eberl, 1999). Die Befunde deuten daraufhin, dass dCirl die Sensitivität der Chordotonalneurone gegenüber mechanischen Reizen moduliert und dadurch das Input-Output Verhältnis einstellt. Adaptation der molekularen Mechanotransduktionsmaschinerie durch dCirl könnte die molekulare Grundlage für diesen Prozess darstellen, eine Hypothese die durch genetische Interaktionsanalysen gestützt wird. Schlussfolglich enthüllen die experimentellen Befunde dieser These eine unerwartete Funktion von Latrophilin/dCirl bei der Mechanoperzeption und implizieren eine generelle modula-torische Rolle für aGPCR bei der Wahrnehmung mechanischer Reize

The auxins are an important class of plant hormones involved in many aspects of plant development The most common naturally occurring auxin is indole-3-acetic acid, or IAA. In Arabidopsis, up to 95% of the IAA pool is found conjugated to small molecules such as sugars and amino acids. However, the genes and enzymes involved in IAA conjugate metabolism are not yet well understood. A mutant, iar1, that is resistant to the inhibitory effects of multiple IAA-amino acid conjugates on root elongation was identified. The IAR1 gene encodes a protein with numerous transmembrane domains and several histidine-rich regions. The IAR1 protein has homologs in other organisms, including Drosophila, C. elegans, and mammals, and is similar in molecular structure to the ZIP family of zinc transporters from Arabidopsis and yeast. Plant reproduction requires precise control of the transition to flowering in response to environmental cues. We have isolated a late-flowering Arabidopsis mutant, fkf1, that is rescued by vernalization or gibberellin treatment. The mutant also exhibits a light-dependent hypocotyl elongation defect. We used a positional approach to clone FKF1, which encodes a novel protein with an N-terminal PAS domain similar to the flavin-binding region of certain photoreceptors, an F-box motif characteristic of proteins that target ubiquitin-mediated degradation, and six kelch repeats predicted to fold into a beta-propeller. FKF1 mRNA levels oscillate with a circadian rhythm and the fkf1 deletion mutation alters the rhythmic expression of other clock-regulated genes, implicating FKF1 in regulation of the circadian clock.

The objective of this dissertation was to identify and characterize diversity within the expressed primary immunoglobulin heavy chain (IgH) repertoire in cattle. Determinations relied heavily upon different comparative analysis strategies focussing on both germline and expressed IgH gene segment sequences. We determined that the early fetal expressed IgH repertoire is constituted by as few as 2–3 VH genes and a single JH gene (multiple D). VH gene use increases with age, though IgH expression is restricted to members of a single VH family and primarily one JH gene (>300 sequences analyzed). All isolated germline VH genes also belong to a single VH family, corresponding to that in the expressed repertoire. Therefore, germline-encoded VH (and JH) sequence polymorphism is low, making limited contributions to overall IgH sequence diversity. In sharp contrast, bovine CDR3 regions exhibit extremely high levels of heterogeneity both in terms o f sequence and hypervariable lengths. A major fraction of IgH diversification occurs after rearrangement, most likely via untemplated somatic hypermutation. Nucleotide substitutions within the JH-Cμ intron, which does not support gene conversion due to a lack of known donor sequences, were consistent with USH on multiple levels, including hotspot targeting, the ratio of transitions to transversions, preferential nucleotide substitutions and potential strand bias. Sequence diversity levels varied with time among immunologically relevant tissues. Early fetal spleen and late fetal ileum appear to be two important sites of B cell diversification during their respective developmental stages. Patterns of IgH expression suggest that spleen is the early site of substantial gene rearrangement, and source of B cell emigrants, which subsequently populate other peripheral tissues including liver, ileum, and bone marrow.