Development requires the acquisition of different cell fates. A major conserved pathway required for cell fate determination is the Notch signalling pathway. Neuralized is a key regulator of the Notch pathway and is essential for embryonic development in Drosophila melanogaster. I have been studying the role of Neuralized during Drosophila development, focusing on the regulation of this protein. Neuralized is an E3 ubiquitin ligase that targets Notch ligands for ubiquitination and endocytosis in the signal sending cell. This endocytic event is required for signal transduction, and cells lacking Neuralized fail to signal through Notch. I have identified a conserved interaction between Neuralized and phosphoinositides that is essential for the ability of Neuralized to promote ligand endocytosis and Notch signalling. Interactions between Neuralized and phosphoinositides are not required for ligand ubiquitination, identifying a role for Neuralized in downstream aspects of ligand trafficking. I have also determined that Neuralized is dynamically regulated through a combination of tissue specific expression, subcellular trafficking, protein interactions and posttranslational modification. Neuralized contains two related protein domains of unknown function called Neuralized homology repeats (NHR). To gain insight into the function of the NHR domain, I characterized another NHR containing protein, CG3894. CG3894 is required for development and preliminary data indicate that NHR domains dimerize, suggesting a possible interaction between Neuralized and CG3894. The study of Neuralized in Drosophila has contributed to our understanding of this essential protein both at a developmental and cellular level, and has provided a means through which to ask questions about regulation of Notch signalling in a relatively simple context. Given the importance of Notch signalling to development, and contributions that aberrations in signalling make to cancer and diseases of the nervous system, expanding our understanding of the regulation of Notch signalling is essential to understanding how this important pathway functions.

The experiments described in this thesis report were aimed at studying the functions of Sox2 and Serrate1 during the development of the neurosensory elements of the inner ear. First, we have described the expression pattern of Sox2 during inner ear development and compared to that of Sox3 and Serate1. Secondly, we have shown the results of plasmid based in ovo electroporation experiments, designed to manipulate gene expression exogenously, and to study the gain of function of Sox2 and Serrate1. Effects on cell fate and downstream targets were assessed by in situ hybridization immunohistochemistry and quantitative real-time PCR (qRT-PCR).The results show that Sox2 is expressed in the neurosensory domain of the otic epithelium during the neurogenic period of otic development and, later on, during the development of the prosensory patches and sensory organs. As differentiation proceeds, Sox2 is excluded from differentiated neurones and hair cells, but remains expressed in the supporting cells of the sensory organs. Sox3 is co-expressed with Sox2 in the neurogenic domain of the otic cup. But Sox3 is then down-regulated and only Sox2 expression persists in the sensory precursors, where it is co-expressed with the Notch ligand Serrate1. The expression domain of Serrate1 is initially nested within Sox2, however, later in development Sox2 becomes restricted within the boundaries of Serrate1 expression, a process that is concomitant to the formation of the sensory patches. These expression patterns suggest: 1) that Sox2 correlates with neurosensory fate in the otic placode, 2) that neurogenesis is associated with Sox2 and Sox3 and 3) that sensory development is associated with Sox2 and Serrate1.Gain of function studies show that Serrate1 regulates prosensory fate and sensory organ development by maintaining Sox2 expression in restricted domains of the otocyst, without affecting neurogenesis. Serrate1 operates in a Notch-dependent manner, consistently with a mechanism of lateral induction that includes the induction of its own expression and downstream targets of the Notch signalling pathway Hes1, Hey1 and Hey2. Similar studies on Sox2 indicate that it specifies neurosensory fate in the otic epithelium. However, high concentrations of Sox2 suppress sensory fate and promote neuronal fate. Besides, Sox2 prevents cell differentiation though the cooperation with Notch and BMP signalling pathways.We like to propose a model in which an extended neural competence is early established in the otic placode with the early expression of Sox2 and Sox3 genes. The cooperation between Sox2 and Sox3 then provides a high concentration of SoxB1 protein and promote neuronal fate. In parallel, Serrate1 maintains Sox2 expression in restricted domains, after Sox3 down-regulation. These domains retain the neurosensory competence and thereby develop as sensory patches. / Los experimentos descritos en esta tesis tuvieron por objetivo estudiar la función de Sox2 y Serrate1 en el desarrollo de los elementos neurosensoriales del oído. En primer lugar describimos el patrón de expresión de Sox2 durante el desarrollo del oído y lo comparamos con el de Sox3 y Serrate1. En segundo lugar, mostramos los resultados de experimentos de electroporación in ovo, diseñados para manipular exógenamente la expresión génica y estudiar la ganancia de función de Sox2 y Serrate1. Los efectos sobre el destino celular y las dianas moleculares se analizaron mediante hibridación in situ, inmunocitoquímica y real-time PCR (qRT-PCR).Los resultados muestran que Sox2 se expresa en el dominio neurosensoerial del epitelio ótico durante la fase de neurogénesis y, más adelante, durante el desarrollo de los parches prosensoriales y los órganos sensoriales. Con la diferenciación, Sox2 es excluido de las neuronas diferenciadas y las células ciliadas, pero permanece expresado en las células de soporte. Sox3 se coexpresa con Sox2 en el dominio neurogénico de la copa ótica. Pero entonces, la expresión de Sox3 se reduce y sólo Sox2 persiste en los precursores sensoriales, en donde se co-expresa con el ligando de Notch Serrate1. El dominio de expresión de Serrate1 está inicialmente contenido en el de Sox2, sin embargo, más adelante, Sox2 se restringe dentro de los límites de Serrate1, un proceso que es concomitante con la formación de los parches sensoriales. Estos experimentos sugieren que : 1) Sox2 se correlaciona con el destino neurosensorial de la placoda ótica, 2) la neurogénesis está asociada con Sox2 y Sox3, y 3) el desarrollo sensorial está asociado a la expresión de Sox2 y Serrate1Los estudios de ganancia de función muestran que Serrate1 regula el destino prosensorial y el desarrollo de los órganos sensoriales mediante el mantenimiento de la expresión de Sox2 en dominios restringidos del otocisto, sin afectar a la neurogénesis. Serrate1 opera en un modo dependiente de Notch, consistente con un mecanismo de inducción lateral que comprende la inducción de su propia expresión y la de las dianas de Notch Hes1, Hey1 and Hey2. Estudios similares sobre Sox2 indican que Sox2 especifica el destino neurosensorial en el epitelio ótico. Sin embargo, las concentraciones altas de Sox2 suprimen el destino sensorial y promueven el destino neuronal. Además, Sox2 previene la diferencoiación celular mediante la cooperación con Notch y Bmp. Se propone un modelo en el cual la competencia neural se establece tempranamente en la placoda ótica mediante la expresión temprana de Sox2 y Sox3. La cooperación entre Sox2 y Sox3 provee una alta concentración de factores SoxB1 que promueven el destino neuronal de los progenitores. En paralelo, Serrate1 mantiene la expresión de Sox2 en dominios restringidos tras la supresión de Sox3. Estos dominios, retienen el potencial neurosensorial y, más adelante, se desarrollan como parches sensoriales.

This thesis is investigating how the effective notch method can be used for fatigue assessment of welded joints.  The effective notch method is based on a finite element analysis where the joint is modeled with all notches fictitiously rounded with a radius of 1 mm. Analyses are performed on a cruciform fillet welded joint where parameters such as, load case, steel plate thickness and weld size, are varied. The achieved lifetime estimations are then compared to calculations with other fatigue assessment methods, linear elastic fracture mechanics and the nominal method. The goal is to draw conclusions about pros and cons of the effective notch method. The results are also compared to experimental fatigue tests performed on the same geometry. The results indicates that the effective notch method tends overestimating the lifetime, especially when the steel plate thickness is small. This leads to a non conservative method that is dangerous to use as guidance when designing. The estimations are though better when considering a toe crack then when considering a root crack. Due to a large scatter in experimental test results, it is hard to validate a fatigue assessment method in an absolute sense. That is also the case for the effective notch method, and more results from experimental fatigue tests are needed before the effective notch method can be fully used. For relative analysis, when variations of the same design needs to be compared, the effective notch can be a very powerful tool. This is because of the flexibility for different geometries that this method grants.

Superconducting microelectronics (SME) technology has the potential of realizing very high speed digital receivers capable of performing direct digitization of radio frequency signals with very low power consumption. The SME receiver is implemented on a single chip using Niobium based low temperature superconductive (LTS) Josephson Junction (JJ) technology by HYPRES. Analogue RF filters are still required at the receiver front end and are key components of the overall superconductor digital receiver. SME receivers usually require two types of RF filters; a wideband bandpass filter and a bandstop filter (a notch filter). The notch filter is required to eliminate interference and unwanted signals in the passband. In this thesis, design of highly miniaturized lumped element wideband and bandstop filters is investigated and some challenges are addressed. The filters are fabricated by the HYPRES process and therefore can be integrated with the SME receiver on the same chip. In a wideband filter, the coupling between the adjacent resonators is high. Achieving such a strong coupling is one of the challenges of designing wideband filters. The wideband filters realized with distributed elements usually suffer from very low spurious frequency. As the bandwidth of the filter becomes wider, the spurious peak of the second harmonic gets closer to the passband of the filter. In the first part of this work, the possibility of realizing lumped element superconducting bandpass filters (BPF) with a relative bandwidth of 80% is investigated. In the second part of the thesis, design and realization of lumped element superconducting bandstop filters (BSF) is discussed. The challenge for designing a bandstop filter is providing a good match over a wide frequency range. So narrowband inverters cannot be used. Instead, usually λ/4 matched transmission lines provide 90° phase shift between the resonators of a notch filter. The possibility of replacing the long transmission line with other means or eliminating the inverters and using both shunt and series resonators are investigated. Having both series and shunt resonators introduces some new challenges that are addressed in the thesis and discussed thoroughly. A tunable notch resonator is presented. The tunability is provided by a superconducting MEMS varactor that is realized in our group by doing some post processing on the device fabricated by HYPRES. The tunability range of the device at cryogenic temperatures is investigated. A 3-pole tunable BSF is also designed that uses the same tunable resonators. The tunability of the filter is investigated through simulation.

The thesis aims to investigate the mechanical behavior and properties of a centrally notched hybrid Magnesium/Carbon-Fiber/PEEK laminate at elevated temperature. The high performance hybrid composite laminates of 0.5mm Magnesium sheets sandwiched by Carbon-Fiber/PEEK (APC-2) guasi-isotropic and cross-ply laminates were fabricated. The Magnesium sheets were polished and cleaned by acetone, then underwent the surface treatment by CrO3-base solvent etchants, cured by the improved diaphragm curing process. The finished laminates were cut into the specimen than drilled a 4mm diameter hole in the center of specimen. At first, the ultimate strength, stiffness and stress-strain diagram were obtained due to static tension tests at elevated temperature, such as 25¢XC(RT), 75¢XC, 100¢XC, 125¢XC, and 150¢XC. Compare of them, the notched quasi-isotropic ones drop almost 50% in strength, and the notched cross-ply ones are half of unnotched ones. The two lay-up notched specimens are slightly below the unnotched ones in stiffness. The strength of the specimens are decrease as temperature rise. As the temperature rise the stiffness of quasi-isotropic ones drop, but it just change little in cross-ply ones. Then the notched specimen fatigue life and load-cycle (P-N) curves were obtained by tension-tension fatigue test. The P-N curves were adopt to prevent the stress concretion of the notched specimen. Consider the same loading, notched specimens has worse fatigue behavior, but in the same load ratio, the normalized P-N curves of the unnotched ones were below the notched ones means notched ones has better fatigue behavior. Recording the specimen image by video camera during the testing process, the cracks at the edge of hole were found. However delamination was not found. Necking was observed in quasi-isotropic specimens, but not in cross-ply. Observed by optical microscopy, the improved surface treatment will decrease the probability of delamination from 20% to less than 10% after hot press.

Notch signaling is important for cell-cell signaling during development. Notch signaling is highly conserved across all metazoans and failure in Notch signaling is causative in many human diseases. In the Drosophila eye, activation of the Notch pathway requires Lqf (Drosophila Epsin)-dependent and Clathrin-dependent internalization of the Notch receptor ligands, Delta or Serrate, by the signal-sending cells. However, it is unclear why ligand must be internalized into the signal-sending cells to activate Notch signaling in the signal-receiving cells. Evidence suggests that in addition to Clathrin and Epsin, Auxilin is essential for signaling and is indirectly required for internalization of the Notch receptor ligand Delta. Auxilin functions in uncoating Clathrin-coated vesicles to maintain a pool of free Clathrin and Epsin in the cell. auxilin mutants were used as an entryway to identify previously unknown components of the Notch signaling pathway. An F1, FLP/FRT, EMS screen was performed and enhancers of an auxilin mutant rough eye defect were isolated. The enhancers ultimately formed one complementation group on the 2nd chromosome and fourteen complementation groups on the 3rd chromosome. Three of the 3rd chromosome complementation groups were each identified as Delta, lqf, or hsc70. A single allele was identified as faf. Delta and Epsin have known roles in signaling cells to activate Notch as described above. Hsc70 is an ATPase that functions with Auxilin to uncoat Clathrin-coated vesicles and Faf is a deubiquitinating enzyme that maintains levels of active Epsin in the cell. These results suggest I have isolated mutations in genes closely tied to Notch signaling or functioning directly with Auxilin. Mutations in two genes previously undescribed in Notch signaling in the developing Drosophila eye were also isolated from the screen and identified. The second chromosome complementation group was identified as α-adaptin. α-Adaptin is a subunit of the heterotetrameric Clathrin adaptor protein AP-2. One of the third chromosome complementation groups was identified as crumbs. Crumbs is an integral membrane protein that functions at adherens junctions and in establishing apical/basal polarity in cells. Characterizing roles for α-Adaptin and Crumbs during Notch signaling may elucidate the purpose for Delta internalization to activate Notch signaling. / text

No description available.

The choroid plexus is an organ within each brain ventricle comprised of elaborate folds of epithelium (CPe) and vasculature. It performs numerous functions essential for brain development and health, including secretion of cerebrospinal fluid (CSF) and acting as the blood-CSF barrier. Functionality requires: (1) that CPe and vasculature develop in register and in close proximity, so that the CPe ensheaths the vasculature at a high surface area to volume ratio, which permits efficient CSF secretion; and (2) that CPe barrier integrity is sustained throughout choroid plexus expansion. Genetic experiments in mouse embryos have identified a central role for Sonic hedgehog (Shh) in coordinating these developmental challenges. Specifically, Shh is secreted by differentiated CPe and drives choroid plexus expansion. In the absence of Shh, a hypoplastic choroid plexus forms, which is deficient in CPe, vasculature, and villous folds. Two choroid plexus cell populations respond to Shh: (1) rhombic lip-resident CPe progenitor cells and (2) vascular pericytes. Here, I present evidence that canonical Shh signaling to CPe progenitors alone is insufficient to fully drive their proliferation at normal rates. Rather, Shh-triggered pericytes appear to secondarily boost CPe progenitor cell proliferation, in addition to acting in vascular development. Shh-triggered pericytes also appear necessary for formation of the characteristic folds of the choroid plexus. Thus, pericytes coordinate the expansion of choroid plexus epithelium and vasculature. Notch signaling was also explored and was found to inhibit the differentiation of CPe progenitors, maintaining them in a proliferative state. Notch activation in CPe progenitors leads to invaginated tubules from the overproliferating CPe progenitor domain, without associated vascular growth or villous folds. Folding morphogenesis may thus be regulated by vascular components such as pericytes, and require that vascular growth match CPe growth. To identify Shh-induced pericyte signaling programs that might underlie these developmental processes, expression profiling was performed on dsRed-labeled pericytes isolated from Shh-deficient versus wild-type choroid plexuses. Candidate genes, including several involved in lipid metabolism, were identified. Collectively, this work points to pericytes as central in orchestrating the coordinated elaboration of multiple choroid plexus cell types, producing the complex tissue architecture required for efficient CSF production.

Το φυλοσύνδετο γονίδιο wiser (CG32711) είναι απαραίτητο για την ανάπτυξη της Drosophila melanogaster. Η μελέτη μιας θερμοευαίσθητης, θανατογόνου μετάλλαξης που ονομάζεται wisertsl αποκάλυψε ότι το γονίδιο wiser εμπλέκεται μεταξύ άλλων στην ανάπτυξη των φτερών. Η μετάλλαξη wisertsl οφείλεται σε ένα P στοιχείο (7E P) που βρίσκεται 490 bp ανοδικά του σημείου έναρξης της μεταγραφής του γονιδίου wiser. 95 bp καθοδικά του 7E P στοιχείου υπάρχει μια P{lacW} ένθεση υπεύθυνη για τη θανατογόνο μετάλλαξη PL26. Οι μεταλλάξεις wisertsl και PL26 είναι αλληλόμορφα του ίδιου γονιδίου ενώ 12000 περίπου βάσεις ανοδικά του γονιδίου wiser και 490 bp ανοδικά του γονιδίου trf2 υπάρχει μια άλλη P{lacW} ένθεση που είναι υπεύθυνη για τη θανατογόνο μετάλλαξη PL28. Οι PL26 και PL28 δεν δείχνουν συμπληρωματικότητα με τη μετάλλαξη wisertsl όσον αφορά το θανατογόνο φαινότυπο στους 29ºC. Όμως το διαγονίδιο UAS-wiser δε διασώζει το θανατογόνο φαινότυπο του PL28. Τα αποτελέσματα της παρούσας εργασίας αποκάλυψαν ότι: 1) Το γονίδιο wiser αλληλεπιδρά με το γονίδιο dpp. Εκτοπική έκφραση του διαγονιδίου (UAS wiser) υπό τον έλεγχο του οδηγού στελέχους apGAL4, μειώνει την έκφραση του dpp στην περιοχή του εμβρυικού δίσκου που θα δώσει τμήμα του θώρακα (notum). 2) Σε ομόζυγα wisertsl άτομα η έκφραση των γονιδίων dpp, dad, omb και salm (όπως αποκαλύπτεται από την έκφραση των αντίστοιχων –lacZ διαγονιδίων) μειώνεται στον εμβρυικό δίσκο του φτερού. Τα παραπάνω γονίδια είναι απαραίτητα για την ανάπτυξη του προσθοπίσθιου άξονα του εμβρυικού δίσκου του φτερού που σημαίνει ότι και το γονίδιο wiser εμπλέκεται στο σχηματισμό του. 3) Το γονίδιο wiser αλληλεπιδρά με το γονίδιο Notch (N) καθώς N wisertsl /wisertsl θηλυκά έχουν εντονότερα φαγωμένα φτερά. 4) Οι μεταλλάξεις wisertsl και PL28 είτε αφορούν και οι δύο το γονίδιο wiser ή η PL28 αφορά το γονίδιο trf2 που σημαίνει ότι και αυτό εμπλέκεται στο σχηματισμό του φτερού. / The X- linked wiser (CG32711) gene is a vital gene for the development of Drosophila melanogaster. The study of a temperature sensitive lethal mutation, named wisertsl, revealed that the wiser gene is implicated among others in the development of wings. The wisertsl mutation is due to a wild P element (7E P) located 490 bp upstream of the presumed transcription start site of the gene wiser at the region 7Ε. 95 bp downstream of the 7E P element is located a P{lacW} responsible for the lethal mutation PL26 and ~ 12000 bp upstream of the gene wiser and 490 bp upstream of the gene trf2 exists another P{lacW} insertion which is responsible of the lethal mutation PL28. The mutations PL26 and PL28 do not show complementation with the wisertsl mutation as regards the lethal phenotype at 29°C. However, while the transgene UAS-wiser saves the lethal phenotypes of wisertsl and PL26 it does not save the lethal phenotype of the mutation PL28. The present data study revealed that: 1) The wiser gene interacts with the dpp gene. Ectoping expression of the UAS wiserCDS construct under the control of apGAL4 driver, reduced the dpp expression (revealed by dpp-lacZ) in the notum territory of the wing imaginal disc. 2) In the homozygous wisertsl individuals the expression of dpp, dad, salm and omb genes (revealed by the corresponding -lacZ strains) is reduced in the wing imaginal disc. The above genes are implicated in the development of the anterior-posterior (A/P) axis of the wing imaginal disc. 3) The wiser gene interacts with the Notch (N) gene. N wisertsl/wisertsl females have stronger notching phenotype. 4) The induction of mitotic clones revealed that the mutation PL28 either concerns an enhancer of the wiser gene or the gene trf2. At the late case the gene trf2 must affect the development of the wings as well.

Both Notch and the Wnt pathways are key regulators in maintaining the homeostasis in the intestine. Defects on the key tumor suppressor adenomatous polyposis coli, APC a gene in the Wnt pathway is most frequently mutated in colorectal cancer. Previous studies have indicated that there is a crosstalk between these two pathways. We investigate if there is correlation by first using bioinformatics to find Lef1/Tcf sites in several of the Notch pathway gene promoters. Bioinformatically we found that a lot of the genes contained theses sites controlled by the APC's destruction target β-catenin. By using semi quantitative PCR and western blot we found that Hes 1, Hes 7, JAG 2, MAML 1, Notch 2, NUMB, NUMBL, RFNG and LFNG was downregulated in HT29 colon cancer cells carrying a vector containing wild type APC. All but JAG 2 contains at least one Lef1/Tcf site in their promoter region. The results were verified in HT29 cells transfected with siRNA against β-catenin. We also investigated what would happen to the Lef1/Tcf target gene program of the Wnt pathway, if the Notch pathway was inhibited with the gamma-secretase inhibitor DAPT. Results showed no downregulution of β-catenin or its target gene Cyclin D1.Taken together, these results demonstrate that the Wnt pathway can be placed upstream of the Notch pathway and regulates the latter through β-catenin and the Lef1/Tcf target gene program. However, preliminary results indicate that there is no regulation of APC/β-catenin by the Notch pathway.