"Adhesion Molecules have been associated with a number of neurological and psychological disorders (humans), and implicated in various developmental processes (animals). Better understanding the development of the nervous system and the roles of adhesion molecules in it may be crucial to better understanding these disorders. LIGs, Leucine Rich Repeat and ImmunoGlobulin containing transmembrane proteins, represent a novel class of such adhesion molecules and have been implicated in various neuronal processes, including neurite outgrowth, axonal pathfinding, neuronal regeneration and survival. Two such LIGs are Kek1 and Kek2, members of a Drosophila LIG family, which have been reported to function in axonal pathfinding and synaptic plasticity, respectively. It is unclear what their roles in these processes are, as well as if other members of the Drosophila LIG family have similar roles. Current studies aim to survey the Kekkon family function in the nervous system, looking to identify new phenotypes and/or to elucidate the mechanisms underlying previously identified phenotypes. To achieve this goal, tissue specific inducible RNAi technique was employed. Validating of a number of transgenic RNAi stocks obtained was necessary and showed that all stocks obtained promoted specific and efficient knock down of target gene. Next an assessment of RNAi knockdown efficacy in developing nervous system was carried out and knockdown was shown to be weak if not in the presence of Dicer-2 co-misexpression. A number of screens for general behavioral phenotypes were performed including ubiquitous, neural, and imaginal discs knockdown. These uncovered possible effects of kek1 neural knockdown, as well as possible interaction of Kek1 with neurotactin, neuroglian and kek2. NMJ analysis of Kek5 and Kek6 was also carried out and preliminary results indicate possible interaction of kek5 in NMJ, although no statistical significance was detected."

Cyanobacteria are solar-powered cell factories that can be engineered to supply us with renewable fuels and chemicals. To do so robust and well-working biological parts and tools are necessary. Parts for controlling gene expression are of special importance in living systems, and specifically promoters are needed for enabling and simplifying rational design. Synthetic biology is an engineering science that incorporates principles such as decoupling, standardization and modularity to enable the design and construction of more advanced systems from simpler parts and the re-use of parts in new contexts. For these principles to work, cross-talk must be avoided and therefore orthogonal parts and systems are important as they are decoupled by definition. This work concerns the design and development of biological parts and tools that can enable synthetic biology in cyanobacteria. This encompasses parts necessary for the development of other systems, such as vectors and translational elements, but with a focus on transcriptional regulation. First, to enable the development and characterization of promoters in different cyanobacterial chassis, a broad-host-range BioBrick plasmid, pPMQAK1, was constructed and confirmed to function in several cyanobacterial strains. Then, ribosome binding sites, protease degradation tags and constitutive, orthogonal promoters were characterized in the model strain Synechocystis PCC 6803. These tools were then used to design LacI-regulated promoter libraries for studying DNA-looping and the behaviour of LacI-mediated loops in Synechocystis. Ultimately, this lead to the design of completely repressed LacI-regulated promoters that could be used for e.g. cyanobacterial genetic switches, and was used to design a destabilized version of the repressed promoter that could be induced to higher levels. Further, this promoter was used to implement an orthogonal transcriptional system based on T7 RNAP that was shown to drive different levels of T7 promoter transcription depending on regulation. Also, Gal4-repressed promoters for bacteria were engineered and examined in Escherichia coli as an initial step towards transferring them to cyanobacteria. Attempts were also made to implement a light-regulated one-component transcription factor based on Gal4. This work provides a background for engineering transcription and provides suggestions for how to develop the parts further.

No description available.

Les nanoparticules (NP) de poly(acide lactique) (PLA) sont des vecteurs biodégradables prometteurs pour la vaccination et la délivrance thérapeutique. Cependant leur évaluation in vivo n'est pas toujours couronnée de succès. Un des écueils réside dans la difficulté à suivre la prise en charge cellulaire de ces nanomatériaux à l'échelle d'un organisme, ces NP étant indécelables dans les tissus profonds. L'objectif de cette thèse est de valider l'utilisation des NP de PLA comme plateforme d'imagerie et de vectorisation d'actifs chez Drosophila Melanogaster et d'analyser ainsi le devenir de NP dans un corps entier. Le modèle drosophile a été choisi pour son faible encombrement, sa facilité d'élevage, la diversité des lignées transgéniques et la puissance des outils génétiques à disposition. Il permet également de mener des études mécanistiques in vivo dans un laps de temps restreint. Nous avons évalué in cellulo et in vivo la toxicité de ces NP afin d'établir des conditions optimales expérimentales. Ensuite le potentiel des NP de PLA a été évalué in cellulo sur des cellules de drosophile transfectées transitoirement par un plasmide porteur du gène GFP sous le contrôle du promoteur UAS. Les NP vectorisant le gène gal4 ou la protéine GAL4 permettent de confirmer par simple observation en microscopie à fluorescence l'efficacité de délivrance de molécules actives dans la cellule via la fixation de la protéine GAL4 sur le promoteur UAS. Enfin, ces formulations ont été administrées par voie orale à des drosophiles transgéniques UAS-RFP pour confirmer les résultats précédents in vivo. GAL4 est un outil prometteur pour le suivi indirect de NP dans des organismes transgéniques / The biodegradable NanoParticles (NPs) of PolyLactic Acid) (PLA) are promising vectors for vaccination and therapeutic delivery. However, their in vivo evaluation is not always successful. NPs being undetectable in deep tissues, one of the challenges is the difficulty to follow the cellular uptake of these nanomaterials at the organism level. The aim of this thesis is to validate the use of PLA NPs as an imaging and drug vectorization platform in Drosophila melanogaster, and to analyze their fate in the whole fly body. The Drosophila model was chosen for its small footprint, the ease of breeding, the variety of transgenic lines, and the power of genetic tools available. Tt also allows to carry out in vivo mechanistic studies in a limited time window. We evaluated in cellulo and in vivo toxicity of these NP to determine optimal experimental conditions. Then the potential of PLA NPs was evaluated in cellulo on transiently transfected Drosophila cells by a plasmid carrying the GFP gene under the control of the UAS promoter. A simple observation by fluorescence microscopy of NPs vectorizing the gal4 gene or the GAL4 protein can confirm the effective delivery of active molecules into the cell through the binding of GAL4 protein to the UAS promoter. Finally, these formulations were orally administered to transgenic Drosophila UAS-RFP to confirm the previous in vivo results. GAL4 is a promising tool for indirect monitoring of NPs in transgenic organisms

Genomic and proteomic investigations have yielded, and continue to produce, a large amount of information about genes and their protein products. In contrast, the evidence bearing on physiological roles of specific proteins is much more scarce. To address the functional part of biological inquiry, one would like to perturb, at will and selectively, the function of any protein of interest in vivo and to analyze the resulting phenotypic effects, thereby probing the protein’s role in a cell. Ideally, a method for doing so should be applicable both to individual gene products and to a large collection of them. Gene knockouts, a powerful tool to study gene function, have limitations in the study of development when the early phenotypes are cell- or organismal- lethal. Conditional mutants are particularly useful for analysis of genes whose functions are essential for the organism’s viability. A conditional mutant retains the function of a gene under one set of conditions, called permissive, and shows an inactive phenotype under a different set of conditions, called nonpermissive; the latter must be still permissive for the wild type (wt) allele of a gene. Conditional mutants make possible the analysis of physiological changes that follow controlled inactivation of a gene or gene product and can be used to address the function of any gene. Temperature sensitive (ts) mutants are an important class of conditional mutants whose phenotype is similar to that of wt at lower (permissive) temperature, but show low or reduced level of activity above a certain temperature called restrictive temperature, while the wt gene shows a similar phenotype at both the temperatures. Ts mutants provide an extremely powerful tool to study gene expression in vivo and in cell culture. They provide a reversible mechanism to lower the level of a specific gene product simply by changing the temperature of growth of the organism. Ts mutants are typically generated by random mutagenesis; either by ultraviolet light, a chemical mutagen or by error-prone PCR followed by often laborious screening procedures. Therefore, they are cumbersome to make, especially in the case of organisms with long generation times. Keeping in view the importance of ts mutants in biology, Varadarajan et al. 1996, had developed an algorithm to predict ts mutants at predicted, buried sites of a globular protein from its amino acid sequence. Experimental tests of the algorithm were carried out on the CcdB toxin of Escherichia coli to further refine and improve the method (Chakshusmathi et al. 2004). Based on this result simple rules for the design of ts mutants were suggested. This thesis aims at validating and improving on these rules and to find out if ts mutants of a protein can also be generated by perturbing functionally important residues. In addition, it is currently unclear with what frequency ts mutants of a protein isolated in one organism will show a ts phenotype in a completely different organism. This thesis makes preliminary efforts to address this issue. The model system chosen to carry out these studies is a protein called Gal4, which is a yeast transcriptional activator. This protein is biologically relevant as it has been used for ectopic gene expression in diverse organisms including yeast, fruitflies, zebrafish, mice and frogs (Ornitz et al. 1991; Brand and Perrimon 1993; Rahner et al. 1996; Andrulis et al. 1998; Scheer and Camnos-Ortega 1999; Hartley et al. 2002). The introductory chapter (Chapter 1) discusses the importance of ts mutants and our understanding and progress in this field so far, relevant for the work reported in this thesis. Chapter 2 describes generation of ts mutants of Gal4 in yeast. Full length Gal4 (fGal4) is an 881-aa protein. To simplify the construction of ts Gal4, we have designed a functional truncated Gal4 (miniGal4 or mGal4) of 197 residues. Five residues (9, 10, 15, 18 and 23) of the Gal4 DNA binding domain, which are in close contact with the DNA, were randomized in mGal4. Based on average hydrophobicity and hydrophobic moment, 68, 69, 70, 71, and 80 are the only residues in the region 1-150 that are predicted to be buried at the 90% confidence level. Of these five sites, residues 68, 69 and 70 were chosen for mutagenesis. At these three sites, four stereochemically diverse substitutions (Lys, Ser, Ala and Trp) were made. In a separate set of experiments each predicted, buried residues were also individually randomized in both mini and in full length Gal4 (fGal4). In all cases, we have been successful in isolating ts mutants in more than one position. At both permissive and restrictive temperatures, the activity of the Gal4 ts mutants is substantially lower than the wt. However, at the restrictive temperature, the activity of the ts Gal4 is lowered below the threshold required for reporter gene expression. This view of how ts mutants function is quite different from the general notion that the ts and wt behave similarly at permissive temperatures. Chapter 3 deals with transferability of two of the ts constructs mutated at DNA binding residues (R15W and K23P) to Drosophila. Two fGal4 encoding DNA fragments carrying the mutations were cloned into P element vectors under control of Elav and GMR promoters and several transgenic Drosophila lines were generated. These were crossed to various UAS reporter lines and progeny were characterized for reporter gene expression as a function of temperature. We show that both of these yeast ts mutants also show a ts phenotype in Drosophila. We have compared our ts Gal4 system with a popularly used system (TARGET) (McGuire et al. 2003) used for conditional gene expression in Drosophila. Our ts Gal4 mutants appear to provide tighter control at the restrictive temperature and a more uniform and rapid induction of gene expression upon shifting from the restrictive to the permissive temperature than the TARGET system with the promoters and the reporters we have used. Although cold sensitive (cs) mutants are often more useful than ts mutants, for reasons currently unclear, cs mutants are much more difficult to isolate than ts mutants. In Chapter 4, we have attempted to convert the ts phenotypes observed with Gal4 mutants in Drosophila and CcdB mutants in E. coli (Chakshusmathi et al. 2004) to cs phenotypes by increasing the expression level of these mutant proteins selectively at higher temperature. Several ts mutants of CcdB have been previously reported (Chakshusmathi et al. 2004). For converting the ts phenotype observed by E. coli toxin CcdB mutants (Chakshusmathi et al. 2004) to a cs phenotype, the arabinose inducible plasmid pBAD24CcdB and its mutant derivatives were used. By inducing expression of the mutant protein at higher temperature with arabinose, while keeping the basal level of expression without arabinose at lower temperature, we have been able to show cold sensitive behavior by these CcdB ts mutants in E. coli. For producing a cs phenotype with Gal4 mutants in Drosophila, we have used a P element vector where the GMR element is placed in-between hsp70 binding sites. This driver results in enhanced expression of downstream genes at 30 relative to 18°C because of the presence of the hsp elements (Kramer and Staveley 2003). Ts mutants at DNA binding and buried residues of fGal4 were cloned into this vector and several transgenic lines for each construct were obtained. The Gal4 mutants at exposed DNA binding residues but not at buried residues show a cs phonotype when they were crossed to various UAS-reporters lines. The buried residue mutants are likely to be destabilized and their degradation pathway might differ in yeast and in Drosophila. Because of this, these mutants might not be showing the desired cs phenotype in Drosophila. Although mGal4 and fGal4 have very similar activities in yeast, it was necessary to examine if they also had identical activities in Drosophila. Determining their relative activities in Drosophila is the aim of Chapter 5. To this end, mGal4 was cloned into P element vectors under control of hsp70 or GMRhs promoters and transgenic flies were generated. The transgenic lines were crossed to various UAS-reporters and reporter gene activities in the progeny were characterized. Although mGal4 and fGal4 showed similar activity in yeast, in Drosophila for reasons that are currently unclear, mGal4 was considerably less active than fGal4. As some of the fGal4 mutants showed a cs phenotype under GMRhs driver as shown in the earlier chapter (Chapter 4), several ts mutants of mGal4 in yeast in buried and as well as at the DNA binding residues were transferred to Drosophila under hs and GMRhs promoter. The transgenic lines obtained were tested for cold sensitivity by crossing with various UAS-reporter lines. However, in all cases mutant mGal4 showed an inactive phenotype in Drosophila. We suggest that this is because the intrinsic activity of these mGal4 mutants is substantially weaker than wt mGal4 even at permissive temperature in yeast. The further lowering of activity in Drosophila pushes the activity below the threshold required for reporter gene expression resulting in an inactive phenotype. The concluding chapter (Chapter 6) summarizes the conclusions drawn from this entire study and provides insights into possible mechanisms responsible for ts and cs phenotypes. The mutant phenotypes of Gal4 in yeast and in Drosophila suggest that ts phenotypes appear to result from a threshold effect. Such mutations lower the activity and/or level of the protein relative to the wt at all temperatures. Since maximal stability temperatures are rarely in excess of room temperature, with an increase in temperature, the activity of an already marginally active mutant can fall below the threshold required for function resulting in a temperature sensitive phenotype. The strategies we used for producing ts mutants have several advantages over standard approaches of generating ts alleles by random mutagenesis. We anticipate that conclusions of this study would be useful for generation of ts mutants of other globular proteins in diverse organisms. We also show that exposed, functional residues involved in protein: ligand or protein: protein interactions appear to be attractive candidate sites for generating ts mutants that are transferable between organisms. In addition, the active site mutants of fGal4 in Drosophila, which show ts and cs phenotypes depending on the Drosophila promoter chosen for expression, can be used for conditional and reversible expression of a number of other genes using the Gal4-UAS system (Brand and Perrimon 1993).

The mammalian genome encodes five collapsin response mediator protein (CRMP) isoforms. Cell culture studies have shown that the CRMPs mediate growth cone dynamics and neuron polarity through associations with a variety of signal transduction components and cytoskeletal elements. CRMP is also a member of a protein family including the presumably ancestral dihydropyrimidinase (DHP) protein that catalyzes the second step in pyrimidine degradation. In Drosophila, CRMP and DHP proteins are produced by alternatively spliced transcripts of the CRMP gene. The alternative protein forms have a 91% sequence identity, but unique expression patterns. CRMP is found exclusively in neuronal tissues and DHP is ubiquitously expressed in non-neuronal tissues. Comparative analysis of CRMP homologous sequences from insect taxa show CRMP alternative splicing is a common feature and probably represents the ancestral state of this gene family. To investigate the regulatory role of CRMP, loss-of-function mutations of CRMP that lack both proteins were isolated; homozygous animals display DHP-null phenotypes but exhibit no overt developmental or neurological defects. To determine possible interactions of Drosophila CRMP with signaling pathways in which mammalian CRMP has been shown to act, the UAS-GAL4 system was utilized. Phenotypes produced by misexpression of a variety of UAS signal transduction mediator responders were modified in a CRMP mutant background. The modification entails enhancement or suppression of a specific phenotype in a direction that corresponds to the hypothesized involvement of mammalian CRMP in signaling pathways that regulate growth cone dynamics. These data suggest that Drosophila CRMP has a role in cell signaling pathways similar to the role of the mammalian CRMPs. Furthermore, recent findings demonstrate that CRMP plays an important role in learning and memory of mice, leading to the assessment of new phenotypes in the Drosophila CRMP mutants. Tests utilizing the Pavlovian olfactory conditioning assay reveal that loss of CRMP function leads to significant learning, 3 hour memory, and long term memory deficits. Preliminary data also suggest that Drosophila CRMP may be required for normal circadian locomotor rhythms. Collectively, the data presented here demonstrate CRMP’s role in adult behavioral processes and regulating signaling events comparable to mammalian CRMP signaling.

A fatty acid amide is precisely as the name suggests: A fatty acid (CHn-COOH), in which the hydroxyl group of the carboxylic acid is displaced by an amine functional group from a biogenic amine (R-NH2), ultimately forming an amide bond. Furthermore, these fatty acid amides can be composed of a variety of different acyl chain lengths donated by the fatty acid and a myriad of different biogenic amines. Thus, these molecules can be subdivided in a number of different ways including the separation of short chain (acetyl to heptanoyl) and long chain (palmitoyl to arachidonoyl) and also based off the biogenic amine type. The long chain fatty acid amides quickly gained the interest of the scientific community through the discovery of anandamide (N-arachidonoylethanolamide), which was found to be the endogenous ligand for the cannabinoid receptor-1 (CB1) found in the mammalian brain. This particular neural molecule is an N-acylethanolamide, which is one specific classification of long chain fatty acid amide. However, there exist other types of long chain fatty acid amides including the N-acylglycines, primary fatty acid amides (PFAMs) and N-acylarylalkylamides. Yet, despite the type of fatty acid amide, it has been shown many of these types of molecules are synthesized using a type of N-acyltransferase. These N-acyltransferases are believed to be members of the GCN5-related superfamily of N-acyltransferases (GNAT), which share the feature of being able to accept acyl-CoA thioester substrates. This dissertation will discuss and demonstrate the extraction of all types of the aforementioned classifications of long chain fatty acid amides but will have a particular focus on the N-acylarylalkylamides. Elucidating more about the biosynthetic pathways and metabolic routes of the long chain fatty acid amides could lead to the development of potential therapeutics and pest control agents. We have determined Drosophila melanogaster arylalkylamine N-acyltransferase like 2 is responsible for the in vivo biosynthesis of N-acyldopamines. We have also demonstrated Bombyx mori is another suitable model systems for the study of long chain fatty acid amides, as three insect arylalkylamine N-acyltrasnferase from Bombyx mori (Bm-iAANAT) were found to share some homology in primary sequence (25-29%) to AAANTL2 in Drosophila melanogaster. We show herein that one of these enzymes is able to catalyze the formation of long chain N-acylarylalkylamides in vivo. The change in the transcription of these enzymes was tracked to try and understand if these enzymes serve a focused purpose in the physiological development of the insect. If it is found one of these Bm-iAANAT are crucial for growth, it may elucidate a general function of the enzyme, which may be able to inhibit growth of specific insects that are known pests, while not targeting endangered insects like Apis melliferra (honey bee). Understanding this would help in the eventual creation of targeted insecticides on specific insect pests Furthermore, a novel panel of fatty acid amides was characterized and quantified in extracts from this organism via LC-QToF-MS, ultimately showing it is very possible the Bm-iAANATs are performing this catalysis in vivo.

Soy contains many bioactive molecules known to elicit anti-cancer effects. One such peptide, Lunasin, has been shown to selectively act on newly transformed cells while having no cytotoxic effect on non-tumorigenic or established cancer cell lines. In this study we attempt to understand the developmental effects of Lunasin overexpression in vivo and create reagents that will help us understand Lunasin’s anti tumorigenic effects in an intact organism. cDNA encoding lunasin and EGFP-lunasin were cloned into pUAST and microinjected into Drosophila embryos. Tissue-specific overexpression of EGFP-Lun in the resulting transgenic lines was accomplished by crossing transgenics to various GAL4 driver lines. Progeny were assessed for phenotypic alterations and no phenotypic abnormalities were observed in tissues expressing EGFP-Lunasin, supporting current studies that show Lunasin does not affect normal cells. Previous studies have localized Lunasin to the nuclear compartment. To test if this was the case for EGFP-Lun, subcellular localization of EGFP-Lun was determined via fluorescence microscopy. Salivary glands from EGFP-Lun expressing individuals were dissected, fixed, and mounted in Vectashield® with the nuclear stain, DAPI. Our results demonstrate that EGFP-Lun, like native Lunasin, is localized to the nucleus. Eight transgenic lines were mapped to specific chromosomes and EGFP-Lun transgenic line GEJ1-L2 was balanced in preparation for use in tumor suppression studies. In summary, we have created and characterized transgenic flies capable of overexpressing Lunasin under the control of the GAL4/UAS system. Localization of EGFP-Lunasin to the nucleus and data on the phenotypic consequence of its overexpression in flies is presented. Finally, reagents created as part of this thesis will aid experiments aimed at understanding the effects of Lunasin on benign and invasive tumors.

京都大学 / 新制・課程博士 / 博士(生命科学) / 甲第24758号 / 生博第499号 / 新制||生||66(附属図書館) / 京都大学大学院生命科学研究科高次生命科学専攻 / (主査)教授 今吉 格, 教授 鈴木 淳, 教授 谷口 雄一 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DGAM

Two problems hampering efforts to produce salt-tolerant plants through constitutive expression of transgenes include: 1. Spatial control. Particular cell-types must respond specifically to salt stress to minimise the amount of Na⁺ delivered to the shoot; and, 2. Temporal control. Transgenes are typically expressed in plants at similar levels through time, irrespective of the stress encountered by the plant, which may exacerbate pleiotropic effects and means that, particularly in low-stress conditions, costly and/or detrimental metabolic processes may be active, thus reducing yield. To address these issues, Gateway® destination vector constructs were developed combining the GAL4 UAS (upstream activating sequence) with the ethanol-inducible gene expression system to drive inducible cell-specific expression of Na⁺ transporter transgenes (or to silence salt transporter transgenes inducibly and cell-specifically). Rice (Oryza sativa L. cv. Nipponbare) GAL4-GFP enhancer trap lines (Johnson et al., 2005: Plant J. 41, 779-789) that express GAL4 and GFP specifically in either the root epidermis or xylem parenchyma (and therefore ‘trap’ cell-type specific enhancer elements) were transformed with this GAL4 UAS – ethanol switch construct, thereby allowing both spatial and temporal control of transgenes. In preliminary experiments, the expression system successfully limited the expression of RFP to specific cell-types after induction with ethanol. Other genes expressed using this system include PpENA1, a Na⁺-extruding ATPase from the moss, Physcomitrella patens, and AtHKT1;1, a Na ⁺ transporter from Arabidopsis thaliana. The two enhancer trap rice lines were also transformed with the GAL4 UAS driving stable expression of AtHKT1;1 and PpENA1 specifically in root epidermal or xylem parenchyma cells. Expression of AtHKT1;1 in root epidermal cells reduced Na⁺ accumulation in the shoots, while expression in the root xylem parenchyma appeared to have little effect on shoot Na⁺ accumulation. Using cryo-scanning electron microscopy (SEM) X-ray microanalysis, the outer cells of the roots of the line expressing AtHKT1;1 in the epidermal cells were found to accumulate higher levels of Na⁺ than the parental enhancer trap line. Additionally, this line had decreased unidirectional ²²Na⁺ influx. Similar results were observed for plants expressing AtHKT1;1 driven by the CaMV 35S / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1325289 / Thesis (Ph.D.) -- University of Adelaide, School of Agriculture, Food and Wine, 2008