The timely and selective proteasomal degradation of proteins is important for the maintenance of proper cellular processes. Prior to proteasomal degradation, proteins destined for degradation are polyubiquitiylated by ubiquitin ligases (E3s). The SKP1-CUL1-F-box protein (SCF) complex is a member of the cullin-RING ligase (CRL) superfamily of modular, multi-protein E3s, in which the F-box protein (FBP) acts as a substrate specificity factor for the recruitment of substrates to the SCF complex. Many of the 69 human FBPs remain uncharacterized. From our current knowledge of FBPs, we know that they regulate a myriad and diverse set of cellular processes and their misregulation is associated with diseases, including cancer. Though many approaches exist for the identification of SCF substrates and/or interactors, most existing genetic and quantitative proteomic methods are not capable of adaptor identification, while interaction proteomic approaches are generally performed in a low throughput manner. To facilitate our characterization of FBPs, we have developed a novel, facile proteomic approach for the identification of interactors, including substrates, of FBPs. In this method, FBPs, which together with SKP1 form the adaptor subunit of the SCF complex, are individually expressed in cells. These cells are subsequently exposed to 3 conditions: left untreated, treated with MLN4924 (neddylation inhibitor) or treated with Bortezomib (proteasome inhibitor). After treatment, cells are lysed, and the lysates are affinity-purified and processed in parallel for proteomic analysis. This approach, which we have named Parallel Adaptor Capture (PAC) proteomics, was successfully applied for the characterization of three novel FBP/substrate interactions: FBXW11 (β-TrCP2)/DEPTOR (Appendix 1), FBXL17/BACH1 (Chapter 2) and FBXO22/KDM4A (Chapter 3). Besides the characterization of these FBPs, PAC proteomics was performed on 19 leucine-rich repeats containing FBPs, the FBXLs, identifying 230 high confidence interacting proteins, including known regulatory proteins and substrates. Deciphering the biological context and significance of these interactions will allow us to understand the importance of FBXLs in the cellular processes in which they regulate. Since CRLs require neddylation to efficiently ubiquitylate their substrates and most CRL substrates are degraded by the proteasome, PAC proteomics, in principle, can also be applied to other CRL adaptors.

Recent research advances in Discrete EVent system Specification (DEVS) as well as cellular space modeling emphasized the need for high performance modeling methodologies and environments. The growing demand for cellular space models has directed researchers to use different implementation formalisms. Many efforts were dedicated to develop cellular space models in DEVS in order to employ the advantage of discrete event systems. Unfortunately, the conventional implementations degrade the performance in large scale cellular models because of the huge volume of inter-cell messages generated during simulation. This work introduces a new multi-layer formalism for cellular DEVS models that assures high performance and ease of user specification. It starts with the parallel DEVS specification layer and derives a high performance cellular DEVS layer using the property of closure under coupling. This is done through converting the parallel DEVS into its equivalent non-modular form which involves computational and communication overhead tradeoffs. The new specification layer, in contrast to multi-component DEVS, is identical to the modular parallel DEVS in the sense of state trajectories which are updated according to the modular message passing methodology. The equivalency of the two forms is verified using simulation methods. Once the equivalency has been ensured, analysis of the models becomes a decisive factor in employing modularity in cellular DEVS models. Non-modular models show significant speedup in simulation runs given that their event list handler is implemented based on analytical and experimental survey that involve actual operation counts. However, the new high performance non-modular specification layer is complicated to implement. Therefore, a third layer of specification is proposed to provide a simple user specification that is automatically converted into the fast complex cellular DEVS specification, which is finally put in the standard parallel DEVS specification. A tool was implemented to automatically accept user's model specification via GUI and generate the models using the new specifications. The generated models are then required to be tested and verified using some automatic DEVS verification methods. As a result, the model development and verification processes are made easier and faster.

Because Drosophila do not grow after initiation of metamorphosis, their final body size is determined by larval growth rate and duration of the larval growth phase. Drosophila metamorphosis is triggered by the steroid hormone ecdysone, which is produced in the prothoracic gland (PG). Ecdysone synthesis requires expression of the "Halloween" genes, which encode ecdysone biosynthetic enzymes. Growth rate is regulated by Insulin-like peptides, which are released from the insulin-producing cells (IPCs). Genetic ablation of the IPCs decreases growth rate and delays onset of metamorphosis, suggesting that ecdysone synthesis is induced by insulin signaling. Inhibiting PI3 Kinase (PI3K), the major effector of insulin signaling, in the PG similarly delays metamorphosis as a consequence of decreased ecdysone synthesis and decreased Halloween gene expression. In contrast, activating PI3K in the PG advances the onset of metamorphosis and increases Halloween gene expression. Here I report that increased insulin signaling, accomplished inhibiting the protein kinase A pathway in the IPCs increases insulin signaling and increases growth rate but also advances the onset of metamorphosis by increasing expression of at least one Halloween gene. Ecdysone synthesis is promoted by a second peptide hormone, PTTH, which activates Halloween gene expression via the Torso receptor followed by Ras and Raf in the PG. Null mutations in the transcription factor broad (br ) prevent torso transcription and thus prevent Halloween gene expression and metamorphosis. Here I identify Br as the mechanistic link between PI3K activity and Halloween gene expression. I found that PI3K activity is required for br expression by inhibiting the downstream kinase GSK-3. I provide evidence that three nuclear hormone receptors, βFTZ-F1, HR3 and E75, link GSK-3 activity with br expression: RNAi-mediated βFTZ-F1 or HR3 knockdown, or E75A overexpression, in the PG prevents br expression. I also found that ectopic Torso pathway activation, accomplished by expressing the constitutively active Rafgof , restores Halloween gene transcription to larvae lacking br or βFTZ-F1 , suggesting that these larvae fail to express Halloween genes because they fail to transcribe torso . These studies identify a potential molecular mechanism linking growth rate with competence to respond to the PITH metamorphic signal and thus initiate metamorphosis.

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<p>The process of cytotoxic T lymphocyte (CTL) killing involves the recognition and destruction of foreign antigens by cytotoxic T cells and is of crucial importance to the defence of the organism against viral infections. Defects in this process can lead to various autoimmune diseases and cancer. The aim of this study was to identify more genes involved in the cell death pathway and to link CTL killing, apoptosis and cancer.</p>

Cellular metals are a relatively new class of engineering materials that can be fabricated with either a random or controlled cellular structure. A controlled cellular structure allows the precise control of the pore geometry and hence subsequent material properties that can be important for some applications such as orthopathic implants. Recently the interest in using magnesium (Mg) as a biodegradable implant in the body has been growing rapidly. However, current methods for fabricating cellular magnesium typically results in a random arrangement of the cellular structure. A novel processing method is developed for the preparation of cellular metals with controlled open-cellular architectures. In particular, this process has been developed for magnesium due to the difficulties associated with powder processing. The fabrication routine utilises a multistage inverse templating technique implemented with assistance of a rapid prototyping (RP) technique. Rapid prototyped polymer performs of desired architectures were infiltrated using a specially designed slurry of NaCl. Removal of the polymer resulted in an accurate negative NaCl template that could be infiltrated with liquid metal using low pressure die casting. Subsequently, the template material is removed, resulting in a controlled cellular structure within Mg. Prior to metal infiltration, the compressive modulus, strength, grain growth and microstructure of the NaCl structure with and without sintering was examined using compression testing and electron backscattered diffraction (EBSD). For the EBSD analyses a new sample preparation technique for the micro porous samples had to be developed for use in the scanning electron microscopy (SEM). The NaCl and the cellular metal were evaluated using SEM and micro-computed tomography (µ-CT). Furthermore, the relationship between the original CAD model and the final NaCl pore morphology was investigated were the surfaces of the RP scaffold and the NaCl template were analysed and compared to the as-cast Mg.

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<p>Traditional <italic>Drosophila</italic> hearts screens have focused on early patterning and development, and adult heart phenotypes have only recently been pursued due to difficulty in accurately measuring cardiac function in adult <italic>Drosophila</italic>. For my dissertation I performed a screen in <italic>Drosophila</italic> using optical coherence tomography (OCT) to phenotype cardiac function in awake, adult <italic>Drosophila</italic>, in order to discover novel disease-causing and disease-modifying genes for heart failure. I initiated a screen of X chromosome deficiency stocks for mutants displaying abnormal cardiac function in the adult, and I identified two mutant strains from the X chromosome with the phenotype of dilated cardiomyopathy. These deficiencies of 125kb and 92kb each correspond to 10 and 16 deleted genes in each, respectively. Interestingly, the candidate genes did not include any sarcomeric proteins, nor any proteins previously implicated in heart function. Utilizing genetic tools including customized deletions, RNAi constructs, and transgenic rescues, I identified the causative gene in each deficiency. I show that cardiomyopathic genes can be identified in adult <italic>Drosophila</italic> using genetics and noninvasive phenotyping methodologies.</p> / Dissertation

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