Erythropoietin (Epo) modulates red blood cell production (erythropoiesis) by binding to its receptor and activating STAT5, a Signal Transducer and Activator of Transcription (STAT) protein implicated in both basal and stress erythropoiesis. Epo concentration in serum changes over three orders of magnitude, as it regulates basal erythropoiesis and its acceleration during hypoxic stress. However, it is not known how STAT5 translates the changes in Epo concentration into the required erythropoietic rates. We addressed this question by studying STAT5 phosphorylation, at the single cell level, in developing erythroblasts. We divided erythroid progenitors in tissue into several flow-cytometric subsets and found that each of them exhibited distinct modes of Stat5 activation, based on their developmental stage. STAT5 activation is bistable in mature erythroblasts, resulting in a binary (or digital), low-intensity STAT5 phosphorylation signal (p-Stat5). In early erythroblasts, and in response to stress levels of Epo, the low intensity bistable p-Stat5 signal is superseded by a high-intensity graded, or analog, signal. The gradual shift from high-intensity graded signaling in early erythroblasts to low intensity binary signaling in mature erythroblasts is due to a decline in STAT5 expression with maturation. We were able to convert mature, digital transducing erythroblasts into analog transducers simply by expressing high levels of exogenous STAT5. We found that EpoR-HM mice, expressing a mutant EpoR that lacks STAT5 docking sites, generate the binary, but not the analog, STAT5 signal. Unlike Stat5-null mice, which die perinatally, the EpoR-HM mice are viable but deficient in their response to stress, demonstrating that while binary STAT5 signaling is sufficient to support basal erythropoiesis, analog signaling is required for the stress response. Bistable systems contain a positive loop, which is important for flipping the switch between the two stable ‘on’ or ‘off’ states. We show that bistable activation is the result of an autocatalytic loop in which active STAT5 promotes further STAT5 activation. The isolated STAT5 N-terminal domain, which is not required for STAT5 phosphorylation, enhanced autocatalysis, converting a high intensity graded signal into a high intensity binary response. The N-terminal domain is known to participate in a radical conformational reorientation of STAT5 dimers inherent in STAT5 activation. We propose that the N-terminal domains of active STAT5 dimers facilitate the conformational reorientation of inactive dimers, in a prion-like autocatalytic interaction that underlies bistability and binary signaling. Together, bistable STAT5 activation, combined with a graded response allow erythropoietic rate to faithfully reflect a wide Epo concentration range, while preventing aberrant signaling.

An animal’s ability to navigate through its natural environment is critical to its survival. Navigation can be slow and methodical such as an annual migration, or purely reactive such as an escape response. How sensory input is translated into a fast behavioral output to execute goal oriented locomotion remains elusive. In this dissertation, I aimed to investigate escape response behavior in the nematode C. elegans. It has been shown that the biogenic amine tyramine is essential for the escape response. A tyramine-gated chloride channel, LGC-55, has been revealed to modulate suppression of head oscillations and reversal behavior in response to touch. Here, I discovered key modulators of the tyraminergic signaling pathway through forward and reverse genetic screens using exogenous tyramine drug plates. ser-2, a tyramine activated G protein-coupled receptor mutant, was partially resistant to the paralytic effects of exogenous tyramine on body movements, indicating a role in locomotion behavior. Further analysis revealed that ser-2 is asymmetrically expressed in the VD GABAergic motor neurons, and that SER-2 inhibits neurotransmitter release along the ventral nerve cord. Although overall locomotion was normal in ser-2 mutants, they failed to execute omega turns by fully contracting the ventral musculature. Omega turns allow the animal to reverse and completely change directions away from a predator during the escape response. Furthermore, my studies developed an assay to investigate instantaneous velocity changes during the escape response using machine based vision. We sought to determine how an animal accelerates in response to a mechanical stimulus, and subsequently decelerates to a basal locomotion rate. Mutant analysis using this assay revealed roles for both dopamine and tyramine signaling. During my doctoral work, I have further established the importance for tyramine in the nematode, as I have demonstrated two additional roles for tyramine in modulating escape response behavior in C. elegans.

The central nervous system (CNS) is composed of two types of cells: neurons that send electrical signals to transmit information throughout the animal and glial cells. Glial cells were long thought to be merely support cells for the neurons; however, recent work has identified many critical roles for these cells during development and in the mature animal. In the CNS, glial cells act as the resident immune cell and they are responsible for the clearance of dead or dying material. After neuronal injury or death, glial cells become reactive, exhibiting dramatic changes in morphology and patterns of gene expression and ultimately engulfing neuronal debris. This rapid clearance of degenerating neuronal material is thought to be crucial for suppression of inflammation and promotion of functional recovery, but molecular pathways mediating these engulfment events remain poorly defined. Drosophila melanogaster is a genetically tractable model system in which to study glial biology. It has been shown that Drosophila glia rapidly respond to axonal injury both morphologically and molecularly and that they ultimately phagocytose the degenerating axonal debris. This glial response to axonal debris requires the engulfment receptor Draper and downstream signaling molecules dCed-6, Shark, and Rac1. However, much remains unknown about the molecular details of this response. In this thesis I show that Drosophila c-Jun kinase (dJNK) signaling is a critical in vivo mediator of glial engulfment activity. In response to axotomy, glial dJNK signals through a cascade involving the upstream MAPKKKs Slipper and TAK1, the MAPKK MKK4, and ultimately the Drosophila AP-1 transcriptional complex composed of JRA and Kayak to initiate glial phagocytosis of degenerating axons. Interestingly, loss of dJNK also blocked injury-induced up-regulation of Draper levels in glia and glial-specific over-expression of Draper was sufficient to rescue phenotypes associated with loss of dJNK signaling. I have identified the dJNK pathway as a novel mediator of glial engulfment activity and show that a primary role for the glial Slipper/Tak1→MKK4→dJNK→dAP-1 signaling cascade is activation of draper expression after axon injury.

The final step of paramyxovirus infection requires the assembly of viral structural components at the plasma membrane of infected cells followed by budding of virions. While the matrix (M) protein of some paramyxoviruses has been suggested to play a central role in the assembly and release of virus particles, the specific viral and host protein requirements are still unclear. Using Newcastle disease virus (NDV) as a prototype paramyxovirus, we explored the role of each of the NDV structural proteins in virion assembly and release. For these studies, we established a virus-like particle (VLP) system for NDV. The key viral proteins required for particle formation and the specific viral protein-protein interactions required for assembly and release of particles were explored in chapter 2. First we found that co-expression of all four proteins resulted in the release of VLPs with densities and efficiencies of release (1.18 to 1.16 g/cm3and 83.8%±1.1, respectively) similar to that of authentic virions. Expression of M protein alone, but not NP, F-K115Q or HN proteins individually, resulted in efficient VLP release. No combination of proteins in the absence of M protein resulted in particle release. Expression of any combination of proteins that included M protein yielded VLPs, although with different densities and efficiencies of release. To address the roles of NP, F and HN proteins in VLP assembly, the interactions of proteins in VLPs formed with different combinations of viral proteins were characterized by co-immunoprecipitation. The co-localization of M protein with cell surface F and HN proteins in cells expressing all combinations of viral proteins was characterized. Taken together, the results show that M protein is necessary and sufficient for NDV budding. Furthermore, they suggest that M protein – HN protein and M protein - NP interactions are responsible for incorporation of HN protein and NP proteins into VLPs and that F protein is incorporated indirectly due to interactions with NP and HN protein. Since the vacuolar protein sorting (VPS) system is involved in the release of several enveloped RNA viruses, chapter 3 describes studies which explored the role of the VPS system on NDV particle release. First, we characterized the effects of three dominant negative mutant proteins of the VPS pathway on particle release. Expression of dominant negative mutants of CHMP3, Vps4 and AIP1 proteins inhibited M protein particle release as well as release of complete VLPs. Mutation of a YANL sequence in the NDV M protein to AANA inhibited particle release while replacement of this sequence with either of the classical late domain motifs, PTAP or YPDL, completely restored particle release. The host protein AIP1, which binds YXXL late domain sequences, is incorporated into M protein particles. These results suggest that an intact VPS pathway is necessary for NDV VLP release and that the YANL sequence is an NDV M protein L domain. The sequence and structure of the Newcastle disease virus (NDV) fusion (F) protein are consistent with its classification as a type 1 glycoprotein. We have previously reported, however, that F protein can be detected in at least two topological forms with respect to membranes in both a cell-free protein synthesizing system containing membranes as well as infected COS-7 cells (J. Virol. 2004 77:1951). One form is the classical type 1 glycoprotein while the other is a polytopic form in which approximately 200 amino acids of the amino terminal end as well as the cytoplasmic domain (CT) are translocated across membranes. Furthermore, we detected CT sequences on surfaces of F protein expressing cells and antibodies specific for these sequences inhibited red blood cell fusion to HN and F protein expressing cells suggesting a role for surface expressed CT sequences in cell-cell fusion. In chapter 4, we extended these findings and found that the alternate form of the F protein can also be detected in infected and transfected avian cells, the natural host cells of NDV. Furthermore, the alternate form of F protein was also found in virions released from both infected COS-7 cells and avian cells by Western analysis. Mass spectrometry confirmed its presence in virions released from avian cells. Two different polyclonal antibodies raised against sequences of the CT domain of the F protein slowed plaque formation in both avian and COS-7 cells. Antibody specific for the CT domain also inhibited single cycle infections as detected by immunofluorescence of viral proteins in infected cells. The potential roles of this alternate form of the NDV F protein in infection are discussed. Virus-like particles (VLPs) generated from different viruses have been shown to have potential as good vaccines. Chapter 5 explored the potential of NDV VLPs as a vaccine for NDV or as a vaccine vector for human pathogens. Significant quantities of NDV VLPs can be produced from tissue culture cells. These VLPs are as pure as virions prepared in eggs. In addition, some rules for incorporation of viral proteins into VLPs were also explored. We found that the cytoplasmic domain of the fusion (F) protein is necessary for its incorporation into VLPs. We found that an HN protein with an HA tag at its carboxyl terminus was incorporated into VLPs. We also found that the HN and F proteins of NDV, strain B1, can be incorporated into VLPs with M and NP of strain AV. The demonstration of specific domains required for protein incorporation into particles is important in using NDV VLPs as a vaccine vector for important human pathogens. In conclusion, this dissertation presents results that show that the M protein plays a central role in NDV assembly and release, a finding that is consistent with findings with other paramyxoviruses. More importantly, this work extends the current knowledge of paramyxovirus assembly and release by providing the first direct evidence of interactions between paramyxovirus proteins. These interactions between viral proteins provide a rational basis for incorporation of viral proteins into particles. This work also provides a clearer understanding of the role of the host vacuolar protein sorting machinery in NDV budding. A clear understanding of virus assembly and budding process contributes to the design of strategies for therapeutic intervention and in the development of safer, more economical and effective vaccines.

RNA interference or RNAi was first discovered as an experimental approach that induces potent sequence-specific gene silencing. Remarkably, subsequent studies on dissecting the molecular mechanism of the RNAi pathway reveal that RNAi is conserved in most eukaryotes. In addition, genes and mechanisms related to RNAi are employed to elicit the regulation of endogenous gene expression that controls a variety of important biological processes. To investigate the mechanism of RNAi in the nematode C. elegans, we performed genetic screens in search of RNAi deficient mutants (rde). Here I report the summary of the genetic screens in search of rde mutants as well as the identification of two novel genes required for the RNAi pathway, rde-3 and rde-8. In addition, we demonstrate that some of the rde genes, when mutated, render the animals developmentally defective, suggesting that these rde genes also function in developmental gene regulation. This work presents novel insights on the components of the RNAi pathway and the requirement of these components in the regulation of endogenous gene expression.

Feeding is one of the most important behaviors for an animal’s survival. At a gross level, it is known that the nervous system plays a major role in the expression of this complex behavior, yet a detailed understanding of the neural circuits directing feeding behavior remains unknown. Here we identify a command neuron in Drosophila melanogaster whose artificial activation, using dTrpA1, a heat-activated cation channel, induces the appearance of complete feeding behavior. We use behavioral, genetic, cellular and optical imaging techniques to show that the induced behavior is composed of multiple motor programs and can function to uptake exogenous, even noxious, material. Furthermore, we resolve the neuron’s location to the subesophageal ganglion, characterize its pre and post-synaptic sites, and determine its responsiveness to sucrose stimulation. Interestingly, the neuron’s dendritic field is proximal to sweet sensing axon terminals and its baseline activity corresponds to the fly’s satiation state, suggesting a potential point of integration between sensory, motor and motivational systems. The identification of a command neuron for feeding in a genetically tractable organism provides a useful model to develop a deeper understanding of the neural control of this ubiquitous and evolutionarily ancient behavior.

Emerging evidence in both vertebrates and invertebrates is redefining glia as active players in the development and integrity of the nervous system. The formation of functional neuronal circuits requires the precise addition of new synapses. Mounting evidence implicates glial function in synapse remodeling and formation. However, the precise molecular mechanisms governing these functions are poorly understood. My thesis work begins to define the molecular mechanisms by which glia communicate with neurons at the Drosophila neuromuscular junction (NMJ). During development glia play a critical role in remodeling neuronal circuits in the CNS. In order to understand how glia remodel synapses, I manipulated a key component of the glial engulfment machinery, Draper. I found that during normal NMJ growth presynaptic boutons constantly shed membranes or debris. However, a loss of Draper resulted in an accumulation of debris and ghost boutons, which inhibited synaptic growth. I found that glia use the Draper pathway to engulf these excess membranes to sculpt synapses. Surprisingly, I found that muscle cells function as phagocytic cells as well by eliminating immature synaptic ghost boutons. This demonstrates that the combined efforts of glia and muscle are required for the addition of synapses and proper growth. My work establishes that glia play a crucial role in synapse development at the NMJ and suggests that there are other glial-derived molecules that regulate synapse function. I identified one glial derived molecule critical for the development of the NMJ, a TGF-β ligand called Maverick. Presynaptically, Maverick regulates the activation of BMP pathway confirmed by reducing the transcription of the known target gene Trio. Postsynaptically, it regulates the transcription of Glass bottom boat (Gbb) in the muscle suggesting that glia modulate the function of Gbb and consequently the activation of the BMP retrograde pathway at NMJ. Surprisingly, I also found that glial Maverick regulates the transcription of Shaker potassium channel, suggesting that glia potentially could regulate muscle excitability and consequently modulate synaptic transmission. Future work will elucidate such hypothesis. My work has demonstrated two novel roles for glia at the NMJ. First is that glia engulfing activity is important for proper synaptic growth. Second is that the secretion of glial-derived molecules are required to orchestrate synaptic development. This further supports that glia are critical active players in maintaining a functional nervous system.

I detta kandidatarbete undersöker vi hur projection mapping (projicering på en yta) kan användas för att skapa en rumslig berättelse eller upplevelse. Utifrån vår egna tolkning av calm technologys (lugn teknologi) principer har vi skapat ett designperspektiv som syftar till att göra designen lugn, genom att teknologin inte ska bli störande för åskådaren. Till vår hjälp har vi använt oss utav Keri Smiths metoder till att utforska och experimentera med omgivningen och material. För att sedan kunna utöka kreativiteten och hitta nya idéer inom projection mapping har vi använt oss av metoder såsom brainstorming, moodboards och skisser. Vi har även utgått från Stolterman och Löwgrens syn på en designprocess. Detta resulterade till tre gestaltningar där alla utgår från naturens lugna atmosfär. Under detta arbete har vi kommit fram till att det finns många olika sätt att berätta med ytor, det behöver inte bara vara ett fysiskt rum med väggar och tak. Ytor kan bli rum genom att manipulera fysiska föremål med projektorer. Genom att lägga på ett extra lager på objekten som till exempel en animation så kan det förstärka objektet i sig eller få en helt ny karaktär. Med enkla medel som olika mönster, färger och ljus. / In this bachelor thesis we will examine how projection mapping (projection on a surface) can be used as a spatial experience. From our own interpretation of calm technology’s principles, will we create a design perspective that could keep the design calm with respect to the technology, and not be a disturbing moment for the viewer. To guide us through this we have taken Keri Smith’s methods to explore and experiment the environment and different materials. To expand the creativity to find new possibilities in projection mapping, we have used methods like brainstorming, moodboards and sketches. To help our process we based our project on Stolterman and Löwgren’s view of the design process. This led to a result of three figurations where all of them are inspired by the atmosphere of our nature. Through this project we have reached that there’s a lot of ways you can narrate with spaces, it doesn't need to be a physical room where you can stand in. Spaces can transform by manipulating physical artifacts. By adding an extra layer onto these artifacts like an animation it can amplify it’s meaning or give it a new character. Only with simple means like different patterns, colors and light.

L'Intelligence Artificielle est présente dans tous les aspects de notre vie à l'ère du Big Data. Elle a entraîné des changements révolutionnaires dans divers secteurs, dont le commerce électronique et la finance. Dans cette thèse, nous présentons quatre applications de l'IA qui améliorent les biens et services existants, permettent l'automatisation et augmentent considérablement l'efficacité de nombreuses tâches dans les deux domaines. Tout d'abord, nous améliorons le service de recherche de produits offert par la plupart des sites de commerce électronique en utilisant un nouveau système de pondération des termes pour mieux évaluer l'importance des termes dans une requête de recherche. Ensuite, nous construisons un modèle prédictif sur les ventes quotidiennes en utilisant une approche de prévision des séries temporelles et tirons parti des résultats prévus pour classer les résultats de recherche de produits afin de maximiser les revenus d'une entreprise. Ensuite, nous proposons la difficulté de la classification des produits en ligne et analysons les solutions gagnantes, consistant en des algorithmes de classification à la pointe de la technologie, sur notre ensemble de données réelles. Enfin, nous combinons les compétences acquises précédemment à partir de la prédiction et de la classification des ventes basées sur les séries temporelles pour prédire l'une des séries temporelles les plus difficiles mais aussi les plus attrayantes : le stock. Nous effectuons une étude approfondie sur chaque titre de l'indice S&P 500 en utilisant quatre algorithmes de classification à la pointe de la technologie et nous publions des résultats très prometteurs / Artificial Intelligence has penetrated into every aspect of our lives in this era of Big Data. It has brought revolutionary changes upon various sectors including e-commerce and finance. In this thesis, we present four applications of AI which improve existing goods and services, enables automation and greatly increase the efficiency of many tasks in both domains. Firstly, we improve the product search service offered by most e-commerce sites by using a novel term weighting scheme to better assess term importance within a search query. Then we build a predictive model on daily sales using a time series forecasting approach and leverage the predicted results to rank product search results in order to maximize the revenue of a company. Next, we present the product categorization challenge we hold online and analyze the winning solutions, consisting of the state-of-the-art classification algorithms, on our real dataset. Finally, we combine skills acquired previously from time series based sales prediction and classification to predict one of the most difficult but also the most attractive time series: stock. We perform an extensive study on every single stocks of S&P 500 index using four state-of-the-art classification algorithms and report very promising results

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