Fibroblast growth factor (Fgf) and Retinoic acid (RA) are known to be involved in patterning the posterior embryo. Work has shown that Fgf can convert anterior tissue into posterior fates and that embryos deficient in Fgf signaling lack posterior trunk and tail structures. Likewise, studies performed on RA have shown that overexpression of RA posteriorizes anterior tissue, while disrupting RA signaling yields a loss of posterior fates. While it appears these signals are necessary for posterior development, the role Fgf and RA play in development of the hindbrain is still enigmatic. A detailed study of the requirements for Fgf and RA in the early vertebrate hindbrain are lacking, namely due to a deficiency in gene markers for the presumptive hindbrain at early developmental stages. In this study, we make use of recently isolated genes, which are expressed in the presumptive hindbrain region at early developmental stages, to explore Fgf and RA regulation of the early vertebrate hindprain. We employed both overexpression and loss of function approaches to explore the role of Fgf in early vertebrate development with an emphasis on the presumptive hindbrain region in zebrafish embryos. By loss of function analysis, we show that Fgf regulates genes expressed exclusively in the hindbrain region (meis3 and hoxbla) as well as genes whose expression domains encompass both the hindbrain and more caudal regions (nlz and hoxb1b), thus demonstrating a requirement for Fgf signaling throughout the anteroposterior axis of the hindbrain (rostral to caudal hindbrain) by mid-gastrula stages. To further characterize early gene regulation by Fgf, we utilized an in vitro system and found that Fgf is sufficient to induce nlz directly and hoxb1b indirectly, while it does not induce meis3 or hoxb1a. Furthermore, in vivo work demonstrates that Fgf soaked beads can induce nlz and hoxb1b adjacent to the bead and meis3at a distance. Given the regulation of these genes in vitro and in vivo by Fgf and their position along the rostrocaudal axis of the embryo, our results suggest an early acting Fgf resides in the caudal end of the embryo and signals at a distance to the hindbrain. We detect a similar regulation of hindbrain genes by RA at gastrula stages as well, suggesting that both factors are essential for early hindbrain development. Interestingly however, we find that the relationship between Fgf and RA is dynamic throughout development. Both signals are required at gastrula stages as disruption of either pathway alone disrupts hindbrain gene expression, but a simultaneous disruption of both pathways at later stages is required to disrupt the hindbrain. We suggest that Fgf and RA are present in limiting concentrations at gastrula stages, such that both factors are required for gene expression or that one factor is necessary for activation of the other. Our results also reveal a changing and dynamic relationship between Fgf and RA in the regulation of the zebrafish hindbrain, suggesting that at segmentation stages, Fgf and RA may no longer be limiting or that they are no longer interdependent. As we have demonstrated that an early Fgf signal is required for gastrula stage hindbrain development, we next questioned which Fgf performed this function. We have demonstrated that the early Fgf signal required for hindbrain development is not Fgf3 or Fgf8, two Fgfs known to be involved in signaling centers at the mid-hindbrain boundary (MHB) and rhombomere (r) 4. We further show that two recently identified Fgfs, Fgf4 and Fgf24 are also insufficient alone or in combination with other known Fgfs to regulate hindbrain gene expression. However, as Fgfs may act combinatorially, we do not rule out the possibility of their involvement in early hindbrain gene regulation. However, as time passes and additional Fgfs are isolated and cloned, the elusive Fgf signal required for early hindbrain development will likely be identified. Taken together, we propose that an early acting Fgf residing in the caudal end of the embryo regulates hindbrain genes together with RA at gastrula stages. We suggest that both Fgf and RA are required for gene expression at gastrula stages, but this requirements changes over time as Fgf and RA become redundant. We also demonstrate that the Fgf required for gastrula stage hindbrain development has yet to be identified.

The observations that substance P (SP) is localized in the anterior pituitary gland (AP) and is regulated by the hormonal status of the animal, as well as the demonstration of SP binding sites in the AP, have led to the idea that SP may participate in the regulation of AP function. Numerous and sometimes contradictory reports of SP effects on AP hormone secretion, particularly on luteinizing hormone (LH), left the question of whether SP acts directly at the level of the AP to regulate LH secretion still unanswered. To investigate a possible physiological function of SP in the AP, the effects of exogenous SP on LH secretion from AP cells from adult and prepubertal male and female rats in short term culture were studied. It was found that SP (100nM-1μM) significantly stimulates LH release in cultured AP cells and that this effect varies as a function of age and sex. SP has no significant effect on LH release from AP cells of male and female prepubertal rats. After day 30 a sharp increase in the response to SP occurs in both sexes. This level of responsiveness continues through adulthood in AP cells from the female rat. In contrast, AP cells from male rats failed to respond during adulthood (over 50 days of age) but were highly responsive during the peripubertal period (30-35 days). The possibility that the responsiveness to SP is influenced by the endocrine status of the animal was investigated by exposing AP cells from responding animals to androgens in vivo and in vitro. It was found that AP cells from female rats treated with androgen were less responsive to 100nM SP but did respond at higher doses of SP. SP effects on AP function were further analyzed in experiments using radioligand binding assays to assess possible changes in SP receptor number or affinity as related to age and sex. In AP membranes from female rats, maximum binding is 8-fold higher (Bmax=4.2 pmo1/mg membrane protein) than in AP membranes from male rats (Bmax=560fmo1/ mg membrane protein). These studies suggest a role for SP as a secondary regulator of LH secretion with possible physiological significance for reproductive function.

This thesis details my examination of several mechanisms for modulation of N-type calcium channels in neonatal rat superior cervical ganglion (SCG) neurons. The first part of this work characterizes cross-talk between two distinct mechanisms of modulation: readily-reversible inhibition induced by activation of heterotrimeric G-proteins (termed G-protein-mediated inhibition), and phosphorylation of the channel by protein kinase C (PKC). Data previously presented by other groups suggested that one effect of activating PKC is to prevent G-protein-mediated inhibition. The goal of this project was to confirm this hypothesis by testing functional competition between these two pathways. My findings show that G-protein-mediated inhibition blocks the effects of activating PKC, and that phosphorylation by PKC blocks G-protein-mediated inhibition, confirming that these two mechanisms are mutually exclusive. In addition, I investigated the effect of activating PKC on whole-cell barium currents in the absence of G-protein-mediated inhibition. When endogenous G-proteins were inactivated by dialyzing the cell with GDP-β-S, a guanine nucleotide that prevents activation of the G-protein's α subunit, activation of PKC with phorbol esters was without obvious effect on whole-cell current amplitude, fast and holding potential-dependent inactivation, and voltage-dependent activation, suggesting that PKC's principal role in modulating these currents is to prevent G-protein-mediated inhibition. From these results, I advanced Bean's 1989 model of reluctant and willing gating (induced by G-protein-mediated inhibition and relief of that inhibition, respectively). In this expanded model, reluctant channels, inhibited by G-proteins, are resistant to phosphylation by PKC (reluctant/P-resistant). Unmodulated channels are called willing/available, as they exhibit willing gating, and are available for either binding to a G-protein or phosphorylation by PKC. Finally, phosphorylation of a willing/available channel by PKC drives the channel into the willing/G-resistant state, in which the channel gates willingly, and is resistant to G-protein-mediated inhibition. These results are published in the Journal of General Physiology(2000; 115:277-286), and are presented in this thesis as Chapter II. In addition to membrane-delimited inhibition, N-type calcium channels are also subject to inhibition via a diffusible second-messenger pathway. In SCG neurons, this inhibition can be observed following stimulation of M1 muscarinic receptors by the agonist oxotremorine-M. Our lab previously hypothesized that the diffusible messenger involved might be the polyunsaturated fatty acid arachidonic acid (AA). To test this hypothesis, our lab examined the effect of bath-applied AA on whole-cell SCG calcium currents, and demonstrated that AA induces inhibition with similar properties as M1 muscarinic inhibition. An analysis of AA's effects on unitary N-type calcium currents, published by Liu and Rittenhouse in Journal of Physiology(2000; 525:391-404), revealed that this inhibition is mediated, at least in part, by both a significant increase in the occurrence of null-activity sweeps and a significant decrease in mean closed dwell time. Based on these results, our lab conducted an examination of AA's effects on whole-cell currents in SCG neurons, and found that AA-induced inhibition is mediated by an increase in holding potential-dependent inactivation and appears independent of AA metabolism. When I examined AA's effects in greater detail, I discovered that, in addition to inhibition, AA also appeared to cause significant enhancement of whole-cell currents. The results characterizing AA's general effects on whole-cell calcium currents in SCG neurons have been published in American Journal of Physiology - Cell Physiology(2001; 280:C1293-C1305). Because my finding that AA enhances whole-cell neuronal calcium currents revealed a novel pathway through which this current can be modulated, I proceeded to characterize this effect. My results showed that enhancement develops significantly faster than inhibition, suggesting different mechanisms or pathways. In addition, dialyzing the cell with BSA, a protein that binds fatty acids, blocked the majority of AA-induced inhibition, but did not reduce enhancement, suggesting that enhancement is independent of inhibition and might be mediated at an extracellular site. Using fatty acid analogs that cannot cross the cell membrane, I confirmed that enhancement occurs extracellularly. My data also indicate that AA-induced enhancement of whole-cell currents does not require metabolism of AA, consistent with enhancement being mediated directly by AA. I also examined the biophysical characteristics of enhancement, and found that both an increase in the voltage sensitivity of activation and an increase in activation kinetics underlie this effect. Finally, using both pharmacological agents and a recombinant cell line, I presented the first demonstration that AA enhances N-type calcium current. These findings are described in detail in a paper recently published in American Journal of Physiology - Cell Physiology(2001; 280:C1306-C1318), and are presented in this thesis as Chapter III. In our investigation of AA's effects on whole-cell calcium currents, we utilized a voltage protocol, in conjunction with pharmacology, to enhance the level of L-type current in these cells. Since whole-cell calcium currents in SCG neurons are comprised of mostly (80-85%) N-type current, with the remaining current comprised of mostly L-type current, this approach allowed us to examine both N- and L-type currents. When currents are recorded in the presence of 1 μM FPL 64174 (FPL), a benzoyl pyrrole L-type calcium channel agonist first described in 1989, stepping the membrane potential to -40 mV following a test pulse to +10 mV generates a slowly-deactivating ("tail") current. This tail current is made up entirely of L-type current, and allows us to readily investigate the effect of various modulatory mechanisms on this current type. Although FPL has been used for almost a decade to study L-type calcium currents, activity of FPL on N-type calcium currents has not been investigated. Because our lab routinely uses micromolar concentrations of FPL to measure whole-cell and unitary calcium currents in neuronal cells, I tested whether FPL has any effects on N-type calcium current. Therefore, I examined the effect of FPL on whole-cell calcium currents in an HEK 293 cell line that expresses recombinant N-type calcium channels. Application of 1 and 10 μM FPL caused significant, voltage-independent inhibition of currents, demonstrating that FPL inhibits N-type calcium current. Thus, at micromolar concentrations, FPL is not selective for L-type calcium current, and any examination of its effects on whole-cell calcium currents should take this into account. The results describing FPL's effects on L- and N-type calcium currents are included in a manuscript currently in preparation, and are presented as Chapter IV.

A rapid cascade of regulatory events defines the differentiated fates of embryonic cells, however, once established, these differentiated fates and the underlying transcriptional programs can be remarkably stable. Here, we describe two proteins, MEP-1, a novel protein, and LET-418/Mi-2, both of which are required for the maintenance of somatic differentiation in C. elegans. MEP-1 was identified as an interactor of PIE-1, a germ-specific protein required for germ cell specification, while LET-418 is a protein homologous to Mi-2, a core component of the nuc1eosome remodeling/histone deacetylase (NuRD) complex. In animals lacking MEP-1 and LET-418, germline-specific genes become derepressed in somatic cells, and Polycomb group (PcG) and SET domain-related proteins promote this ectopic expression. We demonstrate that PIE-1 forms a complex with MEP-1, LET-418, and HDA-1. Furthermore, we show that the overexpression of PIE-1 can mimic the mep-1/let-418 phenotype, and that PIE-1 can inhibit the Histone deacetylase activity of the HDA-1 complex in COS cells. Our findings support a model in which PIE-1 transiently inhibits MEP-1 and associated factors to maintain the pluripotency of germ cells, while at later times MEP-1 and LET-418 remodel chromatin to establish new stage- or cell-type-specific differentiation potential.

Asymmetric cell divisions constitute a basic foundation of animal development, providing a mechanism for placing specific cell types at defined positions in a developing organism. In a 4-cell stage embryo in Caenorhabditis elegansthe EMS cell divides asymmetrically to specify intestinal cells, which requires a polarizing signal from the neighboring P2 cell. Here we describe how the extracellular signal from P2 is transmitted from the membrane to the nucleus during asymmetric EMS cell division, and present the identification of additional components in the pathways that accomplish this signaling. P2/EMS signaling involves multiple inputs, which impinge on the Wnt, MAPK-like, and Src pathways. Transcriptional outputs downstream of these pathways depend on a homolog of β-catenin, WRM-1. Here we analyze the regulation of WRM-1, and show that the MAPK-like pathway maintains WRM-1 at the membrane, while its release and nuclear translocation depend on Wnt/Src signaling and sequential phosphorylation events by the major cell-cycle regulator CDK-1 and by the membrane-bound GSK-3 during EMS cell division. Our results provide novel mechanistic insights into how the signaling events at the cortex are coupled to the asymmetric EMS cell division through WRM-1. To identify additional regulators in the pathways governing gut specification, we performed suppressor genetic screens using temperature-sensitive alleles of the gutless mutant mom-2/Wnt, and extra-gut mutant cks-1. Five intragenic suppressors and three semi-dominant suppressors were isolated in mom-2 suppressor screens. One extragenic suppressor was mapped to the locus ifg-1, eukaryotic translation initiation factor eIF4G. From the suppressor screen using cks-1(ne549), an allele of the self-cleaving nucleopore protein npp-10 was identified as a suppressor of cks-1(ne549)and other extra-gut mutants. Taken together, these results help us better understand how the fate of intestinal cells are specified and regulated in early C. elegans embryos and broaden our knowledge of cell polarity and fate specification.

In my work, I will focus on interpersonal conflict management and ways to prevent them. A description of how to ideally act in conflict situations and achieve the best possible solution to the "right" way.

Les cavités artificielles funéraires sont à la fin de la Préhistoire en Méditerranée occidentale un type d’architecture privilégié. La compréhension de la chaîne opératoire de construction des hypogées est essentielle afin de déterminer quelles techniques et stratégies entrent en jeu dans la mise en place de ces architectures souterraines. C’est dans le cadre de cette thèse qu’une méthode a été développée sur le sujet. Celle-ci se présente en trois axes : l’étude technologique du macro-outillage de creusement, l’analyse à l’aide de la photogrammétrie des traces présentes sur les parois des hypogées et l’expérimentation permettant de confirmer ou d’infirmer les hypothèses émises. Ces trois axes ont été développés dans des contextes différents, en France dans les Bouches-du-Rhône avec l’ensemble monumental de Fontvieille et en Sardaigne avec notamment la nécropole à domus de janas de S’Elighe Entosu. Ces deux contextes offrent la possibilité d’étudier des hypogées possédant des caractéristiques différentes. La comparaison entre les divers contextes sélectionnés en Méditerranée occidentale permet d’entrevoir les différences et similitudes présentes dans les chaînes opératoires de creusement et les choix effectués par les différents groupes humains. Au-delà de la connaissance des techniques employées pour le creusement de ces architectures, cette étude permet également de s’interroger sur le rôle de celles-ci au sein de la communauté par l’investissement que leur mise en place engendre. / At the end of Prehistory in the Western Mediterranean, the rock-cut tombs are a privileged type of architecture. The understanding of the hypogea’s digging process is essential In order to determine which techniques and strategies come into play in the establishment of these structures. In the context of a PhD research work, a method has been developed on the subject. It is presented in three lines of reflection: the technological study of digging macro-tools, the analysis of the traces which are present on the walls of hypogea using photogrammetry, and an experimentation to confirm or refute the assumptions made during the first two axes of the study. These three axes were developed in different contexts, the monumental context of Fontvieille, located in the geographical area of the Bouches-du-Rhône administrative department in France and the necropolis of S'Elighe Entosu in Sardinia, Italy. These two fieldworks allows us to study the different characteristics of the hypogea. The comparison between the various contexts selected in the western Mediterranean allows us to glimpse the differences and similarities present in the digging process chains and the choices made by the different human groups. Beyond the knowledge of the techniques used for the digging of these architectures, this study also makes it possible to question the role of these in the community by the investment that their establishment generates.

C1q TNF Related Protein 3 (CTRP3), is a cytokine that is primarily secreted from adipose tissue, which classifies it as an adipokine. Our previous research has shown that CTRP3 prevents alcoholic fatty liver disease (ALD) in male mice. However, even when accounting for confounding factors such as absolute and relative alcohol intake, females are more sensitive to the effects of consumption compared to male mice. Therefore, the goal of this project was to determine whether CTRP3 prevented ALD in female mice. Methods: Female wild type (WT) and female CTRP3 transgenic over expressing (Tg) mice were fed an ethanol containing liquid diet (5% v/v) for 6 weeks. Daily weight and food intake measurements were taken and external heat-pads were placed under a portion of the cage to facilitate thermoregulation. Hepatic steatosis was determined by total triglyceride quantification and lipid droplet quantitation in liver sections. Data were analyzed by repeated measures ANOVA, t-test, or Log-rank (Mantel-Cox) test as appropriate. Results: There was no difference between WT and Tg mice in food intake or body weight. There was no difference in survival between WT and Tg mice, however, Tg mice trended towards a reduced rate of survival compared with WT mice (78% in WT versus 44% in Tg, p=0.13). Stereological analysis indicated no difference in the percent of lipid liver volume between the two groups (WT 7.2±3.6 vs Tg 5.1±4.1%). This finding was consistent with no difference in total hepatic triglyceride accumulation observed between WT and Tg mice (12.7±4.4 vs. 13.1±6.8 mg triglycerides/gram liver protein). Conclusion: Combined these data indicate that unlike previous studies with male mice, CTRP3 is not protective against alcohol-induced hepatic steatosis in female mice. Combined, these data indicate that the adipokines such as CTRP3 contribute to physiology in a sex-specific manner.

Relational Web tables have become an important resource for applications such as factual search and entity augmentation. A major challenge for an automatic identification of relevant tables on the Web is the fact that many of these tables have missing or non-informative column labels. Research has focused largely on recovering the meaning of columns by inferring class labels from the instances using external knowledge bases. The table context, which often contains additional information on the table's content, is frequently considered as an indicator for the general content of a table, but not as a source for column-specific details. In this paper, we propose a novel approach to identify and extract column-specific information from the context of Web tables. In our extraction framework, we consider different techniques to extract directly as well as indirectly related phrases. We perform a number of experiments on Web tables extracted from Wikipedia. The results show that column-specific information extracted using our simple heuristic significantly boost precision and recall for table and column search.

Synaptic plasticity—the ability of a synapse to change—is fundamental to basic brain function and behavioral adaptation. Studying the mechanisms of synaptic plasticity benefits our understanding of the formation of neuronal connections and circuitry, which has great implications in the field of learning and memory and the studies of numerous human diseases. The Drosophila larval neuromuscular junction (NMJ) system is a powerful system for studying synaptic plasticity. The NMJ consists of at least two different types of motorneurons innervating the body wall muscles. Type I motorneurons controls muscle contraction using glutamate as the neurotransmitter, while type II are modulatory neurons that contain octopamine. Octopamine is a potent modulator of behavior in invertebrates. Nevertheless, its function at the synapse is poorly understood. In my thesis research, I investigated the role of octopamine in synaptic plasticity using the Drosophila NMJ system. Preliminary observations indicate that increased larval locomotion during starvation results in an increase of filopodia-like structures at type II terminals. These structures, which we termed as “synaptopods” in our previous studies, contain synaptic proteins and can mature into type II synapses. I demonstrated that this outgrowth of type II terminals is dependent on activity and octopamine. Mutations and genetic manipulations affecting the production of octopamine decrease synaptopods, whereas increase of type II activity or exogenous application of octopamine increase synaptopods. Interestingly, I found that the type II octopaminergic neurons have an absolute dependence on activity for their innervation of the muscles. Blocking activity in these neurons throughout development results in no type II synapses at the NMJ, whereas blocking activity after the formation of synapses results in gradual degradation of type II terminals. Next, I examined the autoregulatory mechanism underlying the octopamine-induced synaptic growth in octopaminergic neurons. I discovered that this positive-feedback mechanism depends on an octopamine autoreceptor, Octß2R. This receptor in turn activates a cAMP- and CREB-dependent pathway that is required in the octopamine-induction of synaptopods. Furthermore, I demonstrated that this octopaminergic autoregulatory mechanism is necessary for the larva to properly increase its locomotor activity during starvation. Thirdly, I investigated the possibility that type II innervation might regulate type I synaptic growth through octopamine. We found that ablation, blocking of type II activity, or the absence of octopamine results in reduced type I outgrowth, and this paracrine signaling is mediated by Octß2R which is also present in type I motorneurons. Lastly, the function of another octopamine receptor, Octß1R, was examined. In contrast to Octß2R, Octß1R is inhibitory to synaptic growth. I demonstrated that the inhibitory effect of this receptor is likely accomplished through the inhibitory G-protein Goα. Similar to Octß2R, Octß1R also regulates the synaptic growth of both type I and type II motorneurons in a cell-autonomous manner. The inhibitory function of this receptor potentially breaks the positive feedback loop mediated by Octß2R, allowing the animal to reset its neurons when the environment is favorable. In summary, the research presented in this thesis has unraveled both autoregulatory and paracrine mechanisms in which octopamine modulates synaptic and behavior plasticity through excitatory and inhibitory receptors.