The suprachiasmatic nucleus (SCN) of the hypothalamus is the locus of a master circadian clock that is critical in the temporal organization of circadian activity. The SCN coordinates the rhythmic secretion of gonadal hormones, and in turn, reproductive hormones may act on their receptors within the SCN to alter circadian function. Using transgenic mice that over-express androgen receptor (AR) only in neurons, the current study investigated the influence of neural AR on the function of the SCN. In particular, it addressed the effects of androgens on circadian behaviours as well as physiological responses to light within the SCN by measuring Fos response after a phase-shifting light pulse. It was found that transgenic mice demonstrate a smaller increase in Fos expression in response to a light pulse than do wildtypes. Interpretations of our findings, including the possible functional significance of AR within the SCN, are discussed.

The suprachiasmatic nucleus (SCN) of the hypothalamus is the locus of a master circadian clock that is critical in the temporal organization of circadian activity. The SCN coordinates the rhythmic secretion of gonadal hormones, and in turn, reproductive hormones may act on their receptors within the SCN to alter circadian function. Using transgenic mice that over-express androgen receptor (AR) only in neurons, the current study investigated the influence of neural AR on the function of the SCN. In particular, it addressed the effects of androgens on circadian behaviours as well as physiological responses to light within the SCN by measuring Fos response after a phase-shifting light pulse. It was found that transgenic mice demonstrate a smaller increase in Fos expression in response to a light pulse than do wildtypes. Interpretations of our findings, including the possible functional significance of AR within the SCN, are discussed.

Soybean (Glycine max (L.) Merr.) cyst nematode (SCN) (Heterodera glycines Ichinohe), an obligate sedentary endoparasite, is the most economically destructive pathogen in soybean production and causes over $1 billion in annual losses in the United States. Planting resistant cultivars is the primary management method to control SCN for the long-term purpose, but the nature of genetic resistance is little known. The Rhg1 (Resistance to H. glycines) locus on chromosome 18 is found as a major quantitative trait locus (QTL) that contributes resistance to SCN. The chemical mutagen ethylmethane sulfonate (EMS) can be utilized to induce genetic mutations in soybean populations, which screened by an efficient reverse genetic strategy known as Targeting Induced Local Lesions IN Genomes (TILLING) for functional gene analyses. The objective of this study was to analyze the function of SNAP gene (Glyma18g02590) at rhg1 allele from `Forrest' (`Peking'-derived SCN resistant cultivar) using TILLING. Soybean cultivar `Forrest' seeds were mutagenized with EMS and grown to generate M1 plants. M1 plants were self-pollinated to produce approximately 3000 M2 plants. Genomic DNAs were extracted from young leaves of individual M2 plants and quantified to normalize concentration of DNAs. The DNA samples were then pooled eight-fold in 96-well plates for mutations screening by TILLING. Moreover, 12 phenotypic traits including chlorophyll deficiency, leaf shape, branch architecture, seed color, seed weight, fatty acid phenotype were identified in the mutagenized population, analyzed and archived in this study.

Soybean is one of the most important grain legumes grown in US and worldwide, and is a major component of human and animal protein diets. Despite improvements in management practices, and the introduction of improved soybean cultivars, soil borne pathogens continue to cause tremendous yield loss in soybean production each year. Among soil borne pathogens; Soybean Cyst Nematode (SCN) or Heterodera glycines together with Sudden Death Syndrome (SDS) induced by Fusarium virguliforme are responsible for the most damages in soybean fields. The most effective way to control these two pathogens is to develop resistant cultivars. Resistance to any population (HgType) of H. glycines, requires a functional allele at rhg1/Rfs2 locus. The rhg1/Rfs2 gene encodes a receptor-like kinase (RLK) protein. By analysing near isogenic lines (NIL) segregating for rhg1/Rfs2, rhg1-like loci were found at other locations most conservedly on LG B1. While the nature of rhg1 allele was thought to be recessive, heterozygous NIL segregating at the rhg1 locus showed that the resistant allele was dominant. Rhg1 was also inferred to be multigeneic due to absence of recombination between the RLK and other 2 genes. Functional and structural analyses were conducted on the leucine rich repeat (LRR) from RLK protein encoded by GmRLK18-1 within the Rhg1/Rfs2 locus. The LRR of GmRLK18-1 showed a high binding affinity to CLE-like peptides found in both nematode secretions and plant developmental control. Crosslinking assays and native gel analysis of GmRLK18-1-LRR validated its model as a crystal homo-dimer. Larger proteins were also shown to bind the LRR domain, in far-Western analyses both methionine synthase and cyclophilin bound strongly to the LRR domain. Homology and ab-initio modeling of the LRR domain of the GmRLK18-1 was predicted as both a monomer and a homodimer containing intrinsically unstructured regions. Amino acid substitutions found among GmRLK18-1 allotypes A87V, Q115K and H274N were predicted to play crucial roles in protein function and stability. The receptor like kinase (RLK) GmRLK18-1 within the Rhg1/Rfs2 locus underlies a pleiotropic resistance to both SCN and SDS. The resistance allele was shown to be dominant in both heterozygous NILs at Rhg1/Rfs2 and transgenics (hetero- or hemi-zygous). The RLK was found to provide a partial resistance to SCN and importantly a nearly complete resistance to both root and leaf symptoms of SDS. In the presence of Rhg4, the RLK-transgenic plants developed nearly full resistance to SCN. Therefore the RLK was proven to underlie a major portion of the Rhg1/Rfs2 locus.

Title: Hypersensitive and circadian effects of acebutolol administration in Scn1b-/- mice. Rationale: Dravet syndrome (DS) is a severe form of pediatric epilepsy with characterizations of pharmacoresistant seizures and developmental delay. A rarer variant of the DS model is caused by homozygous loss-of-function mutations in SCN1B, which is essential in regulating sodium channel gating, expression, localization, and the firing of action potentials. Mutations in SCN1B result in severe seizures as well as a higher risk of Sudden Unexpected Death in EPilepsy (SUDEP). Factors underlying SUDEP are poorly understood, although cardiac arrhythmias have been implicated. Acebutolol (ACE) is a common beta-blocker used in the treatment of arrhythmias and hypertension. We hypothesized that treating mice with ACE will decrease cardiac arrhythmias and the incidence of SUDEP, prolonging lifespan of Scn1b null mice. Methods: Wild-type (WT) and null (KO) mice were given daily injections of 10 mg/kg ACE or saline starting at postnatal day 15 (after typical seizure onset) either during the day (09:00) or at night (21:00). In the day group, ECG was recorded daily from P13 until animal death. Starting at P15 mice were recorded both pre- and post- injection to analyze the long-term and acute effects of treatment. Results: A modest, but significant, increase in survival curves in our KO animals was observed compared to saline treated mice for those given injections during the day (a 2 day increase in median survival). In addition, in this group, the onset of animal death was delayed. To investigate the timing of drug delivery, a subset of mice was given injections at night. In this group there was actually a decrease in lifespan, with an earlier onset of death compared to saline treated mice. On a daily basis from P13, the heart rate (HR) of KO mice was significantly lower than WT but remained steady until the day prior to animal death. HR the day prior to death consistently dropped ~50% (average 414 bpm to 193 bpm) in our saline group; this was prevented in KO animals treated with ACE (421 bpm). Analysis of acute recordings following ACE administration showed that KO mice had a significantly larger reduction in heart rate compared to WT (38% vs. 11%). Further analysis of heart rate variability in these recordings demonstrated that RMSSD (a measure of vagal control of the heart) was reduced in KO mice, with differences in both baseline and following ACE administration. Conclusions: Leading up to death, we believe it is possible ACE assisted in decreased cardiovascular deficits that could lead to SUDEP and contributed to the modestly increased lifespan. In addition, our results demonstrate the importance of timing in delivery of drugs targeted at SUDEP. Finally, these results suggest that there is a possible hypersensitivity to beta-adrenergic blockade in Scn1b-/- mice. Funding: This work was supported by a grant from the Research Development Committee at East Tennessee State University and NIH grant R21NS116647 to C.R.F.

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The Centre d'expertise hydrique du Québec (CEHQ) operates a distributed hydrological model (MOHYSE), which integrates a snow model (SPH-AV), for the management of dams in the south of Québec. It appears that the estimation of the water quantity of snowmelt in spring remains a variable with a large uncertainty. This research aims to evaluate the potential of remote sensing data for the characterization of snow and ultimately to develop methods of integration of satellite data in the snow model for the improvement of the simulations of spring floods. Remote sensing snow cover area (SCA) products (MODIS[subscript SCN] & IMS) are compared with snow depth surveys at Environment Canada stations and initial simulations of the models. Thru these comparisons, an effective method of integration (seuil[subscript ÉEN]) of remote sensing SCA products, based on the hypothesis that satellites can not identify small amount of snow because snow become"dirty" and discontinuous, was developed.The improvement of the Nash coefficient and the root mean square error for spring 2004 to 2007 for the simulations with the approach developed compared with streamflow simulated without remote sensing is 0.11 and 21% on the optimized watershed (du Nord) and 0.13 and 22% on the verification watershed (aux Écorces).The method also relies to improve peaks identification as much as 36% on the du Nord watershed and 19% on the aux Écorces watershed.The study also shows the potential of QSCAT data for the characterization of snow cover. Overall accuracies around 90% are obtained for the detection of melt during the month of April from 2001 to 2007 on both studied watersheds.The relation between the rise of the backscatter coefficient and the snow depth surveys shows good correlation for the 2004 to 2006 years for the Lachute and St-Jérôme stations (0.64 to 0.93), but less interesting results for the St-Hippolyte station (0.29 to 0.73). QSCAT products considering only the descendant orbit give best results.The integration of remote sensing albedo product did not allow improvement in the simulations because of holes in the temporal series caused by cloud cover. Also, the relation between fractional snow cover and snow depth did not show interesting results in an operational context.The study shows the interest to create new remote sensing SCA products more precise on the studied region. Future works should also evaluate the possibility to adapt the seuil[subscript ÉEN] method for a Kalman filter approach. A more spatially extensive study and a better comprehension of the backscatter response in microwaves of the different elements might eventually permit to obtain useful results with QSCAT data.

Mammalian circadian rhythms are controlled by a clock located in the suprachiasmatic nucleus (SCN). The mechanisms through which light phase-shifts the SCN circadian clock are similar to those underlying memory formation and long-term potentiation (LTP). Several secreted proteins, including tissue-type plasminogen activator (tPA), plasminogen, and brain-derived neurotrophic factor (BDNF), have been implicated in this process. These same proteins are important for photic phase-shifts of the SCN circadian clock. Early night glutamate application to SCN containing brain slices resets the circadian clock. Our experiments find that the endogenous tPA inhibitor, plasminogen activator inhibitor 1(PAI-1), blocked these shifts in slices from wildtype mice but not mice lacking its stabilizing protein, vitronectin (VN). Plasmin, but not plasminogen, prevented inhibition by PAI-1. Both plasmin and active BDNF reversed alpha2-antiplasmin inhibition of glutamate-induced shifts. alpha2-Antiplasmin decreased the conversion of inactive to active BDNF in the SCN. Both tPA and BDNF allowed daytime glutamate-induced phase-resetting. Together, these data are the first to demonstrate expression of these proteases in the SCN, their involvement in modulating photic phase-shifts, and their activation of BDNF in the SCN, a potential ‘gating’ mechanism for photic phase-resetting. Using western-blot analyses of SCN tissue maintained in vitro, we find higher tPA, plasmin and mBDNF levels in the SCN at night vs. the day. Also, in vitro glutamate treatment of SCN tissue during early night increases tPA levels to ~2.5 times control levels, while similar treatments during late night and mid-day do not alter tPA expression. Glutamate treatment in the early night does not alter PAI-1, plasmin and BDNF levels. Co-treatment with glutamate and PAI-1 decreases plasmin levels (vs. glutamate treatment alone), while co-treatment with glutamate and alpha2-antiplasmin decreases the amount of pro- and mBDNF in the SCN relative to glutamate treatment alone. We also show that mBDNF levels are significantly lower in tPA knockout mice during both day and night. Together, these results support circadian clock modulation of BDNF and fibrinolytic protein levels in the SCN. They also suggest that glutamate modulates tPA expression in the SCN, while tPA and plasmin modulate BDNF expression.

The suprachiasmatic nucleus (SCN) of the hypothalamus is the master mammalian pacemaker, co-ordinating the multitude of cell-autonomous circadian oscillators across the body to ensure internal synchrony, as well as maintaining an adaptive phase relationship with the light-dark cycle via projections from the retina. Intercellular communication between SCN clock neurons synchronises their oscillations, resulting in coherent output signals to the periphery. Vasoactive intestinal peptide (VIP), a neuropeptide expressed in the retinorecipient ventrolateral region of the SCN, is vital to this circuit-level co-ordination by signalling to its cognate VPAC2 receptor. In addition, VIP is important for the integration of light input into the SCN oscillation. The aims of the work presented in this thesis were to determine the roles of the VIP and VPAC2 cells in controlling circadian rhythmicity, and to elucidate the mechanisms of VIP signalling that underpin these roles. The first two experimental chapters utilise intersectional genetics and viral transduction to address separable roles for the VIP and VPAC2 cell populations. By diphtheria toxin-mediated cell ablation, or by adjusting cell-autonomous periodicity or rhythmicity specifically in these cell populations, I have identified that the VPAC2 cells are important for period setting and rhythmicity of both the SCN ex vivo and mouse behaviour in vivo, while the VIP cells play a vital role in behavioural rhythmicity and phase coherence across the SCN. The next two chapters use application of VIP to SCN slices to address mechanisms of phase-resetting through pharmacological manipulation and microarray analysis. I find that VIP has long lasting effects on all major circadian parameters of the SCN slice oscillation at both the cellular and circuit levels, and that it achieves this through a diversity of molecular pathways, in particular through cAMP/Ca2+ response elements within gene promoters. The final chapter focuses primarily on DUSP4, a negative regulator of the MAP kinase pathway that I have demonstrated to be upregulated by VIP. Here I demonstrate that DUSP4 affects the steady-state period of SCN slices, as well as influences phase shifting characteristics of both slices and mice. To conclude, the work presented here furthers our knowledge of neuropeptidergic communication in mammalian pacemaking. I have undertaken extensive characterisation of the molecular mechanisms through which the VIP neuropeptide influences SCN oscillators, and I have determined differential roles for the VIP and VPAC2 neurons in circadian timekeeping.

An idea of storing contents, such as media files, music files, movie clips, etc. is simple yet challenging in terms of required effort to make it count. Some of the benefits of pre-storing the contents are reduced delay of accessing/downloading a content, reduced load to the centralized servers and of course, a higher data rate. However, several challenges need to be addressed to achieve these benefits. Among many, some of the fundamentals are limited storage capacity, storing the right content and minimizing the costs. This thesis aims to address these challenges. First, a framework for predicting the proper contents that need to be stored to the limited storage capacity is presented. Then, the cost is minimized considering several real-world scenarios. While doing that, all possible collaborations among the local nodes are performed to ensure high performance. Therefore, the goal of this thesis is to come up with a solution to the content storing problems so that the network cost is minimized.