Graduation date: 2003

Plant growth, development, and environmental responsiveness are dependent on hormone-induced gene expression. This dissertation reports multiple interactions between the plant hormones auxin and ethylene and investigates their contribution to the gravitropic response, elongation growth, adventitious root formation, callus and tracheary element initiation and growth, and flower development. Four mutants of tomato (Lycopersicon esculentum, Mill.) altered in either hormone production or hormone response were used to test the involvement of ethylene and auxin. These mutants included diageotropica (dgt) which is auxin-resistant, Never-ripe (Nr), which is ethylene-resistant, epinastic (epi), which overproduces ethylene and lazy-2 (lz-2), which exhibits a phytochrome-dependent reversed-gravitropic response. Additionally, a double mutant between Nr and dgt was constructed and tested. Gravitropism was studied as an exemplary process involving both auxin and ethylene. Mutant analysis demonstrated that ethylene does not play a primary role in the gravitropic response via the currently known ethylene response pathways. However, ethylene can modify the gravitropic response, e.g. the delayed gravitropic response of the dgt mutant can be restored with exceedingly low concentrations of ethylene and ethylene synthesis- and ethylene-action inhibitors can partially inhibit the graviresponse. The role of gravity in tracheary element (TE) production was tested in microgravity (during a space shuttle flight) and in hypergravity (centrifugation). A correlation was found between gravitational force and the production of TEs, with decreased numbers of TEs produced in microgravity and increased numbers produced in response to hypergravity. Increased production of TEs by dgt in both increased and reduced gravity indicates that gravity regulates vascular development via a DGT-dependent pathway involving auxin. Combination of both the Nr and dgt mutations in a double mutant leads to plants which exhibit the reduction of auxin-sensitivity typical of dgt as well as a delay in fruit ripening typical of Nr. The reduced gravitropic response of the dgt mutant was restored to wild-type levels in the double mutant confirming a complex role for ethylene in the gravitropic response. Abnormal floral organ development was observed in a subset of double mutant flowers.These data demonstrate multiple connections between auxin and ethylene during development and provide further insight into their cellular interactions. / Graduation date: 2001

In addition to the well-characterized genomic mechanism of steroid action that uses intracellular receptors, steroid hormones also signal through nongenomic processes that use membrane receptors. A membrane receptor for corticosterone (CORT) has been described in brains of the roughskin newt (Taricha granulosa). This receptor is believed to be a G-protein coupled receptor because corticosterone binding is inhibited by guanyl nucleotides and enhanced by Mg�����. The studies described in this thesis use biochemical, pharmacological and molecular techniques to characterize the newt neuronal membrane glucocorticoid receptor (mGR) in pursuit of its molecular identification. The mGR was successfully solubilized from newt neuronal membranes and conditions were defined that maintained corticosterone binding activity for further study. The solubilized receptor was partially purified using standard chromatographic techniques and an immobilized ligand affinity resin (CORT-Sepharose). These chromatographic studies were combined with the use of a novel photoaffinity ligand (azido-CORT) to biochemically characterize the mGR protein, finding that it is an acidic glycoprotein with an apparent molecular weight of 63 kDa and an isoelectric point of approximately 5.0. Pharmacological studies with mGR showed that a subset of kappa opioid ligands displaced corticosterone from the receptor binding site with K[subscript i] values in the nanomolar to low-micromolar range. The interaction of mGR with kappa opioid ligands was specific because no mu-, delta-, or orphanin-specific opioid ligands were effective at displacing corticosterone from the receptor. These data suggest that the newt neuronal mGR may be a kappa-opioid like receptor. Finally, molecular studies were used to clone a novel newt brain protein, neuronal axonal protein 22 (NAP-22), that was identified in a protein differential display strategy designed to identify mGR. Studies with the cloned and expressed NAP-22 protein suggest that it is not the mGR but, instead, may be a mGR-associated protein. These studies provided new information about the biochemical and pharmacological properties of mGR, and may have discovered a protein that is associated with the newt neuronal mGR. / Graduation date: 1999

Graduation date: 1998

Abstract Gibberellins (GAs) are a class of plant hormones that regulate many aspects of plant growth and development including seed germination, stem elongation and fruit development. To investigate the regulation of GA biosynthesis and the impact of altered GA levels on plant growth and development, transgenic pea (Pisum sativum L. cv. Carneval) plants were generated to overexpress PsGA3ox1 (codes for GA 3-hydroxylase which converts GA20 to bioactive GA1) under the control of the CaMV-35S promoter. Increased expression of the transgene PsGA3ox1 was correlated with altered plant phenotype including longer internodes, larger stipules and tendrils, and longer pods. Transgenic lines also showed upregulation of the GA catabolic genes PsGA2ox1 and/or PsGA2ox2, suggesting that GA1 substrate-induced feedback regulation also occurs to maintain GA homeostasis. Changes in endogenous GAs, quantified using an isotope dilution method, indicated that an increased flux in GA biosynthesis occurred in the expanding internodes, stipules and tendrils of the PsGA3ox1-overexpressor lines. Higher bioactive GA1 levels and growth were correlated with lower PsGA2ox1 transcript levels in elongating internodes, and oscillation of these parameters between adjacent elongating internodes in the PsGA3ox1-overexpression lines suggests that coordination of bioactive GA levels and growth occurs between adjacent internodes. During germination and early seedling growth, GA gene expression studies suggested that PsGA3ox1-overexpression increased the flux through to bioactive GA in the cotyledons, shoots and roots of pea seedlings, resulting in longer shoots but shorter roots. Auxins are a class of plant hormones involved in growth and differentiation of plants that can influence GA biosynthesis and action. The location and action of auxins is in part regulated by auxin carrier proteins. The expression patterns of the putative auxin efflux carrier genes PsPIN1 and PsPIN2 in elongating internodes were correlated with vascular re-patterning events in this tissue, and PsGA3ox1-overexpression appears to increase internode PsPIN1 and PsPIN2 transcript abundance and the formation of the vascular connections between the internode and the axillary buds. Overall, characterization of PsGA3ox1-overexpressor lines in pea demonstrated that bioactive GA levels are tightly regulated in pea tissues for the coordination of plant growth and development. / Plant Science

Undergraduate honors paper--Mount Holyoke College, 2006. Program in Biochemistry. / Includes bibliographical references (leaves 69-74).

The neuropeptides galanin and neuropeptide Y (NPY) play an important role in the reproduction of rodents, e.g. by modulating the release of gonadal hormones, the nutritional status by effects on feeding behavior and also by influencing mating behavior. There are age- and gender- differences in galanin- and NPY- like immunoreactivities (LIs) in brain areas important for higher functions including the hippocampal formation (HiFo) and cortex, that are related to the concentrations of 17β-estradiol. Neuropeptides in general are currently not considered critical in normal integrative neuronal functions but are rather thought to act as slow modulators during periods of stress or injury. In the present thesis we attempted to investigate, if the normal cyclical changes in the female sex-hormone 17β-estradiol can affect neurotransmission in brain areas important for memory, cognition and mood. We studied not only ”long term” (days and weeks) but also ”short-term” (one hour) effects on galanin and NPY concentrations in 17β-estradiol-primed ovariectomized (ovx) rats and mice. Radioimmunoassay (RIA) of galanin-LI in extracts of brain tissues from ”long-term” 17β-estradiol-treated ovx rats showed that its effects on galanin are dependent on boththe dose and on duration. Galanin - and NPY-LI in brain tissues of young ovx rats and mice increased in response to 17β-estradiol treatment in the HiFo, frontal cortex and striatum already within hours. This effect was not blocked by Tamoxifen® in rats. The mechanism of the 17β-estradiol effects on galanin levels in the rat HiFo may be related to decreased release of galanin into the extracellular fluid, since galanin-LI decreased in microdialysis samples two hours after a single injection of 17β-estradiol. Species differences were observed with regards to galanin, possibly due to tissue and species differences in the distribution of estrogen receptors. In the HiFo and caudate nucleus of mice, we found an increase in NPY-transcript after two hours by means of insitu hybridization, perhaps a compensatory up-regulation of NPY mRNA after increased 17β-estradiol-induced release in these areas. Taken together with no effects of Tamoxifen® on the levels on galanin in the HiFo of rats, the short duration, and the fact that the density of classical estrogen receptors seems to be limited in the striatum, we suggest that these effects are mediated through a membrane-related mechanism perhaps not involving the classical ER route. With an antiserum raised against the C-terminal end of the first 16 aminoacids of galanin- the sequence important for binding of intact galanin to its receptor - we found a novel compound which appears to be a homologue to galanin. Chromatographical analysis revealed that it was not galanin(1-29) or the galanin related peptide, galaninlike peptide (GALP), but appeared with immunohistochemistry in the galanin systems in the brain and was further influenced by 17β-estradiol in the HiFo and frontal cortex in a similar manner as galanin(1-29). In conclusion, tissue concentrations of galanin, a putative galanin homologue and NPY can be altered already after one hour by 17β-estradiol treatment e.i. in the HiFo. These ”short-term” effects are most likely to be due to effects on estrogen-primed peptide release which might influence mechanisms important for memory, cognition and mood.

L'expressió de la Kidney Androgen-regulated Protein (KAP) està regulada específicament y diferencialment per andrògens i hormona tiroïdal (T3) en el túbul proximal renal. Hem analitzat la transactivació del promotor del gen del KAP en cèl.lules PCT (pars convoluta) i PR (recta) derivades de ratolins transgènics L-PK/Tag1. Transfeccions transitòries amb diferents contruccions del promotor del KAP indicaven que el fragment de 224 bp era suficient per a mediar l'expressió cèl.lula específica de dit promotor. Assaigs de retardament en gel també mostraven que un putatiu element de resposta a andrògens (ARE), local.litzat a -39 bp de l'inici de transcripció, unia el receptor d'andrògens. La dihidrotestosterona (DHT) estimulava de manera androgen dependent la transactivació del KAP en cèl.lules PCT però en PR i la mutació de la putativa seqüència ARE eliminava dita activació. A més a més, l'IGF-1 però no la T3 augmentava la transactivació androgen dependent de KAP en PCT. Per tant, es suggeria que l'efecte cooperatiu de T3 en l'expressió del KAP existia in vivo podent efectar a l'activació produïda per T3 de l'hormona de creixement (GH) i al seu torn d'IGF-1. Aquests resultats demostraven l'expressió específica del curt fragment de 224 bp del promotor del KAP y suggerien interaccions sinèrgiques entre IGF-1 i andrògens en la regulació del gen del KAP en PCT.L'expressió cortical androgen dependent del KAP està afectada en hipotiroïdisme postnatal. La síntesi puntual de T3 a partir del dia 11 postnatal, comença una resposta cortical feble de KAP que va augmentant cap als dies 15-16, que és quan es produeix un pic fisiològic de T4 i el desenvolupament puberal dels ratolins. Donat que les CCAAT/Enhancer-Binding Proteins (C/EBPs) participen en respostes mitjançades per T3 i que en el promotor del KAP existeixen quatre elements de resposta consens per a C/EBPs, hem analitzat la seva participació en la resposta androgènica de KAP mitjançada per T3. La detecció de p42C/EBPa y p35C/EBPb es troba correlacionada amb l'expressió del KAP, apareixent en extractes renal nuclears de ratolins masles control i hipotiroïdals induïts amb T3 durant els dies 7-21 postnatals, però no en els hipotiroïdals no tractats. Mitjançant transfeccions transitòries es mostrava com C/EBPa i C/EBPb eren capaces d'induir respostes màximes del promotor del KAP i que la deleció de les putatives caixes d'unió a C/EBPs local.litzades a -429/-420 i -457/-448 produïa una gran disminució de l'activitat basal del promotor, irrecuperable amb la sobreexpressió de C/EBPs. Tant la depleció androgènica com al mutació de l'ARE conjuntament amb la deleció de les caixes esmentades augmentava la disminució de l'activitat basal del promotor produïda per la deleció de dites caixes. Per tant, postulem que els andrògens i la T3 mitjançant el control de C/EBPs sinergitzen en la transactivació del gen del KAP. / The kidney androgen-regulated protein (KAP) is specifically expressed and differentially regulated by androgens and triiodothyronine (T3) in proximal tubule. We have analyzed the hormonal dependent transactivation of the KAP gene promoter in PCT (pars convoluta) and PR (recta) cells derived from L-PK/Tag1 transgenic mice. Results from transient transfection studies with different KAP promoter constructs indicated that a 224 bp-truncated fragment was sufficient to mediate cell-specific expression of the KAP promoter. Gel shift assays also showed that a putative androgen-response element (ARE) located at -39 bp from the transcription initiation site was able to bind androgen receptor (AR). Dehydrotestosterone (DHT) stimulated in an androgen-dependent manner the transactivation of KAP in PCT cells but not in PR and mutation of the putative ARE sequence abolished that activation. Moreover, the IGF-1 but not T3 enhanced the androgen-dependent transactivation of KAP in PCT cells. It suggested that the cooperative effect of T3 on KAP expression existing in vivo might be effected by T3 activation of growth hormone (GH) and, in turn, of IGF-1. These results demonstrated the specific expression of a short 224 bp fragment of the KAP promoter and suggested synergistic interactions between IGF-1 and androgens for KAP gene regulation in PCT cells.The cortical androgen-dependent expression of KAP was influenced in postnatal hipothyroidism. The punctual effect of T3 in postnatal day 11 produced cortical expression of KAP gene and it was increased until days 15-16, when there is an fisiological pick of T4 and the puberal development of mice. In this point, we have analyzed the function of CCAAT/Enhancer-Binding Proteins (C/EBPs) on the enhanced androgen-dependent KAP expression by T3 because they participate in T3 responses and there are four consensous C/EBP sequences in KAP promoter. p42C/EBPa and p35C/EBPb were detected when KAP expression existed, that was in nuclear kidney extracts of euthyroid male mice and hipothyroid male mice treated with T3 from day 7 to 21 posnatal, but not in hipothyroid male mice. Transient transfections demostrated that C/EBPa and C/EBPb were able to induce the maximal KAP promoter activity achieved and the delection of putative C/EBP sequences at positions -429/-420 and -457/-448 produced an increment of the decrease observed for KAP promoter activity, that it wasn't recovered by C/EBP overexpression. Androgen deplection or ARE mutation with the deletion of mentioned C/EBP boxes decreased the basal promotor activity more than the mutation of these boxes alone. Because of these results we have postulated that the effect of androgens and T3 on KAP expression were mediated for C/EBPs.

Thèse d'exercice : Médecine. Neurologie : Université de Nantes : 2004. / Bibliogr. f. 95-101.

Thesis (M. Sc.)--York University, 1999. Graduate Programme in Biology. / Typescript. Includes bibliographical references. Also available on the Internet. MODE OF ACCESS via web browser by entering the following URL: http://wwwlib.umi.com/cr/yorku/fullcit?pMQ43394.