Glycogen synthase kinase-3 (GSK-3) alpha and beta are central regulators of key developmental pathways, including Wnt, Hedgehog and Notch, which control stem cell activities and cellular differentiation. Both forms of GSK-3 are also regulated by receptor tyrosine kinases via the PI3K/Akt growth-promoting pathway and are involved in feedback mechanisms that maintain signaling homeostasis. These signaling systems have critical functions in mammary gland development and aberrations in them have been implicated in breast cancer. However, the role of GSK-3 in breast oncogenesis is unclear. Here, I provide evidence that maintenance of appropriate GSK-3 activity is necessary for normal acinar morphogenesis of mammary cells in the ductal/alveolar epithelium. Inadequate GSK-3 levels result in enlarged structures that often lack hollow lumens and resemble early premalignant breast cancer lesions. A potential contribution for PI3K signaling to this phenotype was identified as a PI3K inhibitor partially reversed the observed morphological defects. Mammary epithelial cell (MEC) identity also requires modulation of signals through the Wnt/beta-catenin pathway. GSK-3-depleted mammary glands not only transdifferentiate into squamous epithelium but also develop highly proliferative adenosquamous carcinomas characterized by activated beta-catenin. Furthermore, beta-catenin appears to be required for both cell fate changes and tumorigenesis in the absence of GSK-3 function. Mammary tissues engineered to enable conditional deletion of beta-catenin in a GSK-3-null background also assumed an epidermoid cell fate with ensuing tumor formation albeit with a significantly longer latency and different histopathology. The metaplastic nature of tumors observed is similar to a rare yet aggressive form of human breast disease, metaplastic breast carcinomas (MBCs). Mammospheres (MS) generated from GSK-3 depleted MECs exhibited a less compact morphology compared to those with activated beta-catenin, which also exhibited an expansion of the CD24:CD49f double positive progenitor population and enhanced self-renewal. No MS were formed by MECs lacking GSK-3 and beta-catenin. ErbB2/Neu-mediated mammary tumor progression has been associated with Wnt/beta-catenin pathway activation. Loss of beta-catenin delayed tumor onset in a constitutively active ErbB2 mouse model but did not alter either the luminal characteristics of the ensuing tumors nor their metastatic potential. Collectively these studies indicate GSK-3 plays important roles in mammary gland function thereby suppressing mammary tumor formation.

Glycogen synthase kinase-3 (GSK-3) alpha and beta are central regulators of key developmental pathways, including Wnt, Hedgehog and Notch, which control stem cell activities and cellular differentiation. Both forms of GSK-3 are also regulated by receptor tyrosine kinases via the PI3K/Akt growth-promoting pathway and are involved in feedback mechanisms that maintain signaling homeostasis. These signaling systems have critical functions in mammary gland development and aberrations in them have been implicated in breast cancer. However, the role of GSK-3 in breast oncogenesis is unclear. Here, I provide evidence that maintenance of appropriate GSK-3 activity is necessary for normal acinar morphogenesis of mammary cells in the ductal/alveolar epithelium. Inadequate GSK-3 levels result in enlarged structures that often lack hollow lumens and resemble early premalignant breast cancer lesions. A potential contribution for PI3K signaling to this phenotype was identified as a PI3K inhibitor partially reversed the observed morphological defects. Mammary epithelial cell (MEC) identity also requires modulation of signals through the Wnt/beta-catenin pathway. GSK-3-depleted mammary glands not only transdifferentiate into squamous epithelium but also develop highly proliferative adenosquamous carcinomas characterized by activated beta-catenin. Furthermore, beta-catenin appears to be required for both cell fate changes and tumorigenesis in the absence of GSK-3 function. Mammary tissues engineered to enable conditional deletion of beta-catenin in a GSK-3-null background also assumed an epidermoid cell fate with ensuing tumor formation albeit with a significantly longer latency and different histopathology. The metaplastic nature of tumors observed is similar to a rare yet aggressive form of human breast disease, metaplastic breast carcinomas (MBCs). Mammospheres (MS) generated from GSK-3 depleted MECs exhibited a less compact morphology compared to those with activated beta-catenin, which also exhibited an expansion of the CD24:CD49f double positive progenitor population and enhanced self-renewal. No MS were formed by MECs lacking GSK-3 and beta-catenin. ErbB2/Neu-mediated mammary tumor progression has been associated with Wnt/beta-catenin pathway activation. Loss of beta-catenin delayed tumor onset in a constitutively active ErbB2 mouse model but did not alter either the luminal characteristics of the ensuing tumors nor their metastatic potential. Collectively these studies indicate GSK-3 plays important roles in mammary gland function thereby suppressing mammary tumor formation.

Thy-1 is a glycosylphosphatidylinositol-anchored protein that is expressed on murine T lymphocytes and is involved in T cell-mediated immune responses. In the presence of costimulatory signals, monoclonal antibody (mAb)-induced signaling through Thy-1 is associated with hallmarks of T cell activation, including IL-2 production and T cell proliferation. Thy-1-induced signaling promotes cytotoxic effector molecule expression, but is unable to trigger delivery of the lethal hit to target cells, suggesting that Thy-1 provides an incomplete T cell receptor (TcR)-like signal. However, the effect of Thy-1 signaling on cytokine production and the development of T helper (Th) cell phenotypes (Th1, Th2, Th17) remains unclear. The purpose of this work was to further our understanding of Thy-1-mediated signal transduction and the role that Thy-1 plays in the development of effector T cell responses. I found that, in the context of costimulatory signals, anti-Thy-1 mAb induced significantly less IL-2 production, CD25 expression and T cell proliferation than anti-TcR? mAb. Several key signaling molecules, including protein tyrosine kinases, zeta chain-associated protein-70 and extracellular signal-regulated kinase were activated with delayed kinetics during Thy-1-mediated T cell activation. The delayed signaling kinetics resulted in the delayed acquisition of cytotoxic effector function and also delayed delivery of the lethal hit to target cells. Interestingly, Thy-1-mediated signaling induced significantly more IL-17 and IL-4 synthesis and less IFN-? synthesis in comparison to TcR-mediated signaling. Moreover, Thy-1-activated CD4+ T cells produced high levels of IL-17 and IL-4 but minimal IFN? when restimulated with anti-Thy-1 mAb or anti-TcR? mAb with or without costimulatory signals. The unique ability of Thy-1 signaling to induce IL-17 production correlated with the expression of the Th17 lineage-specific transcription factor, retinoic orphan receptor gamma t. These observations show that Thy-1 signaling differs from TcR signaling in its ability to induce Th cell cytokines. Taken together, my findings show that Thy-1 signaling can provide the full TcR-like signal required for both the differentiation and triggering of Th cells and cytotoxic T lymphocytes, albeit with delayed kinetics in comparison to TcR signaling. They also suggest that Thy-1 signaling may be important in the development of Th2 and Th17 responses.

The Signal Transducer and Activator of Transcription-3 (Stat3) is a transcription factor that is required for transformation by a number of oncogenes, while a constitutively active form of Stat3 alone is sufficient to induce neoplastic transformation. It was previously demonstrated that cell to cell adhesion causes a dramatic increase in the activity of Stat3 in both normal and tumour cells. This hinted for the first time at the possibility that the role of Stat3 may differ upon cellular confluence. To examine such a mechanism, it is important to evaluate the effect of Stat3 downregulation at different time-points relative to confluence. To examine this, two different approaches for Stat3 downregulation were used: (1) the introduction of high levels of peptidomimetics analogs, which block the Stat3-SH2 domain by using a technique of in situ electroporation. (2) Treatment with two platinum compounds that inhibit Stat3 binding to activated receptors and DNA. The results demonstrated that Stat3 downregulation in vSrc or TAg transformed mouse fibroblast cells or in breast carcinoma lines, induced apoptosis which was more pronounced post-confluence at the time of its peak activity. In contrast, in sparsely growing normal mouse fibroblasts, Stat3 inhibition induced merely a growth retardation. However, in densely growing normal fibroblasts, Stat3 inhibition induced apoptosis. At least in part, apoptosis induced by Stat3 inhibition was mediated by p53, as shown by the resistance to cell death by Stat3 downregulation in colon carcinoma cells, HCT116, where the p53 gene is ablated. Overall, our observations point to the possibility that constitutive activation of Stat3 may lead to tumourigenesis by downregulating wt-53 in cancers that do not have p53 mutations. As a result, targeting Stat3 in cancers with wt-p53 may be a promising therapeutic approach for restoring p53 function, thereby inducing p53-mediated apoptosis. Next, we examined the effect of constitutively activated Stat3 as an oncogene. Stat3C expression in rat F111 fibroblasts induced anchorage independence, but to a lower degree compared to other oncogenes, such as vSrc. Surprisingly Stat3C expression increased gap junction intercellular communication, despite the fact that other oncogenes such as vSrc or vRas effectively block gap junctions. / Thesis (Ph.D, Pathology & Molecular Medicine) -- Queen's University, 2009-04-26 01:09:21.654

Many molecules and the complex interactions between them underlie plasticity in the cerebellum. However, the exact relationship between cerebellar plasticity and the different signalling cascades remains unclear. Calcium-calmodulin dependent protein kinase II (CaMKII) regulates many forms of synaptic plasticity, but very little is known about its function during plasticity induction in the cerebellum. The aim of this thesis is to contribute to a better understanding of the molecular mechanisms that regulate the induction of synaptic plasticity in cerebellar Purkinje cells (PCs). The focus of the thesis is to investigate the role of CaMKII isoforms in the bidirectional modulation of plasticity induction at parallel fibre (PF)-PC synapses. For this investigation, computational models that represent the CaMKII activation and the signalling network that mediates plasticity induction at these synapses were constructed. The model of CaMKII activation by calcium-calmodulin developed by Dupont et al (2003) replicates the experiments by De Koninck and Schulman (1998). Both theoretical and experimental studies have argued that the phosphorylation and activation of CaMKII depends on the frequency of calcium oscillations. Using a simplified version of the Dupont model, it was demonstrated that the CaMKII phosphorylation is mostly determined by the average calcium-calmodulin concentration, and therefore depends only indirectly on the actual frequency of calcium oscillations. I have shown that a pulsed application of calcium-calmodulin is, in fact, not required at all. These findings strongly indicate that the activation of CaMKII depends on the average calcium-calmodulin concentration and not on the oscillation frequency per se as asserted in those studies. This thesis also presents the first model of AMPA receptor phosphorylation that simulates the induction of long-term depression (LTD) and potentiation (LTP) at the PF-PC synapse. The results of computer simulations of a simple mathematical model suggest that the balance of CaMKII-mediated phosphorylation and protein phosphatase 2B (PP2B)-mediated dephosphorylation of AMPA receptors determines whether LTD or LTP occurs in cerebellar PCs. This model replicates the experimental observations by Van Woerden et al (2009) that indicate that CaMKII controls the direction of plasticity at PF-PC synapses. My computer simulations support Van Woerden et al’s original suggestion that filamentous actin binding can enable CaMKII to regulate bidirectional plasticity at these synapses. The computational models of intracellular signalling constructed in this thesis advance the understanding of the mechanisms of synaptic plasticity induction in the cerebellum. These simple models are significant tools for future research by the scientific community.

The serious nature of MDD has intensified the need to identify and elucidate new neurobiological targets for antidepressant drug action. Depression presents with evidence for degenerative pathology that relates to disturbances in excitatory glutamatergic pathways, particularly the N-methyl-D-aspartate (NMDA) receptormediated release of the pleiotropic molecule, nitric oxide (NO), and cyclic guanosine monophosphate (cGMP). The contribution of the glutamate-NO/cGMP pathway may realize great importance as a fundamental substrate underlying the pathophysiology of major depression. In the next generation of antidepressant drugs, the nitric oxide pathway could playa dynamic role in addressing urgent therapeutic needs. In this study, we have used a genetic model of depression, the Flinders Sensitive Line (FSL) rat, to investigate the surrogate markers of the NO/cGMP pathway. The aim was to determine whether the depressive-like behaviour of the hypercholinergic FSL rat is accompanied by altered activation of the NO/cGMP pathway. To this end, the extent to which the FSL and Flinders Resistant Line (FRL) rats differ neurochemically with regard to basal hippocampal and frontal cortical NOS-activity, as well as nitric oxide (NO) and cGMP accumulation, were determined. Additionally, select behavioural assessments were performed to confirm the anxiogenic phenotype of the FSL strain. For neurochemical determinations a sensitive fluorometric reversed phase highperformance liquid chromatographic (HPLC) assay was developed to analyze total nitrite and nitrate in brain tissue. Nitrate was enzymatically converted to nitrite before derivatization with 2,3-diaminonaphthalene (DAN). The stable and highly fluorescent product, 2,3-naphthotriazole (NAT), was quantified. Secondly, the quantity of the amino acid L-citrulline was measured by HPLC with electrochemical detection after o-phthalaldehyde (OPA) derivatization. L-citrulline formation was used as an index for nNOS activity. Finally, a direct, competitive enzyme immunoassay kit was used to determine the downstream activity of the NO-pathway in brain tissue. FSL rats were compared to FRL rats with respect to sensitivity to serotonin 5-HT1A . receptor-mediated hypothermia under our lab-conditions. The Open Field Test (OFT) behavioural assessment was performed to compare FSL with FRL groups under baseline conditions according to their level of inherent anxiety. The parameters used to measure anxiety were number of line crosses (locomotor activity), time spent in middle blocks and social interaction time between pairs of rats. As an additional behavioural assessment, the Forced Swim Test (FST) was performed to assess behavioural restraint measured as time of immobility. Basal cGMP levels in the frontal cortex were found to be significantly less in FSL than in FRL rats, whereas the levels in the hippocampus did not differ significantly. No other significant differences with respect to NO and nNOS activity were apparent in either of the brain areas. The hypothermia test confirmed a significantly greater decrease in temperature in the FSL rat than the FRL rat. The FST did not confirm any differences in immobility time between the two rat strains. In the OFT, FSL rat groups exhibited behaviour that indicated significantly more anxiety than FRL rats. Under basal conditions, FSL rats do not present with significant changes in markers of the NO cascade in the hippocampus and frontal cortex compared to FRL controls, including NOS activity as well as NO accumUlation. However, cGMP levels were found to be significantly lower in the frontal cortex of FSL rats versus FRL rats, although not in the hippocampus. Since the FSL rat is known to be hypercholinergic, these data support an interaction between the NO/cGMP pathway and the cholinergIc system in the frontal cortex but not hippocampus of FSL animals. The mechanisms and implications of such a mutual involvement need further clarification. Further, this anatomical differentiation may have important implications for understanding the role of NO in the depressive-like behaviour of the FSL rat and, indeed, may reveal more on the neurobiology and treatment of depression. Through the performed behavioural assessments, the FSL and FRL rats were successfully separated with respect to their anxiety phenotype as well as their heightened response to serotonergic challenge, thus confirming a contribution of both the serotonergic and cholinergic systems to the depressogenic nature of these animals. As concluding remark can be said that under normal basal conditions markers of the NO/cGMP signalling cascade are not altered in FSL vs FRL rats, although cGMP levels are reduced in the frontal cortex of FSL rats, supportive of an NO-independent mechanism of cGMP regulation, possibly involving ACh. / Thesis (M.Sc. (Pharmacology)--North-West University, Potchefstroom Campus, 2009.

The serious nature of MDD has intensified the need to identify and elucidate new neurobiological targets for antidepressant drug action. Depression presents with evidence for degenerative pathology that relates to disturbances in excitatory glutamatergic pathways, particularly the N-methyl-D-aspartate (NMDA) receptormediated release of the pleiotropic molecule, nitric oxide (NO), and cyclic guanosine monophosphate (cGMP). The contribution of the glutamate-NO/cGMP pathway may realize great importance as a fundamental substrate underlying the pathophysiology of major depression. In the next generation of antidepressant drugs, the nitric oxide pathway could playa dynamic role in addressing urgent therapeutic needs. In this study, we have used a genetic model of depression, the Flinders Sensitive Line (FSL) rat, to investigate the surrogate markers of the NO/cGMP pathway. The aim was to determine whether the depressive-like behaviour of the hypercholinergic FSL rat is accompanied by altered activation of the NO/cGMP pathway. To this end, the extent to which the FSL and Flinders Resistant Line (FRL) rats differ neurochemically with regard to basal hippocampal and frontal cortical NOS-activity, as well as nitric oxide (NO) and cGMP accumulation, were determined. Additionally, select behavioural assessments were performed to confirm the anxiogenic phenotype of the FSL strain. For neurochemical determinations a sensitive fluorometric reversed phase highperformance liquid chromatographic (HPLC) assay was developed to analyze total nitrite and nitrate in brain tissue. Nitrate was enzymatically converted to nitrite before derivatization with 2,3-diaminonaphthalene (DAN). The stable and highly fluorescent product, 2,3-naphthotriazole (NAT), was quantified. Secondly, the quantity of the amino acid L-citrulline was measured by HPLC with electrochemical detection after o-phthalaldehyde (OPA) derivatization. L-citrulline formation was used as an index for nNOS activity. Finally, a direct, competitive enzyme immunoassay kit was used to determine the downstream activity of the NO-pathway in brain tissue. FSL rats were compared to FRL rats with respect to sensitivity to serotonin 5-HT1A . receptor-mediated hypothermia under our lab-conditions. The Open Field Test (OFT) behavioural assessment was performed to compare FSL with FRL groups under baseline conditions according to their level of inherent anxiety. The parameters used to measure anxiety were number of line crosses (locomotor activity), time spent in middle blocks and social interaction time between pairs of rats. As an additional behavioural assessment, the Forced Swim Test (FST) was performed to assess behavioural restraint measured as time of immobility. Basal cGMP levels in the frontal cortex were found to be significantly less in FSL than in FRL rats, whereas the levels in the hippocampus did not differ significantly. No other significant differences with respect to NO and nNOS activity were apparent in either of the brain areas. The hypothermia test confirmed a significantly greater decrease in temperature in the FSL rat than the FRL rat. The FST did not confirm any differences in immobility time between the two rat strains. In the OFT, FSL rat groups exhibited behaviour that indicated significantly more anxiety than FRL rats. Under basal conditions, FSL rats do not present with significant changes in markers of the NO cascade in the hippocampus and frontal cortex compared to FRL controls, including NOS activity as well as NO accumUlation. However, cGMP levels were found to be significantly lower in the frontal cortex of FSL rats versus FRL rats, although not in the hippocampus. Since the FSL rat is known to be hypercholinergic, these data support an interaction between the NO/cGMP pathway and the cholinergIc system in the frontal cortex but not hippocampus of FSL animals. The mechanisms and implications of such a mutual involvement need further clarification. Further, this anatomical differentiation may have important implications for understanding the role of NO in the depressive-like behaviour of the FSL rat and, indeed, may reveal more on the neurobiology and treatment of depression. Through the performed behavioural assessments, the FSL and FRL rats were successfully separated with respect to their anxiety phenotype as well as their heightened response to serotonergic challenge, thus confirming a contribution of both the serotonergic and cholinergic systems to the depressogenic nature of these animals. As concluding remark can be said that under normal basal conditions markers of the NO/cGMP signalling cascade are not altered in FSL vs FRL rats, although cGMP levels are reduced in the frontal cortex of FSL rats, supportive of an NO-independent mechanism of cGMP regulation, possibly involving ACh. / Thesis (M.Sc. (Pharmacology)--North-West University, Potchefstroom Campus, 2009.

Mathematical modeling is an important tool in improving the understanding of complex biological processes. However, mathematical models are often faced with challenges that arise due to the limited knowledge of the underlying biological processes and the high number of parameters for which exact values are unknown. The method of generalized modeling is an alternative modeling approach that aims to address these challenges by extracting information about stability and bifurcations of classes of models while making only minimal assumptions on the specific functional forms of the model. This is achieved by a direct parameterization of the Jacobian in the steady state, introducing a set of generalized parameters which have a biological interpretation. In this thesis, the method of generalized modeling is extended and applied to different problems from cell biology. In the first part, we extend the method to include also the higher derivatives at the steady state. This allows an analysis of the normal form of bifurcations and thereby a more specific description of the nearby dynamics. In models of gene-regulatory networks, it is shown that the extended method can be applied to better characterize oscillatory systems and to detect bistable dynamics. In the second part, we investigate mathematical models of bone remodeling, a process that renews the human skeleton constantly. We investigate the connection between structural properties of mathematical models and the stability of steady states in different models. We find that the dynamical system operates from a stable steady state that is situated in the vicinity of bifurcations where stability can be lost, potentially leading to diseases of bone. In the third part of this thesis, models of the MAPK signal transduction pathway are analyzed. Since mathematical models for this system include a high number of parameters, statistical methods are employed to analyze stability and bifurcations. Thereby, the parameters with a strong influence on the stability of steady states are identified. By an analysis of the bifurcation structure of the MAPK cascade, it is found that a combination of multiple layers in a cascade-like way allows for additional types of dynamic behavior such as oscillations and chaos. In summary, this thesis shows that generalized modeling is a fruitful alternative modeling approach for various types of systems in cell biology. / Mathematische Modelle stellen ein wichtiges Hilfmittel zur Verbesserung des Verständnisses komplexer biologischer Prozesse dar. Sie stehen jedoch vor Schwierigkeiten, wenn wenig über die zugrundeliegende biologischen Vorgänge bekannt ist und es eine große Anzahl von Parametern gibt, deren exakten Werte unbekannt sind. Die Methode des Verallgemeinerten Modellierens ist ein alternativer Modellierungsansatz mit dem Ziel, diese Schwierigkeiten dadurch anzugehen, dass dynamische Informationen über Stabilität und Bifurkationen aus Klassen von Modellen extrahiert werden, wobei nur minimale Annahmen über die spezifischen funktionalen Formen getätigt werden. Dies wird erreicht durch eine direkte Parametrisierung der Jacobimatrix im Gleichgewichtszustand, bei der neue, verallgemeinerte Parameter eingeführt werden, die eine biologische Interpretation besitzen. In dieser Arbeit wird die Methode des Verallgemeinerten Modellierens erweitert und auf verschiedene zellbiologische Probleme angewandt. Im ersten Teil wird eine Erweiterung der Methode vorgestellt, bei der die Analyse höherer Ableitungen im Gleichgewichtszustand integriert wird. Dies erlaubt die Bestimmung der Normalform von Bifurkationen und hierdurch eine spezifischere Beschreibung der Dynamik in deren Umgebung. In Modellen für genregulatorische Netzwerke wird gezeigt, dass die so erweiterte Methode zu einer besseren Charakterisierung oszillierender Systeme sowie zur Erkennung von Bistabilität verwendet werden kann. Im zweiten Teil werden mathematische Modelle zur Knochenremodellierung untersucht, einem Prozess der das menschliche Skelett kontinuierlich erneuert. Wir untersuchen den Zusammenhang zwischen strukturellen Eigenschaften verschiedener Modelle und der Stabilität von Gleichgewichtszuständen. Wir finden, dass das dynamische System von einem stabilen Zustand operiert, in dessen Nähe Bifurkationen existieren, welche das System destabilisieren und so potentiell Knochenkranheiten verursachen können. Im dritten Teil werden Modelle für den MAPK Signaltransduktionsweg analysiert. Da mathematische Modelle für dieses System eine hohe Anzahl von Parametern beinhalten, werden statistische Methoden angewandt zur Analyse von Stabilität und Bifurkationen. Zunächst werden Parameter mit einem starken Einfluss auf die Stabilität von Gleichgewichtszuständen identifizert. Durch eine Analyse der Bifurkationsstruktur wird gezeigt, dass eine kaskadenartige Kombination mehrerer Ebenen zu zusätzliche Typen von Dynamik wie Oszillationen und Chaos führt. Zusammengefasst zeigt diese Arbeit, dass Verallgemeinertes Modellieren ein fruchtbarer alternativer Modellierungsansatz für verschiedene zellbiologische Probleme ist.

In Schizosaccharomyces pombe, the MAPK pathway Sty1 is activated upon several stress situations, like osmotic and oxidative stress, stationary phase, UV radiation or heat shock. Since the modulation of gene expression is one of the main outputs of this response, we focused this Thesis work on the charactherization of the transcription regulation by the activation of the Sty1 pathway and through the transcription factors Atf1 and Pcr1. Moreover, we extend our field of interest investigating how stress–related chromatin remodelers are affecting the stress defence transcription of the cells. / En Schizosaccharomyces pombe, la vía de la MAPK Sty1 es activada ante diferentes situaciones de estrés, como son el estrés oxidativo u osmótico, fase estacionaria, radiación UV o choque de calor. Al ser la modulación de la expresión génica uno de los más importantes objetivos de esta respuesta, hemos focalizado el trabajo de esta Tesis doctoral en la caracterización de la regulación transcripcional mediada por la activación de la ruta de Sty1 y los factores de transcripción Atf1 y Pcr1. Además, hemos ampliado nuestra área de interés investigando el papel de remodeladores de cromatina relacionados con la respuesta a estrés y cómo a participan en la transcripción estrés-dependiente.

[Truncated abstract] The phloem long distance translocation system is not only involved in the transport of nutrients and photo-assimilates to different organs of the plant, but it also appears to be important for the transport of information molecules including growth-regulators, proteins and RNA. Translocation of signals appears to be involved in the coordination of developmental processes and also in the response of the plant to environmental cues. Much of the information about macromolecules in phloem comes from analyses of exudates collected from the stylets of sap sucking insects or from incisions made to the vasculature. Among the legumes, members of the genus Lupinus exude phloem 'freely' from incisions made to the vasculature at most organs of the plant. This feature was exploited in this study to document some of the macromolecules present in exudate of L. albus and which might represent potential mobile signals. Phloem exudate was collected mainly from the sutures of developing pods and from inflorescence racemes. Two-dimensional polyacrylamide gel electrophoresis and tandem mass spectrometry were used to identify 83 proteins in exudate. Analysis of a cDNA library constructed from exudate identified 609 unique transcripts. Both proteins and mRNA were classified into functional groups. The largest group was related to general and energy metabolism, suggesting some metabolic activity probably to support the sieve element (SE). Other significant functional groups were represented by proteins and transcripts involved in protein synthesis, turnover and sorting, and in redox homeostasis. Proteins in these categories could play a role in maintaining the functions and stability of proteins in SE. Macromolecules involved in signalling such as transcripts encoding proteins mediating calcium levels and the Flowering locus T (FT) protein were also identified in phloem exudate of L. albus. FT protein has been recently identified as a mobile signal that induces flowering. ... The hen1 mutant accumulates low, sometimes even undetectable levels of miRNA due to the lack of methylation. No translocation of the five miRNA assayed under nutrient replete (non stress) conditions was observed. Translocation of miR395 in response to sulphur (S) deficiency was also investigated, and while conclusive evidence of translocation was not obtained, the data suggested some movement from roots to shoots (possibly in xylem) of a signal in response to S-deficiency. Future work is required to provide greater insight into the translocation path and identity of this S-deficiency signal. This study suggests that not all miRNA identified in phloem exudates are mobile, which raises the question about their biological relevance in SE and how they reached this location (e.g. through the action of a non-selective transport mechanism). However, there is also the possibility that miRNA are translocated only in response to specific internal or external cues not tested in this study. This is the first study that provides information on macromolecules present in the phloem exudate of a member of the Fabaceae. The information obtained from this work, provides a basis for future studies in the identification of potential mobile signals that may play a role in a communication network that traffics information around the plant, regulating its various developmental processes and responding to environmental cues.