The hypothalamic tuberoinfundibular dopaminergic (TIDA) neurones secrete dopamine, which inhibits prolactin secretion. TIDA neurone numbers are deficient in Ames (df/df) and Snell (dw/dw) dwarf mice, which lack prolactin, growth hormone and thyroid-stimulating hormone. Prolactin therapy initiated before 21 days maintains normal-sized TIDA neurone numbers in df/df mice and, when initiated as early as 7 days, maintains the maximum TIDA neurone numbers observed in dw/dw development, which are decreased compared to those in normal mice. The present study investigated the effect of prolactin dose and species on TIDA neurone development. Snell dwarf and normal mice were treated with saline, 5 μg of ovine prolactin (oPRL), 50 μg of oPRL, or 50 μg of recombinant mouse prolactin (rmPRL) beginning at 3days of age. Brains were analysed at 45 days using catecholamine histofluorescence, and immunohistochemistry for tyrosine hydroxylase or bromodeoxyuridine. Normal mice had greater (P ≤ 0.01) TIDA neurones than dw/dw, regardless of treatment. TIDA neurones in 50 μg oPRL-treated dw/dw mice were greater (P ≤ 0.05) than those in 5 μg oPRL- and rmPRL-treated dw/dw mice, which were greater (P ≤ 0.01) than those in saline-treated dw/dw mice. Fifty microgram oPRL-treated dw/dw mice also had greater (P < 0.01) TIDA neurone numbers than the maximum numbers observed in untreated dw/dw mice development. Among saline, 5 μg oPRL and 50 μg oPRL treatments, but not rmPRL, A14 neurone numbers were higher (P ≤ 0.01) in normal compared to in dw/dw mice. The mechanism of TIDA neurone recruitment was investigated using bromodeoxyuridine (BrdU) treatment at intervals after 21 days. Mice treated with rmPRL, but not oPRL, had increased BrdU incorporation in the periventricular area surrounding the third ventricle and median eminence and in the arcuate nucleus. The data obtained in the present study indicate that oPRL, but not rmPRL, when given at a high enough dose, induces TIDA neurone differentiation in dw/dw mice. This supports neurotrophic effects of prolactin on TIDA neurones in early postnatal development that extends beyond maintenance of the cell population.

No description available.

Animals deficient in complement components C1q, C4, C3, and CR1/2 have severely impaired antibody responses. C1q is primarily activated by antibody-antigen complexes. Antigen-specific IgM in complex with an antigen is able to enhance the antibody response against that antigen. This is dependent on the ability of IgM to activate complement. Naïve mice have very low amounts of specific antibodies and therefore it is surprising that classical pathway activation plays a role for primary antibody responses. It was hypothesized that natural IgM, present in naïve mice, would bind an antigen with enough affinity to activate C1q. To test this, a knock-in mouse strain, Cm13, with a point mutation in m heavy chain, making its IgM unable to activate complement was constructed. Surprisingly, the antibody responses in Cm13 were normal. Puzzled by the finding that the ability of IgM to activate complement was required only for some effects, the immunization protocol was changed to mimic an infectious scenario. With this regime, Cm13 mice had an impaired antibody response compared to wildtype (WT) mice. The antibody response in WT mice to these repeated low-dose immunizations was also enhanced. These observations suggest that IgM-mediated enhancement indeed plays a physiological role in initiation of early antibody responses. IgM-mediated enhancement cannot however compensate for the dependecy of T-cell help. Although IgM from WT mice enhanced the antibody response, the T-cell response was not enhanced. The connection between classical pathway activation and CR1/2 is thought to be generation of ligands for CR1/2. In mice, CR1/2 are expressed on B cells and follicular dendritic cells (FDC). Although CR1/2 are crucial for a normal antibody response, the molecular mechanism(s) are not understood. To investigate whether CR1/2 must be expressed on B-cells or FDC to generate a normal antibody response, chimeric mice between WT and CR1/2-deficient mice were constructed. The results show that CR1/2+ FDC were crucial for the generation of antibody responses. In the presence of CR1/2+ FDC, both CR1/2+ and CR1/2- B cells were equally good antibody producers. However, for an optimally enhanced antibody response against IgM-antigen complexes, both B cells and FDC needed to express CR1/2.

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / There are many studies showing that the salinity may directly affect the nitrate uptake, from their osmotic effect, nature of the salt and its ionic composition. However, little is known about the mechanisms related to the salt ability to inhibit the nitrate acquisition indirectly. This study was carried with aim to induce inhibition of NO3- influx in cowpea roots of indirect form triggered by a negative feedback mechanism, caused by the increase in the pool of free amino acids in the tissue, induced by salt stress.For this, were done three isolated studies and continuous.The exogenous glutamine application promoted an increase in the free amino acids content.The presence of glutamine decreased significantly the nitrate acquisition.The free ammonium can also be listed as a key-compound in the role of nitrate influx regulation, since use of the MSX (Methionine sulfoximine) promoted the increase of NH4+ content and also reduced nitrate influx, but, in a lesser degree when compared to treatment with AZA (Azaserine). Salt stress caused a reduction in NO3- influx by decrease in the growth of plants induced by salt. The data indicated which this reduction in the influx is triggered by increase of amino acids content, mainly the glutamine, that is main likely compound to act as signal in the N-feedback regulation. / Existem muitos estudos mostrando que a salinidade pode afetar a absorÃÃo de nitrato de forma direta, a partir do seu efeito osmÃtico, da natureza do sal e de sua composiÃÃo iÃnica. Entretanto, pouco se sabe sobre os mecanismos relacionados com a capacidade do sal em inibir a aquisiÃÃo de nitrato de forma indireta. O presente estudo teve como objetivo induzir a inibiÃÃo do influxo de NO3- em raÃzes de feijÃo-caupà de forma indireta desencadeada por um mecanismo de feedback negativo, provocado pelo aumento no pool de aminoÃcidos livres no tecido, induzido por estresse salino. Para isso, foram realizados trÃs estudos isolados e contÃnuos. A aplicaÃÃo de glutamina exÃgena promoveu um aumento no conteÃdo de aminoÃcidos livres. A presenÃa de glutamina reduziu significativamente a aquisiÃÃo de nitrato. O amÃnio livre tambÃm pode ser listado como um composto-chave no papel da regulaÃÃo do influxo de nitrato, pois a utilizaÃÃo do MSO (Metionina sulfoximina) promoveu o aumento do conteÃdo de NH4+ e tambÃm reduziu o influxo de nitrato, porÃm em menor grau quando comparado ao o tratamento com AZA (Azaserina). O estresse salino causou uma reduÃÃo no influxo de NO3-, pela diminuiÃÃo no crescimento das plantas induzida pelo sal. Adicionalmente, estes dados indicaram que esta reduÃÃo no influxo està ligada ao aumento do teor de aminoÃcidos, principalmente a glutamina, que à o principal componente para atuar como sinal na regulaÃÃo por N-feedback.

The developmental processes of lymphopoiesis generate mature B lymphocytes from hematopoietic stem cells through increasingly restricted intermediates. Networks of transcription factors regulate these cell fate choices and are composed of both ubiquitously expressed and B lineage-specific factors. E-protein transcription factors are encoded by the three genes E2A, E2-2 (SEF2-1), and HEB. The E2A gene is required for B cell development and encodes the alternatively spliced proteins E12 and E47. During B lymphocyte development, the cells have to pass several checkpoints verifying the functionality of their antigen receptors. Early in the development, the expression of a pre-B cell receptor (pre-BCR) with membrane-bound immunoglobulin (Ig) heavy chain protein associated with surrogate light chain (SLC) proteins is a critical checkpoint that monitors for functional Ig heavy chain rearrangement. Signaling from the pre-BCR induces survival and a limited clonal expansion. Here it is shown that pre-BCR signaling rapidly down-regulates the SLCs l5 and VpreB and also the co-receptor CD19. Ca2+ signaling and E2A were shown to be essential for this regulation. E2A mutated in its binding site for the Ca2+ sensor protein calmodulin (CaM), and thus with CaM-resistant DNA binding, makes l5, VpreB and CD19 expression resistant to the inhibition following pre-BCR stimulation. Thus, Ca2+ down-regulates SLC and CD19 gene expression upon pre-BCR stimulation through inhibition of E2A by Ca2+/CaM. A general negative feedback regulation of the pre-BCR proteins as well as many co-receptors and proteins in signal pathways from the receptor was also shown. After the ordered recombination of Ig heavy chain gene segments, also Ig light chain gene segments are recombined together to create antibody diversity. The recombinations are orchestrated by the recombination activating gene (RAG) enzymes, other enzymes that cleave/mutate/assemble DNA of the Ig loci, and the transcription factor Pax5. A key feature of the immune system is the concept that one lymphocyte has only one antigen specificity that can be selected for or against. This requires that only one of the alleles of genes for Ig chains is made functional. The mechanism of this allelic exclusion has however been an enigma. Here pre-BCR signaling was shown to down-regulate several components of the recombination machinery including RAG1 and RAG2 through CaM inhibition of E2A. Furthermore, E2A, Pax5 and the RAGs were shown to be in a complex bound to key sequences on the IgH gene before pre-BCR stimulation and instead bound to CaM after this stimulation. Thus, the recombination complex is directly released through CaM inhibition of E2A. Upon encountering antigens, B cells must adapt to produce a highly specific and potent antibody response. Somatic hypermutation (SH), which introduces point mutations in the variable regions of Ig genes, can increase the affinity for antigen, and antibody effector functions can be altered by class switch recombination (CSR), which changes the expressed constant region exons. Activation-induced cytidine deaminase (AID) is the mutagenic antibody diversification enzyme that is essential for both SH and CSR. The AID enzyme has to be tightly controlled as it is a powerful mutagen. BCR signaling, which signals that good antibody affinity has been reached, was shown to inhibit AID gene expression through CaM inhibition of E2A.  SH increases the antigen binding strength by many orders of magnitude. Each round of SH leads to one or a few mutations, followed by selection for increased affinity. Thus, BCR signaling has to enable selection for successive improvements in antibodies (Ab) over an extremely broad range of affinities. Here the BCR is shown to be subject to general negative feedback regulation of the receptor proteins as well as many co-receptors and proteins in signal pathways from the receptor. Thus, the BCR can down-regulate itself to enable sensitive detection of successive improvements in antigen affinity. Furthermore, the feedback inhibition of the BCR signalosome and most of its protein, and most other gene regulations by BCR stimulation, is through inhibition of E2A by Ca2+/CaM. Differentiation to Ab-secreting plasmablasts and plasma cells is antigen-driven. The interaction of antigen with the membrane-bound Ab of the BCR is critical in determining which clones enter the plasma cell response. Genome-wide analysis showed that differentiation of B cells to Ab-secreting cell is induced by BCR stimulation through very fast regulatory events, and induction of IRF-4 and down-regulation of Pax5, Bcl-6, MITF, Ets-1, Fli-1 and Spi-B gene expressions were identified as immediate early events. Ca2+ signaling through CaM inhibition of E2A was essential for these rapid down-regulations of immediate early genes after BCR stimulation in initiation of plasma cell differentiation.

[Truncated abstract] This study describes the results of a four and a half year investigation examining local regulation of ion transport through pumping epithelial cells. The study focussed on the standard isolated toad skin preparation, made famous by Hans Ussing. Originally, the objective was to perform some simple manipulations on the isolated toad skin, a standard and well-tested epithelial layer, which, according to the literature, was a well-behaved and stable preparation. The purpose of doing these toad skin experiments was to gain familiarity with the experimental techniques, such as measuring the open-circuit voltage (Voc) and the short-circuit current (Isc) across an epithelium. In the process, the experimental information that was obtained was to assist in the development and refinement of a mathematical model of a single pumping epithelial cell . . . Finally, it should be emphasised the toad skin was a convenient tissue model for exploring more general issues such as: (i) how pumping epithelial cells may adjust to changes in the extracellular environment by locally regulating their membrane conductances; (2) how the topology of a cell can influence its function (i.e. the topology can determine whether a cell is optimised for salt transport or water transport). (3) how different cells, with different functions, may be positioned in apposition in a pumping epithelial tissue so that gradients generated by one cell type can be utilised by another. From a broader perspective, it is likely that such issues are also applicable to other pumping epithelia, and ultimately, may assist in understanding how these epithelia function.

Living cells are made up of networks of interacting genes, proteins and other bio-molecules. Simple interactions between network components in forms of feedback regulations can lead to complex collective dynamics. A key task in cell biology is to gain a thorough understanding of the dynamics of intracellular systems and processes. In this thesis, a combined approach of mathematical modelling, computational simulation and analytical techniques, has been used to obtain a deeper insight into the dynamical aspects of a variety of feedback systems commonly encountered in cells. These systems range from model system with detailed available molecular knowledge to general regulatory motifs with varying network structures. Deterministic as well as stochastic modelling techniques have been employed, depending primarily on the specific questions asked. The first part of the thesis focuses on dissecting the principles behind the regulatory design of the Tryptophan Operon system in Escherichia coli. It has evolved three negative feedback loops, namely repression, attenuation and enzyme inhibition, as core regulator mechanisms to control the intracellular level of tryptophan amino acid, which is taken up for protein synthesis. Despite extensive experimental knowledge, the roles of these seemingly redundant loops remain unclear from a dynamical point of view. We aim to understand why three loops, rather than one, have evolved. Using a large-scale perturbation/response analysis through modelling and simulations and novel metrics for transient dynamics quantification, it has been revealed that the multiple negative feedback loops employed by the tryptophan operon are not redundant. In fact, they have evolved to concertedly give rise to a much more efficient, adaptive and stable system, than any single mechanism would provide. Since even the full topology of feedback interactions within a network is insufficient to determine its behavioural dynamics, other factors underlying feedback loops must be characterised to better predict system dynamics. In the second part of the thesis, we aim to derive these factors and explore how they shape system dynamics. We develop an analytical approach for stability and bifurcation analysis and apply it to class of feedback systems commonly encountered in cells. Our analysis showed that the strength and the Hill coefficient of a feedback loop play key role in determining the dynamics of the system carrying the loop. Not only that, the position of the loop was also found to be crucial in this decision. The analytical method we developed also facilitates parameter sensitivity analysis in which we investigate how the production and degradation rates affect system dynamics. We find that these rates are quite different in the way they shape up system behaviour, with the degradation rates exhibiting a more intricate manner. We demonstrated that coupled-loop systems display greater complexity and a richer repertoire of behaviours in comparison with single-loop ones. Different combinations of the feedback strengths of individual loops give rise to different dynamical regimes. The final part of the thesis aims to understand the effects of molecular noise on dynamics of specific systems, in this case the Tryptophan Operon. We developed two stochastic models for the system and compared their predictions to those given by the deterministic model. By means of simulations, we have shown that noise can induce oscillatory behaviour. On the other hand, incorporating noise in an oscillatory system can alter the characteristics of oscillation by shifting the bifurcation point of certain parameters by a substantial amount. Measurement of fluctuations reveals that that noise at the transcript level is most significant while noise at the enzyme level is smallest. This study highlights that noise should not be neglected if we want to obtain a complete understanding of the dynamic behaviour of cells.

No description available.

Mouse embryonic stem cells (mESCs) can be maintained in a proliferative and undifferentiated state over many passages (self-renewal) while retaining the potential to give rise to every cell type of the organism (pluripotency). Autocrine FGF4/Erk signalling has been identified as a major stimulus for fate decisions and lineage commitment in these cells. Recent findings on serum-free culture conditions with specific inhibitors (known as 2i) demonstrate that the inhibition of this pathway reduces transcription factor heterogeneity and is vital to maintain ground state pluripotency of mESCs. We suggest a novel mathematical model to explicitly integrate FGF4/Erk signalling into an interaction network of key pluripotency factors (namely Oct4, Sox2, Nanog and Rex1). The envisaged model allows to explore whether and how proposed mechanisms and feedback regulations can account for different expression patterns in mESC cultures. We demonstrate that an FGF4/Erk-mediated negative feedback is sufficient to induce molecular heterogeneity with respect to Nanog and Rex1 expression and thus critically regulates the propensity for differentiation and the loss of pluripotency. Furthermore, we compare simulation results on the transcription factor dynamics in different self-renewing states and during differentiation with experimental data on a Rex1GFPd2 reporter cell line using flow cytometry and qRT-PCR measurements. Concluding from our results we argue that interaction between FGF4/Erk signalling and Nanog expression qualifies as a key mechanism to manipulate mESC pluripotency. In particular, we infer that ground state pluripotency under 2i is achieved by shifting stable expression pattern of Nanog from a bistable into a monostable regulation impeding stochastic state transitions. Furthermore, we derive testable predictions on altering the degree of Nanog heterogeneity and on the frequency of state transitions in LIF/serum conditions to challenge our model assumptions.

Mouse embryonic stem cells (mESCs) can be maintained in a proliferative and undifferentiated state over many passages (self-renewal) while retaining the potential to give rise to every cell type of the organism (pluripotency). Autocrine FGF4/Erk signalling has been identified as a major stimulus for fate decisions and lineage commitment in these cells. Recent findings on serum-free culture conditions with specific inhibitors (known as 2i) demonstrate that the inhibition of this pathway reduces transcription factor heterogeneity and is vital to maintain ground state pluripotency of mESCs. We suggest a novel mathematical model to explicitly integrate FGF4/Erk signalling into an interaction network of key pluripotency factors (namely Oct4, Sox2, Nanog and Rex1). The envisaged model allows to explore whether and how proposed mechanisms and feedback regulations can account for different expression patterns in mESC cultures. We demonstrate that an FGF4/Erk-mediated negative feedback is sufficient to induce molecular heterogeneity with respect to Nanog and Rex1 expression and thus critically regulates the propensity for differentiation and the loss of pluripotency. Furthermore, we compare simulation results on the transcription factor dynamics in different self-renewing states and during differentiation with experimental data on a Rex1GFPd2 reporter cell line using flow cytometry and qRT-PCR measurements. Concluding from our results we argue that interaction between FGF4/Erk signalling and Nanog expression qualifies as a key mechanism to manipulate mESC pluripotency. In particular, we infer that ground state pluripotency under 2i is achieved by shifting stable expression pattern of Nanog from a bistable into a monostable regulation impeding stochastic state transitions. Furthermore, we derive testable predictions on altering the degree of Nanog heterogeneity and on the frequency of state transitions in LIF/serum conditions to challenge our model assumptions.