The grey squirrel (Sciurus carolinensis) has wildtype and melanic (dark) colour morphs. Melanism is associated with variations in the melanocortin-1 receptor (MC1R) gene in a number of species. The MC1R protein is a G-protein coupled receptor, predominantly expressed in melanocytes, where it is a key regulator of pigment production. To investigate the genetic and molecular basis of melanism, the MC1R genes of the wildtype and melanic grey squirrel were sequenced. The wildtype (MC1R-wt) and melanic (MC1RΔ24) variants of the MC1R were then functionally characterised in a cell-based assay. The MC1R gene of the grey squirrel was found to have a 24 base pair (bp) deletion associated with melanism. The MC1R is typically activated by its agonist, the alpha-melanocyte stimulating hormone (α-MSH), which stimulates dark pigment production by raising intracellular cAMP levels. Conversely, the MC1R is inactivated by its inverse agonist, the agouti signalling protein (ASIP), which stops dark pigment production by lowering intracellular cAMP levels. To investigate the effects that the 24 bp deletion have on receptor function, MC1R-wt and MC1RΔ24 genes were transfected into HEK293 cells. Cells expressing either MC1R-wt or MC1RΔ24 were stimulated with α-MSH or ASIP and intracellular cAMP levels were measured. Unstimulated MC1RΔ24 cells showed higher basal activity than the MC1R-wt cells. Both MC1R-wt and MC1RΔ24 cells responded to α-MSH with a concentration-dependent increase in intracellular cAMP. However, while the MC1Rwt cells responded to ASIP with a concentration-dependent decrease in intracellular cAMP, MC1RΔ24 cells responded with an increase in cAMP. Melanism in the grey squirrel is associated with a 24 bp deletion in the MC1R. Cells expressing MC1RΔ24 have higher basal levels of cAMP than MC1R-wt cells. ASIP acts as an inverse agonist to the MC1R-wt but as an agonist to the MC1RΔ24. As MC1RΔ24 cells have higher levels of cAMP, and higher levels of cAMP lead to dark pigment production, the 24 bp deletion is the likely molecular cause of melanism in the grey squirrel.

A coloração dos vertebrados deve-se a presença de pigmentos, sintetizados e/ou armazenados em células denominadas células pigmentares cutâneas. A mudança de cor nos vertebrados é principalmente regulada por α-MSH e uma família de enzimas melanossômicas, que incluem tirosinase e as proteínas relacionadas à tirosinase 1 e 2 (TRP-1 e TRP-2, respectivamente). Sua ação está ligada à dispersão dos melanossomos ou síntese de melanina, processos que resultam em escurecimento do animal, enquanto a agregação ou inibição de síntese leva ao seu empalidecimento. Opsinas, como a melanopsina e a rodopsina, além de presentes na retina, podem ser expressas em células pigmentares cutâneas, intermediando foto-respostas de proliferação e de dispersão de melanossomos. O objetivo deste trabalho foi investigar a expressão temporal da rodopsina, tirosinase e do receptor MC1R, bem como os efeitos do tratamento com α-MSH 10-7 M, 10-8 M e 10-9 M por 24 horas sobre esses parâmetros, em melanócitos B16 de Mus musculus, mantidos em escuro constante. Através de PCR em tempo real (quantitativo) demonstrou-se que α-MSH 10-7 M não modula os níveis de mRNA para o receptor MC1R quando comparado com o grupo controle, contudo há uma evidente tendência de redução dos níveis do transcrito. Todavia, na concentração de 10-8 M, observou-se um aumento estatisticamente significativo no nível do transcrito na hora 20 quando comparado ao grupo controle e na concentração de 10-9 M o tratamento mostrou uma diminuição estatisticamente significativa no nível do transcrito entre o grupo controle e o tratado para cada ponto temporal analisado. Para a rodopsina, foi demonstrado que &alpha-MSH 10-7 M modula os níveis do mRNA quando comparado ao grupo controle, mostrando uma diminuição estatisticamente significativa na hora 0 e 16. Na concentração de 10-8 M houve um aumento estatisticamente significativo nos níveis do transcrito na hora 4 quando comparado ao grupo controle. Já, na concentração de 10-9 M, o hormônio induziu um robusto aumento no nível do transcrito quando comparado ao grupo controle para cada ponto temporal analisado. Nossos resultados são pioneiros em demonstrar a modulação de rodopsina por α-MSH, pois não há dados na literatura, seja em retina ou em outros tecidos, que tenham investigado essa ação do hormônio melanotrópico. O mesmo padrão foi observado para a tirosinase, demonstrando uma diminuição estatisticamente significativa na concentração de 10-7 M na hora 0 e um aumento significativo na concentração de 10-8 M na hora 8 e na concentração de 10-9 M na hora 12 e 8. Através de PCR em tempo real (quantitavo) nós demonstramos que α-MSH apresenta uma modulação dose-dependente para o transcrito do mRNA do receptor MC1R, tirosinase e rodopsina, mas não sincronizou a expressão desses genes, que permaneceram arrítmicos / In vertebrates, skin color is given by pigments, synthesized and/or stored in cutaneous pigment cells. The vertebrate color change is mainly regulated by α-MSH and a family of melanosome enzymes, which includes tyrosinase and tyrosinaserelated proteins 1 and 2 (TRP-1 and TRP-2, respectively). α-MSH action is associated with melanosome dispersion or melanin synthesis, processes which lead to skin darkening, whereas melanin aggregation or synthesis inhibition results in skin lightening. Opsins, such as melanopsin and rhodopsin, may be expressed in skin pigment cells, besides being present in the retina, and mediate non visual photoresponses such as cell proliferation and melanosome dispersion. The aim of this study was to investigate the temporal expression of rhodopsin, tyrosinase and the receptor MC1R, as well as the effects of 10-7 M, 10-8 M and 10-9 M α-MSH for 24 hours in Mus musculus B16 melanocytes, kept in constant darkness. Using real time PCR (quantitative) we demonstrated that 10-7 M α-MSH does not modulate MC1R mRNA levels, as compared to the control group, although a tendency to reduction was evident. On the other hand, at the concentration of 10-8 M, we observed a statistically significant increase of the transcript level at the hour 20, as compared to the control group and at the concentration of 10-9 M the treatment showed a statistically significant decrease of the transcript level for each temporal point analyzed. For rhodopsin, we showed that 10-7 M α-MSH modulates mRNA levels, as compared to the control group, demonstrating a statistically significant decrease at the hour 0 and 16. At the concentration of 10-8 M there was a statistically significant increase of transcript levels at the hour 4, as compared to the control group. The hormone at 10-9 M induced a robust increase of the transcript levels, as compared to the control group, for each time point analyzed. Our results are pioneering in demonstrating the regulation of rhodopsin by α-MSH, since there are no data in the literature which report the action of melanotropic hormone on rhodopsin in either the retina or other tissues. Similar pattern was observed for the tyrosinase gene, demonstrating a statistically significant decrease in the concentration of 10-7 M at the hour 0 and a significant increase in the concentration of 10-8 M at the hour 8 and in the concentration of the 10-9 M at the hour 12 and 8. Using real time PCR (quantitative) we demonstrated that α-MSH shows a dose-dependent modulation for mRNA transcripts of the MC1R receptor, tyrosinase and rhodopsin, but the hormone was not able to synchronize the expression of these genes, which remained arhythmic

A coloração dos vertebrados deve-se a presença de pigmentos, sintetizados e/ou armazenados em células denominadas células pigmentares cutâneas. A mudança de cor nos vertebrados é principalmente regulada por α-MSH e uma família de enzimas melanossômicas, que incluem tirosinase e as proteínas relacionadas à tirosinase 1 e 2 (TRP-1 e TRP-2, respectivamente). Sua ação está ligada à dispersão dos melanossomos ou síntese de melanina, processos que resultam em escurecimento do animal, enquanto a agregação ou inibição de síntese leva ao seu empalidecimento. Opsinas, como a melanopsina e a rodopsina, além de presentes na retina, podem ser expressas em células pigmentares cutâneas, intermediando foto-respostas de proliferação e de dispersão de melanossomos. O objetivo deste trabalho foi investigar a expressão temporal da rodopsina, tirosinase e do receptor MC1R, bem como os efeitos do tratamento com α-MSH 10-7 M, 10-8 M e 10-9 M por 24 horas sobre esses parâmetros, em melanócitos B16 de Mus musculus, mantidos em escuro constante. Através de PCR em tempo real (quantitativo) demonstrou-se que α-MSH 10-7 M não modula os níveis de mRNA para o receptor MC1R quando comparado com o grupo controle, contudo há uma evidente tendência de redução dos níveis do transcrito. Todavia, na concentração de 10-8 M, observou-se um aumento estatisticamente significativo no nível do transcrito na hora 20 quando comparado ao grupo controle e na concentração de 10-9 M o tratamento mostrou uma diminuição estatisticamente significativa no nível do transcrito entre o grupo controle e o tratado para cada ponto temporal analisado. Para a rodopsina, foi demonstrado que &alpha-MSH 10-7 M modula os níveis do mRNA quando comparado ao grupo controle, mostrando uma diminuição estatisticamente significativa na hora 0 e 16. Na concentração de 10-8 M houve um aumento estatisticamente significativo nos níveis do transcrito na hora 4 quando comparado ao grupo controle. Já, na concentração de 10-9 M, o hormônio induziu um robusto aumento no nível do transcrito quando comparado ao grupo controle para cada ponto temporal analisado. Nossos resultados são pioneiros em demonstrar a modulação de rodopsina por α-MSH, pois não há dados na literatura, seja em retina ou em outros tecidos, que tenham investigado essa ação do hormônio melanotrópico. O mesmo padrão foi observado para a tirosinase, demonstrando uma diminuição estatisticamente significativa na concentração de 10-7 M na hora 0 e um aumento significativo na concentração de 10-8 M na hora 8 e na concentração de 10-9 M na hora 12 e 8. Através de PCR em tempo real (quantitavo) nós demonstramos que α-MSH apresenta uma modulação dose-dependente para o transcrito do mRNA do receptor MC1R, tirosinase e rodopsina, mas não sincronizou a expressão desses genes, que permaneceram arrítmicos / In vertebrates, skin color is given by pigments, synthesized and/or stored in cutaneous pigment cells. The vertebrate color change is mainly regulated by α-MSH and a family of melanosome enzymes, which includes tyrosinase and tyrosinaserelated proteins 1 and 2 (TRP-1 and TRP-2, respectively). α-MSH action is associated with melanosome dispersion or melanin synthesis, processes which lead to skin darkening, whereas melanin aggregation or synthesis inhibition results in skin lightening. Opsins, such as melanopsin and rhodopsin, may be expressed in skin pigment cells, besides being present in the retina, and mediate non visual photoresponses such as cell proliferation and melanosome dispersion. The aim of this study was to investigate the temporal expression of rhodopsin, tyrosinase and the receptor MC1R, as well as the effects of 10-7 M, 10-8 M and 10-9 M α-MSH for 24 hours in Mus musculus B16 melanocytes, kept in constant darkness. Using real time PCR (quantitative) we demonstrated that 10-7 M α-MSH does not modulate MC1R mRNA levels, as compared to the control group, although a tendency to reduction was evident. On the other hand, at the concentration of 10-8 M, we observed a statistically significant increase of the transcript level at the hour 20, as compared to the control group and at the concentration of 10-9 M the treatment showed a statistically significant decrease of the transcript level for each temporal point analyzed. For rhodopsin, we showed that 10-7 M α-MSH modulates mRNA levels, as compared to the control group, demonstrating a statistically significant decrease at the hour 0 and 16. At the concentration of 10-8 M there was a statistically significant increase of transcript levels at the hour 4, as compared to the control group. The hormone at 10-9 M induced a robust increase of the transcript levels, as compared to the control group, for each time point analyzed. Our results are pioneering in demonstrating the regulation of rhodopsin by α-MSH, since there are no data in the literature which report the action of melanotropic hormone on rhodopsin in either the retina or other tissues. Similar pattern was observed for the tyrosinase gene, demonstrating a statistically significant decrease in the concentration of 10-7 M at the hour 0 and a significant increase in the concentration of 10-8 M at the hour 8 and in the concentration of the 10-9 M at the hour 12 and 8. Using real time PCR (quantitative) we demonstrated that α-MSH shows a dose-dependent modulation for mRNA transcripts of the MC1R receptor, tyrosinase and rhodopsin, but the hormone was not able to synchronize the expression of these genes, which remained arhythmic

The kinetic schemes, also known as Boltzmann schemes are based on the moment-method-strategy, where an upwind scheme is first developed at the Boltzmann level and after taking suitable moments we arrive at an upwind scheme for the governing Euler or Navier-Stokes equations. The Kinetic Flux Vector Splitting (KFVS)scheme, which belongs to the family of kinetic schemes is being extensively used to compute inviscid as well as viscous flows around many complex configurations of practical interest over the past two decades. To resolve many flow features accurately, like suction peak, minimising the loss in stagnation pressure, shocks, slipstreams, triple points, vortex sheets, shock-shock interaction, mixing layers, flow separation in viscous flows require an accurate and low dissipative numerical scheme. The first order KFVS method even though is very robust suffers from the problem of having much more numerical diffusion than required, resulting in very badly smearing of the above features. However, numerical dissipation can be reduced considerably by using higher order kinetic schemes. But they require more points in the stencil and hence consume more computational time and memory. The second order schemes require flux or slope limiters in the neighbourhood of discontinuities to avoid spurious and physically meaningless wiggles or oscillations in pressure, temperature or density. The limiters generally restrict the residue fall in second order schemes while in first order schemes residue falls up to machine zero. Further, pressure and density contours or streamlines are much smoother for first order accurate schemes than second order accurate schemes. A question naturally arises about the possibility of constructing first order upwind schemes which retain almost all advantages mentioned above while at the same time crisply capture the flow features. In the present work, an attempt has been made to address the above issues by developing yet another kinetic scheme, known as the low dissipative modified KFVS (m-KFVS) method based on modified CIR (MCIR) splitting with molecular velocity dependent dissipation control function. Different choices for the dissipation control function are presented. A detailed mathematical analysis and the underlying physical arguments behind these choices are presented. The expressions for the m-KFVS fluxes are derived. For one of the choices, the expressions for the split fluxes are similar to the usual first order KFVS method. The mathematical properties of 1D m-KFVS fluxes and the eigenvalues of the corresponding flux Jacobians are studied numerically. The analysis of numerical dissipation is carried out both at Boltzmann and Euler levels. The expression for stability criterion is derived. In order to be consistent with the interior scheme, modified solid wall and outer boundary conditions are derived by extending the MCIR idea to boundaries. The cell-centred finite volume method based on m-KFVS is applied to several standard test cases for 1D, 2D and 3D inviscid flows. In the case of subsonic flows, the m-KFVS method produces much less numerical entropy compared to first order KFVS method and the results are comparable to second order accurate q-KFVS method. In transonic and supersonic flows, m-KFVS generates much less numerical dissipation compared to first order KFVS and even less compared to q-KFVS method. Further, the m-KFVS method captures the discontinuities more sharply with contours being smooth and near second order accuracy has been achieved in smooth regions, by still using first order stencil. Therefore, the numerical dissipation generated by m-KFVS is considerably reduced by suitably choosing the dissipation control variables. The Euler code based on m-KFVS method almost takes the same amount of computational time as that of KFVS method. Although, the formal accuracy is of order one, the m-KFVS method resolves the flow features much more accurately compared to first order KFVS method but the numerical dissipation generated by m-KFVS method may not be minimal. Hence, the dissipation control vector is in general not optimal. If we can find the optimal dissipation control vector then we will be able to achieve the minimal dissipation. In the present work, the above objective is attained by posing the minimisation of numerical dissipation in m-KFVS method as an optimal control problem. Here, the control variables are the dissipation control vector. The discrete form of the cost function, which is to be minimised is considered as the sum of the squares of change in entropy at all cells in the computational domain. The number of control variables is equal to the total number of cells or finite volumes in the computational domain, as each cell has only one dissipation control variable. In the present work, the minimum value of cost function is obtained by using gradient based optimisation method. The sensitivity gradients of the cost function with respect to the control variables are obtained using discrete adjoint approach. The discrete adjoint equations for the optimisation problem of minimising the numerical dissipation in m-KFVS method applied to 2D and 3D Euler equations are derived. The method of steepest descent is used to update the control variables. The automatic differentiation tool Tapenade has been used to ease the development of adjoint codes. The m-KFVS code combined with discrete adjoint code is applied to several standard test cases for inviscid flows. The test cases considered are, low Mach number flows past NACA 0012 airfoil and two element Williams airfoil, transonic and supersonic flows past NACA 0012 airfoil and finally, transonic flow past Onera M6 wing. Numerical results have shown that the m-KFVS-adjoint method produces even less numerical dissipation compared to m-KFVS method and hence results in more accurate solution. The m-KFVS-adjoint code takes more computational time compared to m-KFVS code. The present work demonstrates that it is possible to achieve near second order accuracy by formally first order accurate m-KFVS scheme while retaining advantages of first order accurate methods.

Least Square Kinetic Upwind Method (LSKUM) belongs to the class of mesh-less method that solves compressible Euler equations of gas dynamics. LSKUM is kinetic theory based upwind scheme that operates on any cloud of points. Euler equations are derived from Boltzmann equation (of kinetic theory of gases) after taking suitable moments. The basic update scheme is formulated at Boltzmann level and mapped to Euler level by suitable moments. Mesh-less solvers need only cloud of points to solve the governing equations. For a complex configuration, with such a solver, one can generate a separate cloud of points around each component, which adequately resolves the geometric features, and then combine all the individual clouds to get one set of points on which the solver directly operates. An obvious advantage of this approach is that any incremental changes in geometry will require only regeneration of the small cloud of points where changes have occurred. Additionally blanking and de-blanking strategy along with overlay point cloud can be adapted in some applications like store separation to avoid regeneration of points. Naturally, the mesh-less solvers have advantage in tackling complex geometries and moving components over solvers that need grids. Conventionally, higher order accuracy for space derivative term is achieved by two step defect correction formula which is computationally expensive. The present solver uses low dissipation single step modified CIR (MCIR) scheme which is similar to first order LSKUM formulation and provides spatial accuracy closer to second order. The maximum time step taken to march solution in time is limited by stability criteria in case of explicit time integration procedure. Because of this, explicit scheme takes a large number of iterations to achieve convergence. The popular explicit time integration schemes like four stages Runge-Kutta (RK4) are slow in convergence due to this reason. The above problem can be overcome by using the implicit time integration procedure. The implicit schemes are unconditionally stable i.e. very large time steps can be used to accelerate the convergence. Also it offers superior robustness. The implicit Lower-Upper Symmetric Gauss-Seidel (LU-SGS) scheme is very attractive due to its low numerical complexity, moderate memory requirement and unconditional stability for linear wave equation. Also this scheme is more efficient than explicit counterparts and can be implemented easily on parallel computers. It is based on the factorization of the implicit operator into three parts namely lower triangular matrix, upper triangular matrix and diagonal terms. The use of LU-SGS results in a matrix free implicit framework which is very economical as against other expensive procedures which necessarily involve matrix inversion. With implementation of the implicit LU-SGS scheme larger time steps can be used which in turn will reduce the computational time substantially. LU-SGS has been used widely for many Finite Volume Method based solvers. The split flux Jacobian formulation as proposed by Jameson is most widely used to make implicit procedure diagonally dominant. But this procedure when applied to mesh-less solvers leads to block diagonal matrix which again requires expensive inversion. In the present work LU-SGS procedure is adopted for mesh-less approach to retain diagonal dominancy and implemented in 2-D and 3-D solvers in matrix free framework. In order to assess the efficacy of the implicit procedure, both explicit and implicit 2-D solvers are tested on NACA 0012 airfoil for various flow conditions in subsonic and transonic regime. To study the performance of the solvers on different point distributions two types of the cloud of points, one unstructured distribution (4074 points) and another structured distribution (9600 points) have been used. The computed 2-D results are validated against NASA experimental data and AGARD test case. The density residual and lift coefficient convergence history is presented in detail. The maximum speed up obtained by use of implicit procedure as compared to explicit one is close to 6 and 14 for unstructured and structured point distributions respectively. The transonic flow over ONERA M6 wing is a classic test case for CFD validation because of simple geometry and complex flow. It has sweep angle of 30° and 15.6° at leading edge and trailing edge respectively. The taper ratio and aspect ratio of the wing are 0.562 and 3.8 respectively. At M∞=0.84 and α=3.06° lambda shock appear on the upper surface of the wing. 3¬D explicit and implicit solvers are tested on ONERA M6 wing. The computed pressure coefficients are compared with experiments at section of 20%, 44%, 65%, 80%, 90% and 95% of span length. The computed results are found to match very well with experiments. The speed up obtained from implicit procedure is over 7 for ONERA M6 wing. The determination of the aerodynamic characteristics of a wing with the control surface deflection is one of the most important and challenging task in aircraft design and development. Many military aircraft use some form of the delta wing. To demonstrate the effectiveness of 3-D solver in handling control surfaces and small gaps, implicit 3-D code is used to compute flow past clipped delta wing with aileron deflection of 6° at M∞ = 0.9 and α = 1° and 3°. The leading edge backward sweep is 50.4°. The aileron is hinged from 56.5% semi-span to 82.9% of semi-span and at 80% of the local chord from leading edge. The computed results are validated with NASA experiments