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Tesis por compendio / [EN] Cryopreserved boar sperm is not used extensively for artificial insemination due to poor fertility rates of the sperm after freezing and thawing. The sperm membrane is damaged when cooled from body temperature to 5 ºC (cold shock), as well as during the freeze-thaw process. Increasing the cholesterol content of boar sperm membranes could increase their post-thaw survival, similarly to other species that are cold shock sensitive. Cholesterol can be easily added to sperm membranes using cholesterol-loaded cyclodextrins (CLC). Treating sperm from different species susceptible to cold-shock with CLC before cryopreservation improves sperm cryosurvival. Egg yolk and glycerol are common constituents of extenders used for boar sperm cryopreservation. However, conventional freezing extenders could not be the appropriate for CLC-treated sperm. The aim of this Thesis is to evaluate cryosurvival of CLC or cyclodextrin-treated boar sperm in three different conditions: using conventional freezing extenders, using extenders with alternative concentrations of glycerol and egg yolk and using amides as cryoprotectants. CLC or methyl- ß-cyclodextrin treatment (1 mg/120 x 106 sperm) prior to cryopreservation using a conventional freezing extenders provided either slight or no benefit, respectively, to post-thaw sperm plasma membrane integrity (+ 8%; P < 0.05) and motility (P > 0.05). In addition, sperm from both, good and poor freezers, responded similarly to CLC treatment (P > 0.05). Reduction in egg yolk concentration from 20 to 10% was detrimental for post-thaw sperm viability, even in semen treated with CLC (- 12%; P < 0.05). On the other hand, it was observed that traditional concentration of glycerol (3%) was not the appropriate to freeze CLC-treated sperm (- 13% viable sperm compared to control; P < 0.05). Thus, CLC-treated sperm showed a higher tolerance (+ 13 % sperm viability; P < 0.05) to high glycerol concentrations (5%) than non-treated sperm. Regarding the efficacy of amides as cryoprotectants, three of the amides (lactamide, acetamide and formamide) produced deleterious effects in fresh boar sperm (P < 0.05). The other amides (methylformamide, dimethylacetamide and dimethylformamide) efficiently improved post-thaw sperm viability (+ 5 to 15 %; P < 0.05) but negatively affected the sperm motility (- 11 to 16% total motile sperm; P < 0.05) and the sperm fertilizing ability in vitro (dimethylformamide: - 64 % penetration rate; P < 0.05), irrespective of the sperm treatment. On the other hand, CLC-treated samples showed better in vitro fertilizing ability than control samples when glycerol was used as cryoprotectant (+ 2 penetrated spermatozoa/oocyte; P < 0.05). The results obtained in this Thesis suggest that conventional freezing protocols should be optimized for CLC-treated boar sperm in order to obtain the benefit of CLC treatment observed in other species sensitive to cold shock. / [ES] Las inseminaciones artificiales en la especie porcina se realizan habitualmente con semen refrigerado, debido a las bajas tasas de fertilidad obtenidas con el semen congelado-descongelado. La membrana del espermatozoide sufre importantes daños cuando es sometida a la fase de enfriamiento desde la temperatura corporal hasta alcanzar los 5 ºC (choque térmico), así como durante el proceso de congelación y descongelación. El aumento del contenido de colesterol en las membranas de los espermatozoides de cerdo podría mejorar su supervivencia tras la descongelación, como sucede en otras especies sensibles al choque térmico. Este incremento en la cantidad de colesterol se puede realizar fácilmente utilizando ciclodextrinas saturadas de colesterol (CLC). El tratamiento con CLC de espermatozoides de varias especies susceptibles al choque térmico antes de la congelación ha conseguido mejorar su supervivencia tras la descongelación. En los protocolos convencionales de congelación de semen porcino se utilizan habitualmente diluyentes de congelación compuestos por yema de huevo y glicerol, sin embargo, puede que estos diluyentes de congelación convencionales no sean los más apropiados para congelar espermatozoides tratados con CLC. El objetivo de esta Tesis es evaluar la supervivencia a la congelación de los espermatozoides porcinos tratados con CLC o ciclodextrinas utilizando diluyentes de congelación convencionales, utilizando concentraciones alternativas tanto de yema de huevo como de glicerol o utilizando amidas en lugar de glicerol como crioprotectores Utilizando diluyentes convencionales, el tratamiento con 1mg de CLC o de metil-ß-ciclodextrina/120 millones de espermatozoides previamente a la congelación proporcionó una leve mejora de la integridad de la membrana plasmática espermática (+ 8%; P < 0,05) y ningún beneficio sobre la movilidad espermática (P > 0,05). Además, la respuesta al tratamiento con CLC fue similar independientemente de si los espermatozoides procedían de verracos buenos o malos congeladores (P > 0,05). Una reducción de la concentración de yema de huevo de un 20 a un 10% fue perjudicial para la supervivencia de los espermatozoides tras la descongelación, incluidos aquellos que habían sido tratados previamente con CLC (- 12% espermatozoides vivos; P < 0,05). Por otro lado, observamos que las concentraciones de glicerol utilizadas habitualmente (3%) no son las más apropiadas para congelar espermatozoides tratados con CLC (- 13 % viabilidad espermática comparando con las muestras control; P < 0,05), ya que éstos mostraron una mayor tolerancia (+ 13 % espermatozoides vivos; P < 0,05) que las muestras control a las concentraciones de glicerol más altas (5%). Con respecto a la eficacia de las amidas como crioprotectores para semen porcino, tres de las amidas (lactamida, acetamida y formamida) produjeron efectos perjudiciales durante su incubación con semen fresco (P < 0,05). El resto de amidas evaluadas (metilformamida, dimetilacetamida y dimetilformamida) mejoraron eficientemente la viabilidad espermática tras la congelación (+ 5 a 15 %; P < 0,05), sin embargo, afectaron negativamente tanto la movilidad espermática (- 11 a 16% móviles totales; P < 0,05) como la capacidad de fecundación in vitro (dimetilformamida: - 64 % en la tasa de penetración; P < 0,05), independientemente de si el semen fue tratado con CLC o no. Por otro lado, las muestras tratadas con CLC mostraron mejor capacidad de fecundación in vitro que las muestras control cuando se utilizó el glicerol como crioprotector (+ 2 espermatozoides penetrados/ovocito; P < 0,05). Los resultados obtenidos en esta Tesis sugieren que sería necesaria la adecuación de los protocolos de congelación convencionales para congelar semen porcino tratado con CLC con el propósito de alcanzar los claros beneficios obtenidos con dicho tratamiento cuando ha sido evaluado en otras especies sensibles al choque térmico. / [CA] Les inseminacions artificials en l'espècie porcina es realitzen habitualment amb semen refrigerat, a causa de les baixes taxes de fertilitat obtingudes amb el semen congelat-descongelat. La membrana de l'espermatozoide pateix importants danys quan és sotmesa a la fase de refredament des de la temperatura corporal fins a arribar als 5 ºC (xoc tèrmic), així com durant el procés de congelació i descongelació. L'augment del contingut de colesterol a les membranes dels espermatozoides de porc podria millorar la seva supervivència després de la descongelació, com succeeix en altres espècies sensibles al xoc tèrmic. Aquest increment en la quantitat de colesterol es pot realitzar fàcilment utilitzant ciclodextrines saturades de colesterol (CLC). El tractament amb CLC d'espermatozoides de diverses espècies susceptibles al xoc tèrmic abans de la congelació ha aconseguit millorar la seva supervivència després de la descongelació. En els protocols convencionals de congelació de semen porcí s'utilitzen habitualment diluents de congelació compostos per rovell d'ou i glicerol, però, pot ser que aquests diluents de congelació convencionals no siguin els més apropiats per congelar espermatozoides tractats amb CLC. L'objectiu d'aquesta Tesi és avaluar la supervivència a la congelació dels espermatozoides porcins tractats amb CLC o ciclodextrines utilitzant diluents de congelació convencionals, utilitzant concentracions alternatives tant de rovell d'ou com de glicerol o utilitzant amides en lloc de glicerol com crioprotectors Utilitzant diluents convencionals, el tractament amb 1 mg de CLC o de metil-ß-ciclodextrina / 120 milions d'espermatozoides prèviament a la congelació va proporcionar una lleu millora de la integritat de la membrana plasmàtica espermàtica (+ 8%; P <0,05) i cap benefici sobre la mobilitat espermàtica (P> 0,05). A més, la resposta al tractament amb CLC va ser similar independentment de si els espermatozoides procedien de verros bons o dolents congeladors (P> 0,05). Una reducció de la concentració de rovell d'ou d'un 20 a un 10% va ser perjudicial per a la supervivència dels espermatozoides després de la descongelació, inclosos aquells que havien estat tractats prèviament amb CLC (- el 12% espermatozoides vius; P <0,05) . D'altra banda, observem que les concentracions de glicerol utilitzades habitualment (3%) no són les més apropiades per congelar espermatozoides tractats amb CLC (- 13% viabilitat espermàtica comparant amb les mostres control; P <0,05), ja que aquests van mostrar una major tolerància (+ 13% espermatozoides vius; P <0,05) que les mostres control a les concentracions de glicerol més altes (5%). Pel que fa a l'eficàcia de les amides com crioprotectors per semen porcí, tres de les amides (lactamida, acetamida i formamida) van produir efectes perjudicials durant la seva incubació amb semen fresc (P <0,05). La resta de amides avaluades (metilformamida, dimetilacetamida i dimetilformamida) van millorar eficientment la viabilitat espermàtica després de la congelació (+ 5 a 15%, P <0,05), però, van afectar negativament tant la mobilitat espermàtica (- 11 a 16% mòbils totals; P <0,05) com la capacitat de fecundació in vitro (dimetilformamida: - el 64% en la taxa de penetració; P <0,05), independentment de si el semen va ser tractat amb CLC o no. D'altra banda, les mostres tractades amb CLC van mostrar millor capacitat de fecundació in vitro que les mostres control quan es va utilitzar el glicerol com crioprotector (+ 2 espermatozous penetrats / oòcit; P <0,05). Els resultats obtinguts en aquesta Tesi suggereixen que seria necessària l'adequació dels protocols de congelació convencionals per congelar semen porcí tractat amb CLC amb el propòsit d'assolir els clars beneficis obtinguts amb el tractament quan ha estat avaluat en altres espècies sensibles al xoc tèrmic. / Blanch Torres, E. (2015). Treating boar sperm with cholesterol-loaded cyclodextrins or cyclodextrins prior to cryopreservation: effects on post-thaw in vitro sperm quality of sperm cryopreserved in different freezing extenders [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/58773 / Compendio

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

Les canaux chlorure sont impliqués dans divers processus indispensables à la vie cellulaire, tel que la stabilisation du potentiel de membrane, la régulation du volume ou du pH, ainsi que dans les transports transépithéliaux de sels. Depuis une douzaine d'années, l'application de la biologie moléculaire à l'étude des canaux chlorure a permis d'identifier plusieurs familles de gènes. La variété fonctionnelle des canaux chlorure peut donc s'expliquer par leur diversité moléculaire. Actuellement, la détermination des rôles physiologiques des canaux chlorure clonés constitue un enjeu majeur de leur étude.<br /><br />Récemment, une stratégie de clonage par homologie nous a permis d'isoler deux nouveaux membres de la famille des ClC, canaux chlorure dépendants du voltage: le xClC-5 et le xClC-3. La caractérisation fonctionnelle et la détermination de la localisation tissulaire et cellulaire de ces protéines a constitué l'objectif de notre étude. Pour cela, l'ovocyte de xénope et la lignée cellulaire HEK 293 ont été utilisés comme systèmes d'expression. L'analyse électrophysiologique, menée par une technique de voltage-clamp en double micro-électrodes, nous a permis de caractériser le xClC-5 du point de vue électrophysiologique et de déterminer sa sensibilité au pH, aux ions métalliques et aux inhibiteurs de tyrosine kinases. L'utilisation d'anticorps nous a permis d'étudier la glycosylation des protéines et d'examiner leur distribution tissulaire et/ou leur localisation cellulaire. Nos résultats sont discutés par rapport à ceux d'autres équipes, en prêtant une attention particulière aux modèles fonctionnels possibles, qui émergent actuellement pour les protéines ClC-5 et ClC-3.

Pulmonary arterial hypertension (PAH) is a rare but fatal disease with an annual mortality rate of 15% despite current therapies. Uncontrolled proliferation of pulmonary artery smooth muscle cells (PASMCs) results in adverse vascular remodeling contributing to PAH. Understanding the mechanisms of PASMC proliferation may identify new targets for treatment. Chloride currents/channels (ICl) are expressed in PASMCs and their roles in proliferation have been suggested based on their importance in resting membrane potential and cell volume regulation. The present study explored the role of ICl in proliferation in rat and human PASMCs. We found that either nonspecific ICl inhibitors (DIDS or NPPB) or a putative specific blocker of swelling-activated ICl (ICl,swell) reduced proliferation of PASMCs cultured in serum-containing media. Patch-clamp studies showed that proliferating PASMCs had increased baseline ICl and ICl,swell in association with depolarized membrane potentials. Quantitative real-time RT-PCR studies identified expressions of CLC-3, a candidate gene of ICl,swell, and several other CLC genes in proliferating PASMCs. While selective knockdown of CLC-3 with lentiviral shRNA reduced PASMC proliferation, it had no effect on ICl,swell. These findings are consistent with the conclusion that ICl regulate proliferation of PASMCs and suggest that selective ICl inhibition may be useful in treating pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) is a rare but fatal disease with an annual mortality rate of 15% despite current therapies. Uncontrolled proliferation of pulmonary artery smooth muscle cells (PASMCs) results in adverse vascular remodeling contributing to PAH. Understanding the mechanisms of PASMC proliferation may identify new targets for treatment. Chloride currents/channels (ICl) are expressed in PASMCs and their roles in proliferation have been suggested based on their importance in resting membrane potential and cell volume regulation. The present study explored the role of ICl in proliferation in rat and human PASMCs. We found that either nonspecific ICl inhibitors (DIDS or NPPB) or a putative specific blocker of swelling-activated ICl (ICl,swell) reduced proliferation of PASMCs cultured in serum-containing media. Patch-clamp studies showed that proliferating PASMCs had increased baseline ICl and ICl,swell in association with depolarized membrane potentials. Quantitative real-time RT-PCR studies identified expressions of CLC-3, a candidate gene of ICl,swell, and several other CLC genes in proliferating PASMCs. While selective knockdown of CLC-3 with lentiviral shRNA reduced PASMC proliferation, it had no effect on ICl,swell. These findings are consistent with the conclusion that ICl regulate proliferation of PASMCs and suggest that selective ICl inhibition may be useful in treating pulmonary arterial hypertension.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / In this paper we present a spectral model to describe the decay of turbulent kinetic energy in the Convective Boundary Layer (CLC) of the earth s surface, where the physical processes that occur generate turbulence of convective origin and mechanics in the air. Using the equations of conservation of time, which describe the dynamics of an element of fluid in a flow, you get an equation for the spectrum of kinetic energy in a homogeneous turbulent flow, but not isotropic. The spectrum of energy is expressed in terms of number of wave vector kappa and time. Each term in this equation of energy balance, describing different physical processes that generate the turbulence. The terms of production or loss of energy by the effect of heat and friction, are written according to the number of Richardson, which is a dimensionless quantity that expresses a relationship between potential energy and kinetic energy of a fluid. The term transfer of kinetic energy by inertial effect between eddies of different wave numbers is parameterized from the Heisenberg model which, based on intuitive arguments, assume that the transfer of energy between eddies with small number of wave for the large number of wavelength is similar to conversion of mechanical energy into heat energy, the effect of molecular viscosity. The number of eddies with wave absorbing higher energy of eddies of wave number with lower. The dynamic equation for the three-dimensional spectrum of kinetic energy obtained was solved by the Adomian decomposition method for the analytical solution of ordinary differential equations or partial, linear or nonlinear, deterministic or stochastic. This technique is to decompose a given equation into a linear part and one non-linear, isolating the operator linear, easily inverted of higher order. The nonlinear term is written as a sum of a special class of polynomials called Adomian polynomials of, and unknown function as a series whose terms are calculated on recursively. The application of the Adomian decomposition method for the solution of differential equation integrated non linear due to the spectrum of kinetic energy, has an analytical solution without linearization, commonly used for simplicity, in problems where processes are highly nonlinear. Moreover, due to rapid convergence of the solution in terms of the Adomian polynomials, the spectrum of kinetic energy was obtained without a large computational effort. From the calculation of the energy spectrum could be determined the variation of turbulent kinetic energy in the CLC and compared with results of numerical simulation in the literature. / No presente trabalho é apresentado um modelo espectral para descrever o decaimento da energia cinética turbulenta na Camada Limite Convectiva (CLC) da superfície terrestre, onde acontecem os processos físicos que geram turbulência de origem mecânica e convectiva no ar. Partindo das equações de conservação de momento, que descrevem a dinâmica de um elemento de fluído em um escoamento, se obtém uma equação para o espectro de energia cinética em um escoamento turbulento homogêneo, mas não isotrópico. O espectro de energia é expresso em termos do vetor número de onda κ e do tempo. Cada termo, nesta equaçaão de balanço de energia, descreve processos físicos distintos que geram a turbulência. Os termos de produção ou perda de energia por efeito térmico e por atrito, são escritos em função do número de Richardson, que é uma grandeza adimensional que expressa uma relação entre a energia potencial e a energia cinética de um fluído. O termo de transferência de energia cinética por efeito inercial entre os turbilhões de diferentes números de onda é parametrizado a partir do modelo de Heisenberg que, baseando-se em argumentos intuitivos, assume que o processo de transferência de energia entre turbilhões com pequeno número de onda para os de número de onda grande, é similar a conversão de energia mecânica em energia térmica, por efeito de uma viscosidade molecular. Os turbilhões com número de onda maior absorvem energia dos turbilhões com número de onda menor. A equação dinâmica para o espectro de energia cinética tridimensional obtida foi resolvida pelo método da decomposição de Adomian para solução analítica de equações diferenciais ordinárias ou parciais, lineares ou não lineares, determinísticas ou estocásticas. Esta técnica consiste em decompor uma dada equação em uma parte linear e outra não-linear, isolando o operador linear, facilmente inversível, de maior ordem. O termo não-linear é escrito como uma soma de uma classe especial de polinômios, denominados Polinômios de Adomian, e a função desconhecida como uma série, cujos termos são calculados de forma recursiva. A aplicação do método de decomposição de Adomian na solução da equação integro-diferencial não linear resultante para o espectro de energia cinética, permitiu uma solução analítica sem a linearização, comumente usada por simplicidade, em problemas onde se têm processos altamente não lineares. Além disso, devido a rápida convergência da solução expressa em termos dos polinômios de Adomian, o espectro de energia cinética foi obtido sem uma grande esforço computacional. A partir do cálculo do espectro de energia pôde-se determinar a variação da energia cinética turbulenta na CLC e comparar com os resultados de simulação numérica existentes na literatura.

Master of Science / Department of Anatomy and Physiology / Daniel C. Marcus / Sensory transduction in the cochlea depends on regulated ion secretion and absorption. Results of whole-organ experiments suggested that Reissner’s membrane may play a role in the control of luminal Cl-. We tested for the presence of Cl- transport pathways in isolated mouse Reissner’s membrane using whole-cell patch clamp recordings and gene transcript analyses using RT-PCR. The current-voltage (I-V) relationship in the presence of symmetrical NMDG-Cl was strongly inward-rectifying at negative voltages, with a small outward current at positive voltages. The inward-rectifying component of the I-V curve had several properties similar to those of the ClC-2 Cl- channel. It was stimulated by extracellular acidity and inhibited by extracellular Cd2+, Zn2+, and intracellular ClC-2 antibody. Channel transcripts expressed in Reissner’s membrane include ClC-2, Slc26a7 and ClC-Ka, but not Cftr, ClC-1, ClCa1, ClCa2, ClCa3, ClCa4, Slc26a9, ClC-Kb, Best1, Best2, Best3 or the beta-subunit of ClC-K, barttin. ClC-2 is the only molecularly-identified channel present that is a strong inward rectifier. This thesis incorporates the publication by K.X. Kim and D.C. Marcus, Inward-rectifier chloride currents in Reissner’s membrane epithelial cells. Biochem. Biophys. Res. Commun. 394 (2010) 434-438, with permission of the publisher Elsevier, and is the first report of conductive Cl- transport in epithelial cells of Reissner’s membrane and is consistent with an important role in endolymph anion homeostasis.

This thesis deals with the differences of various types of working tools of the Kverneland CLC Pro cultivator. Under specific conditions of the agricultural enterprise in the Czech Republic, it compares ordinary turning tine, carbide tine, Knock-on tine and renovated tine. In the introduction, there is a general overview of the properties of the soil, followed by an overview of various methods of soil cultivation, which are also related to various methods of founding plants. Furthermore, I summarized the types of cultivators and tillers, followed by an overview of the types of wear tines with their advantages and disadvantages. In the practical part of the work there is the monitoring of the lifetime of the compared technologies, the time required for their replacement, the cost of their acquisition and the overall economic evaluation.

To take advantage of all buildable ground in city and exploit already existing infrastructure for a more efficient society high demands are therefore placed on logistics around large construction projects. Travel center in Västerås will be raised in the same place as existing buildning, while the business will be operational during the construction and the goal with the project work is to identify and present logistic solutions and evaluate, compare and adapt these to new travel center in Västerås. What is being investigated among other things is, ”What is the problem with construction traffic and logistics in large infrastructure projects in urban areas?”. In order to find information and answers of questions a literature study, case study, study visits and interviews are used. Logistics is the strategy that involves planning, development, coordination, organization and control of material flows from supplier. Construction logistics differ considerably from traditional logistics in, for example, the manufacturing industry, since each project is unique. Among other things, consideration must be given to environmental impacts such as noise environmental impact and sustainability, but also transport distances and social aspects. Examples of logistic strategies include supply chain management, lean production, just in time, construction logistic centers, Vendor-controlled inventory. This study focuses primarily on the problems that arise in urban areas and how deliveries of building materials are handled. Work within the railroads track area also entails challenges and special rules. The planning of new construction of the travel center in Västerås is divided into seven different stages at the time of writing, where the railway acts as a barrier between these. The construction project manager for the project has experience from previous projects in the urban area and believes that solutions must be adapted to projects. It is seldom easy to use the same solution in another location without modifying it according to the project's conditions. Building logistics implicates great complexity as many players are involved. The problem around construction traffic and logistics in large infrastructure projects is that is has negativ impact on the enviroment and it disrupts the trafic. Project ”Slussen” in Stockholm used a logistics center and from there used the method Just In Time to the workplace with materials that would be used within three days. A material storage in another location is a better alternative than a material storage on site and a solution for logistics management for the travel center is to use logistics center in the harbour and Mälarhamnar can act as third party logistics partner in the project.