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El objetivo de esta tesis doctoral es profundizar en el conocimiento de las bases genéticas y moleculares de la enfermedad de Charcot-Marie-Tooth, especialmente de las formas con herencia autosómica recesiva. En este sentido, se incluyen los resultados obtenidos del análisis genético y molecular del gen GDAP1 en una serie de 118 familias de origen español, cuyas mutaciones son las causantes de la forma autosómica recesiva axonal tipo 4A (CMT4A), así como también, el estudio genético y molecular de la enfermedad de CMT en 17 familias españolas de etnia gitana con herencia autosómica recesiva y neuropatía desmielinizante.Detectamos la presencia de seis mutaciones diferentes en el gen GDAP1 en 11 pacientes de nuestra serie de estudio, de las cuales tres habían sido descritas anteriormente (p.Q163X, p.S194X y p.T288fsX3) y caracterizamos tres nuevas mutaciones (p.E114fsX, p.R120W, p.T157P). Hasta el momento CMT4A había sido descrita como una forma clínica grave de CMT axonal con herencia autosómica recesiva, sin embargo en nuestra serie de estudio diferenciamos dos grupos: 1) pacientes portadores de 2 mutaciones y con un patrón de herencia recesivo, y 2) pacientes con una única mutación y con un posible patrón de herencia autosómico dominante, no descrito con anterioridad. Aportamos datos clínicos y electrofisiológicos que nos permitieron establecer una posible relación genotipo-fenotipo. El análisis de haplotipos de la mutación más frecuente en nuestra serie de estudio, p.Q163X, también encontrada en pacientes tejanos de origen hispanoamericano, nos permitió confirmar que dicha mutación tiene un origen ancestral común en la península ibérica, siendo su distribución y frecuencia actual consecuencia de un efecto fundador. En las 107 familias en las que no encontramos mutaciones en el gen GDAP1 excluimos al gen GDAP1L1 como responsable de la enfermedad de CMT en estos pacientes.El análisis genético y molecular de la enfermedad de CMT en las 17 familias de etnia gitana se abordó en primer lugar, mediante el estudio de los loci conocidos asociados con la enfermedad de CMT en población gitana europea: la neuropatía sensitivo-motora hereditaria Lom (HMSN-Lom o CMT4D) y la neuropatía sensitivo-motora hereditaria Russe (HMSN-Russe o CMT4G), y en segundo lugar, la descripción de nuevos loci. Detectamos la mutación p. R148X en el gen NDRG1 en homocigosis como responsable de la neuropatía HMSN-Lom en un paciente de nuestra serie de estudio, y un posible ligamiento al locus HMSN-Russe en otras tres familias, aunque no pudimos confirmar dichos resultados al no estar descrito el gen. En las 13 familias restantes realizamos el análisis genético mediante el estudio de ligamiento y de haplotipos al locus CMT4C, debido a que la mayoría de nuestros pacientes presentaban un fenotipo clínico similar al locus CMT4C descrito en población no gitana. Encontramos en 13 de los 29 cromosomas estudiados la presencia de un haplotipo común mayoritario sugiriendo la existencia de una posible mutación ancestral común, mientras que en el resto de cromosomas se observaron variaciones alélicas respecto del haplotipo común que podía ser el resultado de recombinaciones ancestrales, o bien haplotipos diferentes. Estos resultados sugerían la posibilidad de la existencia de diferentes mutaciones o bien no se asociaban con mutaciones patológicas. Tras el análisis de mutaciones en el gen SH3TC2 caracterizamos dos nuevas mutaciones (p.R1109X y p.C737_P738delinsX) que no habían sido descritas en población no gitana como responsable de la enfermedad de CMT en 10 familias de nuestra serie de estudio. Detectamos, por tanto, que la causa más frecuente en nuestra serie de estudio de población gitana española fue la forma CMT4C y confirmamos la existencia de un único origen para la mutación p.R1109X, resultados que no fueron descritos con anterioridad en población gitana. / The aim of this doctoral thesis is to go in depth into the knowledge of the genetic and molecular inheritance. In this respect, we have included the results achieved from the genetic and molecular analysis of the GDAP1 gene in a series of 118 Spanish families by birth, which mutations are responsible for the axonal recessive form CMT4A, as well as the genetic and molecular study of 17 Spanish Gypsy families diagnosed with a demyelinating CMT disease compatible with an autosomal recessive trait.We found mutations in nine isolated patients and two autosomal dominant families. Eight out of nine isolated patients were homozygotes or compound heterozygotes with autosomal recessive inheritance and one patient carried a de novo dominant mutation. Patients with homozygous or compound heterozygous genotypes showed a severe disease, whereas heterozygous patients from the two autosomal dominant families that segregated the p.R120W mutation showed a milder phenotype. We also report a de novo mutation, p.T157P, which has not been described. Haplotype analysis of the CMT4A locus confirmed a unique origin for the p.Q163X mutation 17 Spanish chromosomes and two Moroccan chromosomes carrying the p.S194X was also confirmed.The molecular and genetic analysis of the CMT disease in the 17 Gypsy families was raised at first by means of the test of those known loci related with CMT disease in European Gypsy population: HMSN-Lom (MIM 601455) and HMSN-Russe (MIM 605285). Second, the description of new loci. We found the p.R148X mutation in the NDRG1 gene to be responsible for the HMSN-Lom neuropathy in one family and also possible linkage to the HMSN-Russe locus in three others. We have also studied the CMT4C locus because of the clinical similarities and showed that in 10 families, the disease is caused by mutations located on the SH3TC2 gene: p.R1109X in 20 of 21 chromosomes and p.C737_P738 in only one chromosome. Moreover, the SH3TC2 p.R1109X is associated with a conserved haplotype and, therefore, may be private founder mutation for the Gypsy population.

Sections 2 and 3 of this thesis describe respectively the construction and testing of a 50 channel automatic Peltier psychrometer which records on a printed paper roll. It was shown that with the Peltier type of psychrometer errors caused either by a sample geometry other than that used during the calibration, or by the diffusive resistance of the sample can be eliminated by reducing the Peltier cooling time, and the reading time below a critical value. There was reasonable agreement between the theoretically and the practically determined estimates of the critical cooling time. When short cooling times are used however contamination of the thermocouple junction by osmotica is liable to cause a more serious error than when longer times are used. The size of these errors was not sufficient to account for the finding of other workers that leaf water potential measurements are almost constant over large range of transpiration rates. Equipment for the simultaneous measurement of water uptake, transpiration and plant turgidity of plants growing in nutrient solution was constructed (section 4) and used (section 3) to examine the movement of water through bean plants exhibiting cyclic variations in transpiration. The hypothesis that such variations in transpiration were caused by a delay in the response of the stomata to a change in the leaf water potential was examined with the aid of a simple mathematical model. The behaviour of the model was similar to that of the plants. The implications of the differences between the two are discussed.

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The plant hormone gibberellin (GA) promotes several processes during Arabidopsis reproductive development, including the transition to flowering, floral organ growth and fertility. GA functions during stamen development to promote degradation of the tapetum cell layer through programmed cell death (PCD) and in post-anthesis pollen development. Bioactive GA is synthesised through a multi-step pathway, in which the last two biosynthetic steps are expressed as conserved multigene families. One of these, the GA 20-oxidases (GA20ox) consists of five paralogues in Arabidopsis, though physiological functions have only been ascribed to two (AtGA20ox1 and -2). Through a reverse genetics approach, this project demonstrates that AtGA20ox1, -2 and -3 account for almost all GA20ox activity in Arabidopsis, with very little evidence of any functions for AtGA20ox4 or -5. Unlike AtGA20ox1, -2, -3 and -4, AtGA20ox5 possesses only partial GA20ox activity, performing the first two out of three sequential catalytic conversions in vitro. Partial functional redundancy occurs between AtGA20ox1, -2 and -3 across Arabidopsis development, although AtGA20ox1 and -2 dominate. Mapping of floral AtGA20ox expression through qPCR and the creation of transgenic GUS reporter lines found that the relationship between these three paralogues is complex, and not explicable through the simple hypothesis of co-expression in the same tissues. During anther development, the reported expression of AtGA20ox1, -2, -3 and -4 is mainly restricted to the tapetum cell layer, and loss of AtGA20ox1, -2 and -3 results in an anther developmental arrest in which the tapetum does not degrade. This project demonstrates that stamen development is dependent on an optimum level of GA, with GA-deficiency restricting filament elongation to prevent pollination and GA-overdose negatively affecting anther development. DELLA repression of GA signalling is necessary for successful pollen development, with two of the five DELLA paralogues, RGA and GAI, critical to this process in the Columbia ecotype.

The ability of plants to respond to environmental cues is crucial for the success of the organism. Light is an important source of energy for the plant as well as providing information for plant growth and development. Plant responses to the electromagnetic spectrum are controlled by a number of different photoreceptors. Phytochromes respond to red and far-red light and are responsible for controlling photomorphogenesis in seedlings (Chen and Chory, 2011). The recently identified UVR8 responds to UV-B light and prevents damage to the plant from these harmful wavelengths (Rizzini et al., 2011). Responses to UVA/blue light are controlled by three different photoreceptors: the cryptochromes that are involved in the circadian clock as well as regulating a number of aspects of photomorphogenesis (Chaves et al., 2011); the Zeitlupe family which control the circadian clock and flowering responses (Demarsy and Fankhauser, 2009) and finally the phototropins which regulate the light-dependent processes that increase photosynthetic efficiency of plants (Christie, 2007, Christie and Murphy, 2013). Within Arabidopsis there are two phototropins, phototropin 1 (phot1) and phototropin 2 (phot2), which share approximately 60% sequence identity at the amino acid level (Christie et al., 2002). The phototropins have also been identified in algae, ferns and mosses as well as higher plants where their mode of action appears to be conserved (Onodera et al., 2005). The phototropins function redundantly to control phototropism, chloroplast movement, leaf flattening and positioning, stomatal opening whilst phot1 alone controls the rapid inhibition of hypocotyl growth upon transfer of dark-grown seedlings to light (Christie, 2007). The phototropins are serine/threonine (Ser/Thr) kinases consisting of two Light, Oxygen and Voltage (LOV) domains in the N terminus and a Ser/Thr kinase at the C terminus (Figure 1). In darkness, the protein associates with the plasma membrane and upon illumination partially internalises to cytosolic strands. Illumination with blue light also results in the the LOV domains forming a covalent linkage with the chromophore, Flavin Mononucleotide (FMN) and the protein undergoes autophosphorylation at a number of serine residues including upstream of the LOV1 domain and between the LOV1 and LOV2 linker region, as well as within the kinase activation loop (Inoue et al., 2008; Sullivan et al., 2008). Whilst the physiological functions of the phototropins from Arabidopsis are well characterised, the function of the membrane association and subsequent internalisation is unknown. Therefore the aims of this project were to identify the mechanism of phot1 association with the membrane and to determine if there are specific regions of membrane interaction within the kinase domain of phot1, since the C-terminus of phot2 including the kinase domain is known to direct localisation to the plasma membrane (Kong et al., 2007). It was therefore of interest to analyse the kinase domain of various phototropins to assess if there are sequences that may direct protein localisation to the plasma membrane. Chapter 3 describes the Lysine Rich Motif (LRM) that was identified and subsequently mutated to assess localisation when transformed in to the phot1-5 phot2-1 double mutant as well as subsequent complementation analysis of the physiological responses controlled by phot1. The analysis suggests that phot1 may interact with the membrane through a lipid interaction that is not mediated by the LRM. The consequences of these results are discussed further in the chapter. Further analysis of the role played by the kinase domain in membrane localisation of phot1 is described in the truncation analysis in Chapter 4. A similar approach to the deletion analysis employed by Kong et al. (2013) was used. A predicted secondary structure was generated to ensure that truncations were performed outside secondary structures that may be important for the protein structure. The insect cell system as well as transient expression in N. benthamaiana provided convenient methods to analyse various truncations of the kinase domain of phot1. The method was also used to analyse the membrane association of phot2. The effect of 1-butanol treatment on Arabidopsis seedlings was also investigated in relation to phot1 associating with the plasma membrane by interaction with lipids such as phosphatdic acid (PA). The work presented in this chapter suggests that there may be more than one region of phot1 that interacts with the membrane and the complications of this are discussed further. In order to investigate the function of internalised phot1 after irradiation, the protein was constitutively targeted to the membrane using a farnesyl tag. A farnesylation sequence targets proteins to the membrane via a lipid modification (Sorek, Bloch and Yalovsky, 2009). The farnesyl-tagged phot1-GFP was transformed into the phot1-5 phot2-1 double mutant and the physiological responses controlled by the phototropin were assessed. The results of targeting phot1-GFP to the membrane are shown in Chapter 6. Constitutive targeting of phot1-GFP to the plasma membrane showed that the soluble protein visualised after blue light illumination is, perhaps surprisingly, dispensable for the physiological responses tested. There may however be a role to play in the fine-tuning of the Arabidopsis response to illumination. Together these studies provide new perceptions in the possible mechanism of membrane attachment of phot1. Finally, investigation of the phototropin from the algae Ostreococcus tauri reveal that, while it can mediate some phot-regulated responses when expressed in the phot1-5 phot2-1 double mutant the protein is not functional in phototropism providing a unique phototropin that could be used to investigate the downstream control of this response. These findings highlight key differences between the mode of action of plant and algal phototropins that have so far gone unrecognised.

A la hora de evaluar el riesgo potencial de compuestos con capacidad tóxica sobre las poblaciones expuestas, resulta de gran importancia considerar la exposición de varias generaciones de organismos al efecto de dichos compuestos. Por ello, en este estudio, se propone la incorporación de un esquema multigeneracional para evaluar la toxicidad de concentraciones subletales del insecticida diazinón y el herbicida molinato en el crustáceo D. magna.Este cladócero, perteneciente al zooplancton filtrador de las aguas dulces asociadas a la región templada del globo, presenta una gran sensibilidad a una amplia gama de compuestos químicos, lo cual, junto con el hecho de disponer de él con facilidad y rapidez gracias a su reproducción partenogenética, ha favorecido su consideración como organismo diana en los ensayos de toxicidad.En el presente trabajo se ha ensayado el efecto de los dos plaguicidas seleccionados sobre la supervivencia, capacidad reproductiva y tamaño corporal de los dáfnidos expuestos. Para ello se realizaron ensayos crónicos de 21 días de duración, exponiéndose varias generaciones de organismos a los plaguicidas seleccionados: una generación parental (F0) y una filial, a través de la primera y tercera camadas de descendientes (F1-1ª y 3ª). Los ensayos crónicos comprendieron una primera fase de exposición a los compuestos ensayados y una segunda fase de recuperación de los descendientes al efecto de los mismos.Además se evaluó el efecto sobre el peso de F0, y sobre el contenido proteico y la actividad acetilcolinesterasa (AChE) de D. magna a nivel multigeneracional en ensayos de 120 horas de duración, registrándose las variaciones en estos parámetros a las 0, 24, 48, 72, 96 y 120 horas de iniciarse el proceso de exposición a los plaguicidas.Los resultados de los ensayos crónicos de toxicidad mostraron una mayor sensibilidad de los descendientes de D. magna expuestos a diazinón y molinato en relación a la de los parentales, siendo mayor el efecto producido por el insecticida. Según estos datos se pensó inicialmente en una posible transferencia de los tóxicos ensayados de la generación F0 a las sucesivas de descendientes. Sin embargo, tras un periodo de recuperación en medio exento de plaguicidas, los descendientes de D. magna registraron valores similares a los valores control para casi todos los parámetros estudiados, observándose una mayor recuperación de la tercera camada.Ambos plaguicidas provocaron un descenso del contenido proteico de D. magna, siendo más acusado el efecto tras la exposición al herbicida, probablemente debido al efecto que éste produjo en la calidad del alga utilizada como alimento de los dáfnidos.Se registró una reducción del peso de los parentales de D. magna tras exposición a diazinón y molinato lo cual se relacionó positivamente con el efecto observado a nivel de la capacidad reproductiva.Ambos plaguicidas inhibieron la actividad AChE de D. magna en todas las generaciones estudiadas, siendo el efecto más acusado (en ambos casos) en la generación parental que en los descendientes, y mayor la inhibición tras exposición a diazinón puesto que es un compuesto diseñado con carácter insecticida.En los descendientes de D. magna expuestos a ambos plaguicidas, al aumentar la concentración del contaminante disminuyó en mayor medida el contenido proteico que la actividad AChE, lo que resultó en algunos casos, en un incremento de la actividad AChE expresada por g de proteína. Tras observar la interferencia del contenido proteico en la expresión de la actividad AChE en D. magna, se recomienda la expresión de esta actividad enzimática por individuo, considerando, al menos, un estudio paralelo del efecto de un compuesto sobre ambos parámetros tratándose de determinaciones efectuadas sobre la totalidad de los organismos expuestos, a diferencia de las evaluaciones efectuadas en vertebrados. / In the present study, a multigenerational chronic toxicity study is proposed in the cladoceran D. magna. The effect of sublethal concentrations of the insecticida diazinon and the herbicide molinate were evaluated in the daphnids.Parental generation (F0) and neonates from the first and third brood (F1-1st and F1-3rd, respectively) were exposed during 21 days to both pesticides, and offspring was also transfered to toxicant free medium (21 days assays) to evaluate its ability to recover from diazinon and molinate effects.On the other hand, the effect of these pesticides were evaluated during 5 days on the acetylcholinesterase (AChE) activity and protein content of all daphnid generations used in this study. Paralelly, weight changes were also evaluated in the parental daphnids (F0).The chronic multigenerational study showed that daphnids from the F1-1st and F1-3rd broods seem not to be adapted to the pesticides and they exhibited more mortality and less reproduction than F0. However, after the recovery period, offspring of D. magna pre-exposed to both pesticides was able to recover the values of the studied parameters and the third brood was the most recovered group. Diazinon effects were higher than those of the herbicide molinate in this 21 day study.Daphnids protein content decreased after exposure to both pesticides, and the effect of molinate was higher. Because molinate is an herbicide the nutritive quality of the algal food of D. magna will be affected.The decreased found in parentals weight after exposure to both pesticides was related to the effect on their reproductive ability.Finally, AChE activity was more inhibited on offspring daphnids exposed to both pesticides compared to the parentals. However, diazinon exposure showed a higher effect in this parameter than molinate because it produces a non reversible inhibition on this enzimatic activity. It was observed an interference of the protein content on the AChE activity expression, so we suggest the calculation of the AChE activity by organism nor by protein content. This suggestion could be important in those studies with small invertebrates where the AChE activity is determinated in the whole organism.