Uncovering and Functional Analysis of Novel Genes and Potential Genetic Modifiers for Neuromuscular Disorders

The inherited Neuromuscular Disorders (NMDs) is an umbrella term that encompasses a plethora of diseases that affect the functioning of the muscles and/or their underlying nervous system control. Although individually uncommon, NMDs collectively are no longer considered rare diseases. Only in Europe, approximately 300,000 people are yearly diagnosed with one NMD. The diagnosis of a neuromuscular condition –successful in less than 50% of the cases- is often devastating to patients and their relatives. No cure is available for most of these disorders, and the few available treatments will at best delay disease progression. To find treatments and to develop methods for the early diagnosis of NMDs is a goal of highest importance, and the recent advances in the NGS technologies are the platform to reach this objective. Particularly, the implementation of WES has not only minimized the time and costs of genetic diagnostics but also provided unique opportunities for the exploration of gene function in NMDs pathogenesis. The uppermost goal of this PhD project was to identify and characterize novel NMDs-causative genes. Thereby, two novel NMD-causative genes were investigated in this thesis. First, CHP1 (Calcineurin Homologous Protein-1) was identified as a novel causing gene of Autosomal Recessive Cerebellar Ataxia (ARCA) and second, VAChT (Vesicular Acetylcholine Transporter, encoded by SLC18A3) was analysed in the context of distal Hereditary Motor Neuropathy (dHMN). Following a combination of WES and linkage analysis, we identified a biallelic 3-bp deletion (p.K19del) in CHP1 that co-segregates with a complex ARCA in two siblings of a consanguineous family exhibiting motor neuropathy, cerebellar atrophy and spastic paraparesis. CHP1 was selected as a top disease candidate since: (I) the mutation affects an amino acid highly conserved across species, (II) a point mutation in murine Chp1, causing aberrant splicing and reduced full-length Chp1 transcripts, leads to Purkinje cells loss and ataxia, (III) CHP1 assists posttranscriptional glycosylation and membrane localization of NHE1, a major neuronal Na+/H+ exchanger, (IV) KO of mouse Nhe1 cause ataxia and loss-of-function mutation in NHE1 (encoded by SLC19A1) cause ataxia-deafness Lichtenstein-Knorr syndrome (LINKS). Therefore, we hypothesized that a mutation in CHP1, as a crucial regulator of NHE1, could impair expression and targeting of the exchanger resembling the pathogenesis in mice and humans. To further uncover other families carrying CHP1 mutations, we performed a focused screening for CHP1 variants in two large ARCA and NMD cohorts (approximately 1000 exomes). No additional variants fulfilling or selection criteria were found, which emphasizes on the scarcity of CHP1 variants and the reduced tolerability of CHP1 for mutations. With the purpose to assess the functional consequences of the CHP1-K19del mutation on protein function, size exclusion chromatography (SEC), protein fractionation, 3D-protein modelling, fluorescence microscopy and in vivo zebrafish modelling were performed. We demonstrated that mutant CHP1 fails to integrate into functional protein complexes and is prone to aggregate, thereby leading to diminished levels of soluble CHP1 and reduced membrane targeting of NHE1 both in neuronal and non-neuronal cells. To analyze the pathogenic consequences of the hypomorphic CHP1-K19del mutation in vivo, we used morpholinos (MOs) to inhibit chp1 translation in zebrafish. Closely resembling the clinical features of the ARCA-affected siblings, chp1 downregulation in zebrafish led to cerebellar hypoplasia, Caudal Primary Motor Neuron (CaP-MN) defects and spastic trunk movements. All defects were ameliorated by co-injection with WT, but not mutant, human CHP1 mRNA, hence demonstrating both the specificity of the chp1-MO-induced phenotypes and validating the effect of CHP1-K19del on protein expression and/or function in vivo. Altogether, our results identified CHP1 as a novel ataxia-causative gene in humans, further expanding the spectrum of ARCA-causative loci, and highlight the crucial role of NHE1 within the pathogenesis of these disorders. Moreover, we conducted functional analyses to ascertain the functional basis of a dHMN presented by a family with cranial nerves palsy and vocal cord paresis as an initial feature of a non-progressive infantile onset dominant dHMN. WES analysis of this family led to the identification of a de novo dominant missense mutation (c.439 G>A, p.D147N) in VAChT. The mutation occurred first in the affected mother and was inherited by her affected daughter. VAChT controls the storage of the neurotransmitter Acetylcholine (ACh) by synaptic vesicles, hence it plays a fundamental role in cholinergic neurotransmission and therefore, in the plethora of processes reliant on it, which include: neuronal development and maturation, synaptic transmission and plasticity, patterning of the neuromuscular junction (NMJ), among others. The potential effect of the D147N mutation on VAChT subcellular distribution was analysed in neuron-like NSC-34 cells transiently overexpressing WT or mutant VAChT-GFP tagged proteins. No significant differences were observed in protein expression or localization, thus a detrimental effect of VAChT-D147N mutation at this level was not possible. This prompted us to further examine potential defects either in MN development and axonal outgrowth. Capitalizing once again on the advantages of the zebrafish for the modelling of human neurodegenerative disorders and further considering the evolutionary conservation of both VAChT and the D147 residue across species, the effect of WT and VAChT-D147N OE on CaP-MN outgrowth was analysed in detail. Although our findings were not conclusive at discerning the pathogenicity of the VAChT–D147N in vivo, we observed an axonal migration phenotype that could potentially underlie impairments at the NMJ level. In the light of the novel association of VAChT mutations as causative of myasthenia syndromes, follow-up studies will be performed in order to conclusively confirm the pathogenicity of the VAChT-D147N in a CaP-MN-independent context. The biological function of CHP1 was of further relevance within the scope of this doctoral project, since CHP1 is currently subject of study as potential modifier of Spinal Muscular Atrophy (SMA). Pre-existing evidence from our research group indicates that Chp1 downregulation –within a certain threshold- restores neurite outgrowth and impaired endocytosis (a key pathway disturbed in SMA) in Smn1-depleted NSC-34 cells. Thereupon, this thesis further aimed to validate reduction of Chp1 as potential SMA protective modifier in a zebrafish model of SMA, as a first in vivo approach. Here, we demonstrate that Chp1 downregulation ameliorates the CaP-MN axonal outgrowth defects of Smn-deficient fish larvae. These findings are in concordance with prior validation studies of two other human SMA modifiers –PLS3 and NCALD- in zebrafish, which despite their different function and mode of action (upregulation or downregulation, respectively) exert similar effects on CaP-MN morphology whereby restoring the CaP-MN phenotype of smn morphants in a highly comparable range. Altogether, our findings together with the preliminary findings aforementioned, strongly support CHP1 reduction as a promising therapeutic target for a combinatorial treatment, i.e. together with SMN restoration, counteracting SMA pathology

Trabajo emocional en los trabajadores de servicio de primera línea de hoteles y restaurantes de lujo en la ciudad de Barranquilla

80 páginas ; ilustraciones con anexos.Esta investigación tiene como fin determinar las emociones que muestran, utilizan y se presentan dentro de las relaciones con clientes y superiores en el ámbito laboral logrando un equilibrio entre estas y utilizándolas como creación en cuanto al diagnóstico, diseño y evolución de acuerdo al ecosistema en el que se encuentran entendiendo de manera más profunda el comportamiento de los trabajadores y sus causas. Dentro de los aspectos relevantes de la investigación se evidencia la expresión emocional orientada al alto nivel de tareas que requieren expresión ajustada de emociones que, aunque son rutinarias, se establecen con presión emocional constante en función de los tiempos y protocolos definidos en cada rol del servicio. La atención requerida se identifica en la necesidad de contar con estrategias emocionales para tomar decisiones orientadas a la solución de necesidades de clientes/ jefes. Los empleadores manejan muchas reglas, parámetros, políticas, formas de actuación que en innumerables ocasiones no corresponden a la naturaleza del comportamiento de sus empleados, lo que implica que estos deban incurrir en un esfuerzo planificado y controlado cambiando sus emociones naturales por las "correctas" desde el punto de vista de la organización con el fin de adaptarse al contexto de las respectivas situaciones.The purpose of this research is to determine the emotions that show, use and present within relationships with clients and managers in the workplace, achieving a balance between them and using them as a creation in terms of diagnosis, design and evolution according to the ecosystem in order to understand more thoroughly the behavior of workers and their causes. Within the relevant aspects of the research, emotional expression is shown, oriented to the high level of tasks that require adjusted expression of emotions that, although they are routine, are established with constant emotional pressure according to the times and protocols defined in each role of the service .. The attention required is identified in the need to have emotional strategies to make decisions aimed at solving the needs of clients / managers. Employers handle many rules, parameters, policies, forms of action that countless times do not correspond to the nature of the behavior of their employees, which implies that they must incur a planned and controlled effort changing their natural emotions for the "right" from the point of view of the organization in order to adapt to the context of the respective situations.Magíster en MercadeoMaestrí

Neurocalcin Delta Knockout Impairs Adult Neurogenesis Whereas Half Reduction Is Not Pathological

Neurocalcin delta (NCALD) is a brain-enriched neuronal calcium sensor and its reduction acts protective against spinal muscular atrophy (SMA). However, the physiological function of NCALD and implications of NCALD reduction are still elusive. Here, we analyzed the ubiquitous Ncald knockout in homozygous (NcaldKO/KO) and heterozygous (NcaldKO/WT) mice to unravel the physiological role of NCALD in the brain and to study whether 50% NCALD reduction is a safe option for SMA therapy. We found that NcaldKO/KO but not NcaldKO/WT mice exhibit significant changes in the hippocampal morphology, likely due to impaired generation and migration of newborn neurons in the dentate gyrus (DG). To understand the mechanism behind, we studied the NCALD interactome and identified mitogen-activated protein kinase kinase kinase 10 (MAP3K10) as a novel NCALD interacting partner. MAP3K10 is an upstream activating kinase of c-Jun N-terminal kinase (JNK), which regulates adult neurogenesis. Strikingly, the JNK activation was significantly upregulated in the NcaldKO/KO brains. Contrary, neither adult neurogenesis nor JNK activation were altered by heterozygous Ncald deletion. Taken together, our study identifies a novel link between NCALD and adult neurogenesis in the hippocampus, possibly via a MAP3K10-JNK pathway and emphasizes the safety of using NCALD reduction as a therapeutic option for SMA

Estimating the global conservation status of more than 15,000 Amazonian tree species

Estimates of extinction risk for Amazonian plant and animal species are rare and not often incorporated into land-use policy and conservation planning. We overlay spatial distribution models with historical and projected deforestation to show that at least 36% and up to 57% of all Amazonian tree species are likely to qualify as globally threatened under International Union for Conservation of Nature (IUCN) Red List criteria. If confirmed, these results would increase the number of threatened plant species on Earth by 22%. We show that the trends observed in Amazonia apply to trees throughout the tropics, and we predict thatmost of the world’s >40,000 tropical tree species now qualify as globally threatened. A gap analysis suggests that existing Amazonian protected areas and indigenous territories will protect viable populations of most threatened species if these areas suffer no further degradation, highlighting the key roles that protected areas, indigenous peoples, and improved governance can play in preventing large-scale extinctions in the tropics in this century

Geographic patterns of tree dispersal modes in Amazonia and their ecological correlates

Unidad de excelencia María de Maeztu CEX2019-000940-MAim: To investigate the geographic patterns and ecological correlates in the geographic distribution of the most common tree dispersal modes in Amazonia (endozoochory, synzoochory, anemochory and hydrochory). We examined if the proportional abundance of these dispersal modes could be explained by the availability of dispersal agents (disperser-availability hypothesis) and/or the availability of resources for constructing zoochorous fruits (resource-availability hypothesis). Time period: Tree-inventory plots established between 1934 and 2019. Major taxa studied: Trees with a diameter at breast height (DBH) ≥ 9.55 cm. Location: Amazonia, here defined as the lowland rain forests of the Amazon River basin and the Guiana Shield. Methods: We assigned dispersal modes to a total of 5433 species and morphospecies within 1877 tree-inventory plots across terra-firme, seasonally flooded, and permanently flooded forests. We investigated geographic patterns in the proportional abundance of dispersal modes. We performed an abundance-weighted mean pairwise distance (MPD) test and fit generalized linear models (GLMs) to explain the geographic distribution of dispersal modes. Results: Anemochory was significantly, positively associated with mean annual wind speed, and hydrochory was significantly higher in flooded forests. Dispersal modes did not consistently show significant associations with the availability of resources for constructing zoochorous fruits. A lower dissimilarity in dispersal modes, resulting from a higher dominance of endozoochory, occurred in terra-firme forests (excluding podzols) compared to flooded forests. Main conclusions: The disperser-availability hypothesis was well supported for abiotic dispersal modes (anemochory and hydrochory). The availability of resources for constructing zoochorous fruits seems an unlikely explanation for the distribution of dispersal modes in Amazonia. The association between frugivores and the proportional abundance of zoochory requires further research, as tree recruitment not only depends on dispersal vectors but also on conditions that favour or limit seedling recruitment across forest types

Local hydrological conditions influence tree diversity and composition across the Amazon basin

Tree diversity and composition in Amazonia are known to be strongly determined by the water supplied by precipitation. Nevertheless, within the same climatic regime, water availability is modulated by local topography and soil characteristics (hereafter referred to as local hydrological conditions), varying from saturated and poorly drained to well-drained and potentially dry areas. While these conditions may be expected to influence species distribution, the impacts of local hydrological conditions on tree diversity and composition remain poorly understood at the whole Amazon basin scale. Using a dataset of 443 1-ha non-flooded forest plots distributed across the basin, we investigate how local hydrological conditions influence 1) tree alpha diversity, 2) the community-weighted wood density mean (CWM-wd) – a proxy for hydraulic resistance and 3) tree species composition. We find that the effect of local hydrological conditions on tree diversity depends on climate, being more evident in wetter forests, where diversity increases towards locations with well-drained soils. CWM-wd increased towards better drained soils in Southern and Western Amazonia. Tree species composition changed along local soil hydrological gradients in Central-Eastern, Western and Southern Amazonia, and those changes were correlated with changes in the mean wood density of plots. Our results suggest that local hydrological gradients filter species, influencing the diversity and composition of Amazonian forests. Overall, this study shows that the effect of local hydrological conditions is pervasive, extending over wide Amazonian regions, and reinforces the importance of accounting for local topography and hydrology to better understand the likely response and resilience of forests to increased frequency of extreme climate events and rising temperatures

Geography and ecology shape the phylogenetic composition of Amazonian tree communities

Aim: Amazonia hosts more tree species from numerous evolutionary lineages, both young and ancient, than any other biogeographic region. Previous studies have shown that tree lineages colonized multiple edaphic environments and dispersed widely across Amazonia, leading to a hypothesis, which we test, that lineages should not be strongly associated with either geographic regions or edaphic forest types. Location: Amazonia. Taxon: Angiosperms (Magnoliids; Monocots; Eudicots). Methods: Data for the abundance of 5082 tree species in 1989 plots were combined with a mega-phylogeny. We applied evolutionary ordination to assess how phylogenetic composition varies across Amazonia. We used variation partitioning and Moran\u27s eigenvector maps (MEM) to test and quantify the separate and joint contributions of spatial and environmental variables to explain the phylogenetic composition of plots. We tested the indicator value of lineages for geographic regions and edaphic forest types and mapped associations onto the phylogeny. Results: In the terra firme and várzea forest types, the phylogenetic composition varies by geographic region, but the igapó and white-sand forest types retain a unique evolutionary signature regardless of region. Overall, we find that soil chemistry, climate and topography explain 24% of the variation in phylogenetic composition, with 79% of that variation being spatially structured (R$^{2}$ = 19% overall for combined spatial/environmental effects). The phylogenetic composition also shows substantial spatial patterns not related to the environmental variables we quantified (R$^{2}$ = 28%). A greater number of lineages were significant indicators of geographic regions than forest types. Main Conclusion: Numerous tree lineages, including some ancient ones (>66 Ma), show strong associations with geographic regions and edaphic forest types of Amazonia. This shows that specialization in specific edaphic environments has played a long-standing role in the evolutionary assembly of Amazonian forests. Furthermore, many lineages, even those that have dispersed across Amazonia, dominate within a specific region, likely because of phylogenetically conserved niches for environmental conditions that are prevalent within regions

Mapping density, diversity and species-richness of the Amazon tree flora

Using 2.046 botanically-inventoried tree plots across the largest tropical forest on Earth, we mapped tree species-diversity and tree species-richness at 0.1-degree resolution, and investigated drivers for diversity and richness. Using only location, stratified by forest type, as predictor, our spatial model, to the best of our knowledge, provides the most accurate map of tree diversity in Amazonia to date, explaining approximately 70% of the tree diversity and species-richness. Large soil-forest combinations determine a significant percentage of the variation in tree species-richness and tree alpha-diversity in Amazonian forest-plots. We suggest that the size and fragmentation of these systems drive their large-scale diversity patterns and hence local diversity. A model not using location but cumulative water deficit, tree density, and temperature seasonality explains 47% of the tree species-richness in the terra-firme forest in Amazonia. Over large areas across Amazonia, residuals of this relationship are small and poorly spatially structured, suggesting that much of the residual variation may be local. The Guyana Shield area has consistently negative residuals, showing that this area has lower tree species-richness than expected by our models. We provide extensive plot meta-data, including tree density, tree alpha-diversity and tree species-richness results and gridded maps at 0.1-degree resolution

The global biogeography of tree leaf form and habit

Understanding what controls global leaf type variation in trees is crucial for comprehending their role in terrestrial ecosystems, including carbon, water and nutrient dynamics. Yet our understanding of the factors influencing forest leaf types remains incomplete, leaving us uncertain about the global proportions of needle-leaved, broadleaved, evergreen and deciduous trees. To address these gaps, we conducted a global, ground-sourced assessment of forest leaf-type variation by integrating forest inventory data with comprehensive leaf form (broadleaf vs needle-leaf) and habit (evergreen vs deciduous) records. We found that global variation in leaf habit is primarily driven by isothermality and soil characteristics, while leaf form is predominantly driven by temperature. Given these relationships, we estimate that 38% of global tree individuals are needle-leaved evergreen, 29% are broadleaved evergreen, 27% are broadleaved deciduous and 5% are needle-leaved deciduous. The aboveground biomass distribution among these tree types is approximately 21% (126.4 Gt), 54% (335.7 Gt), 22% (136.2 Gt) and 3% (18.7 Gt), respectively. We further project that, depending on future emissions pathways, 17-34% of forested areas will experience climate conditions by the end of the century that currently support a different forest type, highlighting the intensification of climatic stress on existing forests. By quantifying the distribution of tree leaf types and their corresponding biomass, and identifying regions where climate change will exert greatest pressure on current leaf types, our results can help improve predictions of future terrestrial ecosystem functioning and carbon cycling