An investigation of the expression profile of mRNA encoding Calpain 3, the causative agent in the inherited human muscular disease Limb Girdle Muscular Dystrophy Type 2A, was conducted in two representative mammalian species, human and mouse. Transcripts encoding Calpain 3 were identified from mammalian tissues other than skeletal muscle. In human Peripheral Blood Mononuclear Cells (PBMCs) these transcripts were identified in both the T-cell and B-cell compartments and in a number of human blood cell lines representing different haematopoietic lineages. Calpain 3 transcripts encoding the murine homologue were also described from mouse PBMCs and from murine tissues involved in haematopoiesis. In addition to the confirmation of Calpain 3 expression in non-skeletal muscle tissues in both these species, transcripts were identified with precise and defined deletions, which mapped to known exon-exon boundaries in the Calpain 3 gene from both species. These deletions constituted the removal by alternative splicing of skeletal muscle-specific components of the Calpain 3 protein known to regulate its function in this tissue. Monoclonal antibodies to the Calpain 3 protein were used to confirm the presence of Calpain 3 protein in non-skeletal muscle tissues of both human and mouse. In humans the expression of Calpain 3 protein was confirmed in PBMCs and in the mouse, Calpain 3 expression was confirmed in tissues of the haematopoietic compartment. In both species the Calpain 3 protein expressed correlated with translation from a transcript lacking the skeletal muscle-specific components generated by alternative splicing. An attempt was made using a Yeast Two Hybrid assay to identify potential regulatory molecules of Calpain 3 in human PBMCs, but without a definitive candidate molecule being found. A developmental model of muscle differentiation (murine C2C12 myoblast cells) was used to ascertain the expression profile of Calpain 3 in the early stages of myofibrillogenesis. Using Quantitative Real Time PCR the expression profile of Calpain 3 was assessed in differentiating C2C12 cells. These results showed that the absolute levels of Calpain 3 transcription were elevated during differentiation and that a temporal Calpain 3 isoform shift occurred during this process. This temporal shift in expression was from transcripts having identical deletions to those seen in the haematopoietic tissues, to full length transcripts representative of skeletal muscle-specific Calpain 3. The identification of Calpain 3 expression outside skeletal muscle tissue is novel and the isoforms expressed in these tissues are structurally more analogous to the ubiquitously expressed calpains. This has implications for LGMD2A where a loss of function of Calpain 3 in non-skeletal muscle tissue could be compensated for by the ubiquitous calpains, thus explaining the lack of any non-muscle tissue pathology in LGMD2A patients.

An investigation of the expression profile of mRNA encoding Calpain 3, the causative agent in the inherited human muscular disease Limb Girdle Muscular Dystrophy Type 2A, was conducted in two representative mammalian species, human and mouse. Transcripts encoding Calpain 3 were identified from mammalian tissues other than skeletal muscle. In human Peripheral Blood Mononuclear Cells (PBMCs) these transcripts were identified in both the T-cell and B-cell compartments and in a number of human blood cell lines representing different haematopoietic lineages. Calpain 3 transcripts encoding the murine homologue were also described from mouse PBMCs and from murine tissues involved in haematopoiesis. In addition to the confirmation of Calpain 3 expression in non-skeletal muscle tissues in both these species, transcripts were identified with precise and defined deletions, which mapped to known exon-exon boundaries in the Calpain 3 gene from both species. These deletions constituted the removal by alternative splicing of skeletal muscle-specific components of the Calpain 3 protein known to regulate its function in this tissue. Monoclonal antibodies to the Calpain 3 protein were used to confirm the presence of Calpain 3 protein in non-skeletal muscle tissues of both human and mouse. In humans the expression of Calpain 3 protein was confirmed in PBMCs and in the mouse, Calpain 3 expression was confirmed in tissues of the haematopoietic compartment. In both species the Calpain 3 protein expressed correlated with translation from a transcript lacking the skeletal muscle-specific components generated by alternative splicing. An attempt was made using a Yeast Two Hybrid assay to identify potential regulatory molecules of Calpain 3 in human PBMCs, but without a definitive candidate molecule being found. A developmental model of muscle differentiation (murine C2C12 myoblast cells) was used to ascertain the expression profile of Calpain 3 in the early stages of myofibrillogenesis. Using Quantitative Real Time PCR the expression profile of Calpain 3 was assessed in differentiating C2C12 cells. These results showed that the absolute levels of Calpain 3 transcription were elevated during differentiation and that a temporal Calpain 3 isoform shift occurred during this process. This temporal shift in expression was from transcripts having identical deletions to those seen in the haematopoietic tissues, to full length transcripts representative of skeletal muscle-specific Calpain 3. The identification of Calpain 3 expression outside skeletal muscle tissue is novel and the isoforms expressed in these tissues are structurally more analogous to the ubiquitously expressed calpains. This has implications for LGMD2A where a loss of function of Calpain 3 in non-skeletal muscle tissue could be compensated for by the ubiquitous calpains, thus explaining the lack of any non-muscle tissue pathology in LGMD2A patients.

An investigation of the expression profile of mRNA encoding Calpain 3, the causative agent in the inherited human muscular disease Limb Girdle Muscular Dystrophy Type 2A, was conducted in two representative mammalian species, human and mouse. Transcripts encoding Calpain 3 were identified from mammalian tissues other than skeletal muscle. In human Peripheral Blood Mononuclear Cells (PBMCs) these transcripts were identified in both the T-cell and B-cell compartments and in a number of human blood cell lines representing different haematopoietic lineages. Calpain 3 transcripts encoding the murine homologue were also described from mouse PBMCs and from murine tissues involved in haematopoiesis. In addition to the confirmation of Calpain 3 expression in non-skeletal muscle tissues in both these species, transcripts were identified with precise and defined deletions, which mapped to known exon-exon boundaries in the Calpain 3 gene from both species. These deletions constituted the removal by alternative splicing of skeletal muscle-specific components of the Calpain 3 protein known to regulate its function in this tissue. Monoclonal antibodies to the Calpain 3 protein were used to confirm the presence of Calpain 3 protein in non-skeletal muscle tissues of both human and mouse. In humans the expression of Calpain 3 protein was confirmed in PBMCs and in the mouse, Calpain 3 expression was confirmed in tissues of the haematopoietic compartment. In both species the Calpain 3 protein expressed correlated with translation from a transcript lacking the skeletal muscle-specific components generated by alternative splicing. An attempt was made using a Yeast Two Hybrid assay to identify potential regulatory molecules of Calpain 3 in human PBMCs, but without a definitive candidate molecule being found. A developmental model of muscle differentiation (murine C2C12 myoblast cells) was used to ascertain the expression profile of Calpain 3 in the early stages of myofibrillogenesis. Using Quantitative Real Time PCR the expression profile of Calpain 3 was assessed in differentiating C2C12 cells. These results showed that the absolute levels of Calpain 3 transcription were elevated during differentiation and that a temporal Calpain 3 isoform shift occurred during this process. This temporal shift in expression was from transcripts having identical deletions to those seen in the haematopoietic tissues, to full length transcripts representative of skeletal muscle-specific Calpain 3. The identification of Calpain 3 expression outside skeletal muscle tissue is novel and the isoforms expressed in these tissues are structurally more analogous to the ubiquitously expressed calpains. This has implications for LGMD2A where a loss of function of Calpain 3 in non-skeletal muscle tissue could be compensated for by the ubiquitous calpains, thus explaining the lack of any non-muscle tissue pathology in LGMD2A patients.

Thesis (Ph. D.)--University of Alabama at Birmingham, 2008. / Title from first page of PDF file (viewed Feb. 13, 2009). Includes bibliographical references.

Thesis (Ph.D.). / Written for the Dept. of Experimental Medicine. Title from title page of PDF (viewed 2009/06/09). Includes bibliographical references.

Thesis (Ph. D.) -- University of Texas Southwestern Medical Center at Dallas, 2003. / Vita. Bibliography: 127-154.

The mammalian Y chromosome contains genes important for male sexual maturity and reproduction. The mouse Y chromosome long arm harbours a number of multi-copy genes whose absence or reduced representation has been linked to sperm defects and offspring sex ratio distortion in favour of females. Understanding the biological mechanisms of sex ratio distortion and related sperm aberrations could not only result in benefits for fertility research, but also in the development of methods for large scale animal breeding pre-implantation sex selection. The distortion has been linked to an intragenomic conflict between the X and Y chromosomes that impacts spermiogenesis. Since the proportion of X- and Y-bearing sperm does not differ in affected animals, and there is no selective destruction of male embryos post-fertilisation, a functional difference must exist between the X- and Y-bearing sperm. This thesis describes the investigation into the physiological and molecular mechanisms of a large Y-chromosome long arm deletion in the mouse model MF1XYRIIIqdel. The examination of physiological characteristics revealed a distinct sperm morphology within the deletion model. Detailed characterisation of sperm shape demonstrated that aberrations consistently occur within specific regions of the sperm head, linking the distorted morphology to particular maturation stages in the sperm cycle. Using sperm fluorescence in situ hybridisation, a novel and detailed comparison of X- and Y- bearing sperm has shown that a subtle distinction in shape also exists between the X- and Y- bearing sperm in the deletion model. Breeding data were examined and showed a skew towards female offspring and a slightly reduced litter size. Sperm enzyme activity assays did not reveal altered hyaluronidase activity in MF1XYRIIIqdel sperm. Physiological differences between X- and Y- bearing sperm must result from differential gene expression, complicated by the syncitial nature of sperm development. To explore this, a detailed molecular characterisation of the MF1XYRIIIqdel phenotype in developing haploid spermatids was performed. Cellular elutriation and fractionation techniques were employed to separate spermatids at different stages of maturation and isolate different subcellular compartments. Differences in the transcriptional profile between these populations were analysed by microarray and RNA sequencing analysis of total and micro RNA. This work yielded a collection of coding and non-coding transcripts which show distinctive expression and compartmentalisation differences between the deletion model and its wild-type counterpart across several sperm maturation stages. Combining these strategies has led to the identification of several gene products potentially implicated in observed physiological differences and the offspring sex ratio skew, providing candidate genes for further research.

Les savanes sont des écosystèmes complexes pilotées par plusieurs mécanismes ascendant (ex: les nutriments du sol ou pluviométrie) ou descendant (ex: feu ou herbivorie), mais l'importance relative de ces mécanismes reste largement débattue. En particulier, le rôle des herbivores brouteurs (browsers) reste mal compris en tant que source de perturbation, et donc de force de pression descendante influant sur la dynamique des savanes. Dans cette étude, deux approches ont été développées pour aborder le rôle des perturbations dans la dynamique des savanes. Dans un première partie, j'ai utilisé une approche comparative inter-site pour explorer les réponses des communautés de plantes, et des principaux traits de ces plantes associés aux feuilles, branches, architecture et défense, aux variations de quatre facteurs : les nutriments dans le sol, la pluviométrie, la pression d'herbivorie et l'intensité du feu. Seize sites de savane, en Afrique du Sud et au Zimbabwe, ont été sélectionnés sur des gradients de chacun de ces facteurs. Les espèces ligneuses dominantes (>80 % de la biomasse) sur chaque site ont été identifiées et échantillonnées, afin de mesurer les traits des feuilles et des branches associés à l'appétence, architecture, ainsi qu'aux défenses physiques et chimiques de ces plantes. Des mesures ont également été faites pour estimer les effets des meso-brouteurs et mega-brouteurs. Des transects ont permis d'estimer la fréquence et l'intensité du feu sur chaque site, et l'effet sur les plantes. En préambule à l'analyse, et devant le manque de protocole standard pour estimer la fertilité des sols dans la littérature écologique, je propose une méthode et un échantillonnage afin de définir de manière robuste la fertilité des sols sur chaque site. Dans cette partie inter-site, huit traits principaux ont été comparés sur le gradient de qualité de sol et de pluviométrie, et bien que quelques relations statistiques existent entre les traits des feuilles, le sol et la pluviométrie, ces relations sont très faibles comparées à celle trouvées dans les méta analyses inter-biomes publiées dans la littérature. Cependant, ces approches interbiome sont dominées par des sites tempérés qui ont des niveaux de perturbations bien inférieurs à ceux des savanes africaines. L'évaluation des effets des meso-brouteurs et mega-brouteurs le long des gradients de sol et de pluviométrie sur vingt traits associés aux défenses structurelles et chimiques des plantes montre que les défenses structurelles sont plus corrélées aux caractéristiques du sol que les défenses chimiques, mais que seules les défenses structurelles sont fortement corrélées à l'impact par les brouteurs. Le niveau d'utilisation des plantes par les mesobrouteurs apparaît plus prévisible en fonction des traits des plantes que celui par les mégabrouteurs. Dans une deuxième partie présentent des résultats de deux études basées sur des expériences d'exclos. Dans le parc national de Kruger, la composition de la communauté, l'abondance et la démographie des ligneux dominants ont été estimées à l'intérieur et à l'extérieur de trois exclos de 40 ans, et les brouteurs apparaissent comme ayant un impact significatif sur la distribution, la densité et la structure des populations des espèces arbustives et arborées ayant des traits préférés : forte concentration en azote foliaire et faible teneur en défenses chimiques. L'interaction entre les effets des brouteurs et du feu semble aussi affecter le recrutement des juvéniles ligneux dans les grandes classes de taille. Dans le parc de Hluhluwe-iMfolozi, cinq exclos ont été utilisé pour tester l'effet des brouteurs sur l'architecture, la croissance, les défenses chimiques et structurelles des jeunes individus de sept espèces d'acacia. Des différences nettes apparaissent entre les espèces d'acacia de savane semi-aride et plus humide dans les traits associés à l'appétence, l'architecture et les défenses … [etc] / Savannas are complex ecosystems affected by several bottom-up (e.g. soil nutrient availability and rainfall) and top-down (e.g. fire and herbivory) drivers. However, the relative importance of bottom-up vs. top-down drivers in influencing savanna dynamics is still widely debated. Within the top-down (disturbance) category of drivers, the role of mammal browsers in particular in driving savanna functioning is still not well understood. Two approaches were adopted to determine the role of disturbance in savannas. Firstly, by using a comparative approach, I attempted to address the so-called ‘savanna problem’ by investigating how savanna woody plant community compositions and key plant traits relating to the leaves, stems, architecture, and defence are influenced by soil nutrient status, rainfall, fire and browsing. Sixteen sites were selected along gradients of these four drivers from savanna parks throughout South Africa and Zimbabwe. The dominant woody species (species that accounted for >80% of standing biomass) at each site were identified and sampled for the key leaf and stem traits relating to plant functioning, palatability, architecture, physical and chemical defences. Measurements were undertaken for each species in order to determine both meso-browser and mega browser impact. Transects were undertaken in order to determine the relative abundance and the effects of fire on each species at each site. Due to the current lack of standardized soil sampling protocols in the ecological literature, and uncertainty around the definition of what denotes a fertile or infertile soil, I propose a number of standardized protocols and sampled according to these established protocols in order to accurately determine the soil nutrient status at each site. Following this, the relationships between climatic variables and soil nutrients with both species means and community weighted means for eight key leaf traits were explored. Although some significant relationships were found between savanna leaf traits of woody plants, climate, soil nutrients and their interactions, these tended to be weaker than those found in meta-analyses. These broad-scale studies usually include sites from many biome types, many of which are from temperate regions where inherent levels of disturbance are typically much lower than in African savannas. The high levels of disturbance typically found in African savannas are thought to partially account for the high within site variability found in leaf traits and the weak relationships found between leaf traits, soil nutrients and rainfall. To assess the importance of resources vs. disturbance in savannas functioning, the effects of soil nutrients, rainfall, fire and both meso-browser and mega-browser impact on twenty savanna woody plant traits relating to plant palatability, chemical and structural defences were explored. Structural defences were found to be more strongly correlated with soil characteristics than chemical defences, while browser impact was found to be strongly correlated with structural defences but not with chemical defences. Actual browser utilisation tended to be more predictable for meso-browsers than mega-browsers. Secondly using an experimental approach, two sets of herbivore exclosures were utilized to directly test how mammal browsers influenced woody species distributions, abundance, population structure and plant traits relating to palatability and defence. The effects of three longterm herbivore exclosures in the Kruger National Park on savanna woody plant community compositions, population demographics and densities were determined. Browsers were found to have significant impacts on species distributions, densities and population structures by actively selecting for species with favourable traits, particularly higher leaf N. An interaction between browsers and fire which limited the recruitment of seedlings and saplings into larger size classes was also demonstrated… [etc]

Submitted by (ana.araujo@ufrpe.br) on 2016-07-06T15:45:14Z No. of bitstreams: 1 Ilzes Celi Cruz Santana.pdf: 709492 bytes, checksum: f1f840db7e78a9caef2e77dfe96a9132 (MD5) / Made available in DSpace on 2016-07-06T15:45:14Z (GMT). No. of bitstreams: 1 Ilzes Celi Cruz Santana.pdf: 709492 bytes, checksum: f1f840db7e78a9caef2e77dfe96a9132 (MD5) Previous issue date: 2006-02-22 / The mammalian fauna of the Caatinga Biome was recently analysed throughout the definition of a National Conservation Strategy and to evaluated the environmental impact. The objective was identify pattern and analyses the adjustment of most common index. A total of 69 mammals was captured in two States (Pernambuco and Ceará) in ten (10) different vegetation types on dry and wet season. For each vegetation type and season were calculated the Capture Effort and the Capture Success. Described Statistical Analysis (sum, mean, percentages, and frequency) was utilized to calculate Abundance and Biological Characteristics of the entirety mammalian fauna. Those characteristic include sex, age classes, occupation and diary activity. Confirmatory Analyses (Qui-square Test and Pearson Coefficient of Correlation) was employed to test differences in mammals’ distribution between vegetation types and seasonality. The searching for pattern used Cluster Analyses (Median Method and Jaccard Coefficient). It created three clusters. For each one it was used Coefficients of Richness (Margalef) and Dominance and the Index of Diversity (Shannon). Ours results shows that clusters with six out of 10 vegetation types – called Habitat X – had adiversified mammalian fauna (α = 10,95 e H´ = 2,32), nevertheless the specie dominance was a marsupial Didelphis albiventris. The second cluster had three out of 10 vegetation type – called Habitat Y – no species were dominant. The third cluster – Habitat Z – with only one vegetation type, was different from the others, mainly due to its proximity to human settlements and plantations. In conclusion we could say that the Caatinga Biome is diverse and complex on its mammalian fauna aspects. It was possible to join vegetation types geographically apart in order to its mammals’ similarities. Habitats X and Y showed low similarities in mammals’species composition (Sxy = 0,30). The method of capture and the small success of capture, despite a significant effort, interfered in our results. An enormous effort should be carried out to keep representative and sustainable samples of this biome, because we know almost nothing about it. / Estudos recentes realizados para definir estratégias nacionais de conservação e para avaliar impactos de grandes empreendimentos sobre o meio ambiente forneceram dados de levantamentos da mastofauna da Caatinga que foram aqui analisados, com o intuito de identificar padrões e avaliar a adequação dos índices comumente utilizados. Um total de 69 mamíferos silvestres foram capturados em excursões de pesquisa que envolveu dois Estados (Pernambuco e Ceará) e estudou dez (10) tipos vegetacionais nas duas estações sazonais (seca e chuvosa). Para cada tipo e estação climática foram utilizadas fórmulas para o cálculo do Esforço de Captura e Sucesso de Captura. Análises da estatística descritiva (somatórios, médias, porcentagens, e freqüência) foram utilizados para os cálculos de abundância e de características biológicas do conjunto da mastofauna inventariada. As características incluíram sexo, classe de idade, hábito quanto ao deslocamento e ocupação e atividade cíclica diária. Análises confirmatórias (Teste Qui-quadrado e Coeficiente de Correlação de Pearson) foram empregadas para testar se a distribuição dos mamíferos difere entre as vegetações e estações sazonais. Na busca por padrões foi utilizada a Análise de Cluster (método da mediana e coeficiente de Jaccard) que gerou três agrupamentos. Para cada um foram utilizados os Coeficientes de Riqueza de Margalef e de Dominância e o Índice de Diversidade de Shannon. Nossos resultados mostraram que o agrupamento que contemplou 6 das 10 vegetações – chamado de habitat X – apresentou uma mastofauna diversificada (α = 10,95 e H´ = 2,32), porém com dominância do marsupial Didelphis albiventris; enquanto que no agrupamento que abrigou 3 vegetações – chamado de habitat Y – nenhumaespécie foi dominante. O terceiro agrupamento – habitat Z – com apenas um tipo de vegetação se distinguiu dos demais, em parte por sua proximidade com assentamentos humanos e áreas agriculturáveis. Como conclusão desse estudo podemos dizer que o Bioma Caatinga é diverso e complexo nos seus aspectos mastofaunísticos. Foi possível agrupar tipos vegetacionais, distantes geograficamente, devido às suas similaridades mastofaunísticas. Na composição das espécies de mamífero, os habitats X e Y apresentaram baixa similaridade (Sxy = 0,30). O método de captura pode ter privilegiado alguns táxons e o pequeno sucesso de captura, apesar do alto esforço apreendido, interferiu nos resultados. Um grande esforço deve ser empreendido para resguardar amostras representativas e sustentáveis desse bioma tão pouco conhecido.

Recently, long non-coding RNAs (lncRNAs) emerged as important regulators of many cellular functions. Many nuclear lncRNAs regulate the expression of geomically proximal or overlapping protein coding genes. Less clear is whether intergenic lncRNAs can regulate transcription by modulating chromatin at genomically distant loci in an RNA-dependent manner. This thesis investigated molecular functions of Dali, an intergenic central nervous system expressed lncRNA conserved in therian mammals. Dali is transcribed from a locus 50 kb downstream of the Pou3f3 transcription factor gene and performs both genomically local and distal RNA-dependent roles. Its depletion disrupts the differentiation of neuroblastoma cells. Locally, Dali regulates transcription of the Pou3f3 locus. Distally, it preferentially binds near to and regulates active promoters across the genome, including by physically associating with the POU3F3 transcription factor. Dali also interacts with the DNMT1 DNA methyltransferase in mouse and human and regulates CpG island-associated promoters by modulating their DNA methylation levels in trans. This work is the first to demonstrate that a lncRNA can regulate the DNA methylation of CpG island-associated promoters in trans and one of the first large scale studies to identify direct transcriptional targets of a lncRNA genome-wide. It also provides a more detailed molecular dissection of the extended Pou3f3 locus and a framework for the prioritisation and comprehensive functional characterisation of nuclear lncRNAs.