Tesi sul tema "Tissue specificity"

Mitochondrial disorders comprise a large group of heterogeneous disorders which are characterized by impairments in the cellular energy production. One of the great challenges of mitochondrial disease is the variety of clinical features present in patients. Mitochondrial disorders affect more than one organ leading to complex multisystem dysfunctions. Tissues, in which the metabolic demand is higher, such as skeletal muscle, neurons, liver or heart are typically affected. Mutations in both mitochondrial DNA (mtDNA) and nuclear DNA (nDNA) often lead to mitochondrial disorders. Although mtDNA encodes key proteins for the normal function of the mitochondrial respiratory chain enzymes, the vast majority of the essential components and proteins needed for the maintenance and replication of the mitochondrial DNA are encoded by the nDNA. Exome sequencing in combination with bioinformatics tools has proven really effective in determining novel alterations in the genomic sequence. One aim of this thesis was to evaluate novel mutations from affected patients with combined respiratory deficiencies. As a result, mutations in C12orf65 and in the novel disease gene MiD49, associated with mitochondrial disorders, are thoroughly presented. Vitamin supplements, pharmacological agents and exercise therapy are common strategies used in patients suffering from mitochondrial disorders. It has been shown that in cell lines of patients suffering from two rare reversible infantile mitochondrial diseases (reversible infantile respiratory chain deficiency and reversible infantile hepatopathy due to TRMU deficiency) supplementation of L-cysteine resulted in an improvement in most respiratory chain complexes activities. During my PhD I studied and proved that L-cysteine supplementation was also beneficial in cells from patients suffering from common forms of mitochondrial disorders such as MELAS and MERRF as the supplementation resulted in improved mitochondrial respiratory chain function. Finally, direct conversion of fibroblasts to neuronal progenitor cells was used to model mitochondrial disorders and study the tissue specificity. This project was very challenging due to the complex characteristics of mitochondrial biology. In summary, this thesis reveals the description of novel genes and mutations associated with combined mitochondrial deficiencies. Furthermore, we detected a positive effect of L-cysteine on a subset of mitochondrial disorders, which can be the base of further therapy development.

In tuberculosis (TB), matrix metalloproteinases (MMPs) have a major role in tissue destruction and cavitation. The composition of each extracellular matrix (ECM) gives specificity to tissues and is important in control of local immune responses by acting as a “molecular postcode”. Hence, it is likely to have important implications in TB. During central nervous system (CNS) infection, the effect of TB on blood-brain barrier (BBB) function is unknown. I hypothesised that the ECM environment may determine the MMP response during innate immune activation in pulmonary and CNS TB. I aimed to investigate the effect of adhesion to the lung’s ECM in regulation of MMP expression and activity. I also aimed to develop a BBB cellular model and investigate the role of Mycobacterium tuberculosis (Mtb)-driven MMP secretion on BBB function. In a model of pulmonary TB, human bronchial epithelial cells (NHBEs) and monocytes were exposed to ECM components and stimulated with conditioned medium from Mtb-infected monocytes (CoMtb) or infected with Mtb. Type I collagen (Coll-I) matrix was shown to decrease MMP-1 mRNA accumulation by 48% and collagenolytic activity compared to NHBEs in the absence of matrix. MMP-1 co-localised with integrin α2β1 resulting in enhanced cell migration in wound healing assays. In contrast, soluble Coll-I led to integrin α2β1 occupancy without clustering and caused a 7-fold increase in collagenolytic activity but decreased migration. In Mtb-infected monocytes, adhesion to ECM components increased MMP-1 secretion by over 60%. Fibronectin and Coll-I also increased MMP-10 by 55% and 90% respectively. Surface expression of integrin αVβ3 was upregulated by Mtb-infection and adhesion to Coll-I. Activation of integrin αVβ3 mimicked the effect of Coll-I MMP secretion, while its inhibition impaired monocyte migration. CoMtb-stimulation of the BBB model decreased trans-endothelial electrical resistance from 154±1.2ohm.cm2 to 111.6±4.7ohm.cm2, while Papp increased 3-fold. Coll-IV and tight junction proteins (TJPs) degradation was also detected. MMP concentrations increased 125-fold for MMP-1 (0.35±2 to 43.8±5.1ng/mL) and 619-fold for MMP-9 (0.072±0.014 to 44.6±8.9ng/ml). Treatment with the MMP inhibitor Ro32-3555 prevented BBB disruption. Neutrophil and monocyte transmigration increased 60% and 80% respectively and returned to control levels with MMP blockade. MMP-9 was shown to be responsible for BBB disruption and its inhibition by antibodies prevented BBB disruption. The Hedgehog pathway was downregulated during infection, resulting in decrease TJPs gene expression, which contributes to BBB dysfunction. In summary, the ECM regulates both epithelial and monocyte expression of MMP-1 via integrins signalling. In the CNS, MMP-9 drives tissue destruction and BBB disruption which may be a potential reversible event in CNS TB immunopathology.

Accidental exposure to arsine (AsH₃) is possible in the semiconductor industry, metal mining, painting and herbicide preparation. First symptoms include intravascular hemolysis and dark red urine (hematuria), followed by abdominal pain, jaundice, and anemia. Exposure to AsH₃ is fatal in up to 25% of the reported human cases, usually caused by acute oliguric renal failure. The mechanism of AsH₃ toxicity in the kidney is unknown and was studied in vitro using established cell lines, primary cells, and isolated kidney. The hypothesis was that AsH₃ cause renal toxicity by its conversion to arsenite (AsIII). Renal cells were more susceptible to As(III) cytotoxic effects on ion homeostasis and cell integrity, but AsH₃ showed oxidative stress-like toxicity. Red blood cells were only susceptible to direct AsH₃ cytotoxicity. Hepatocytes, chosen because liver is also affected by AsH₃, were susceptible to both arsenicals. It was established that AsH₃ produce tissue specific toxicity. The toxicity of the AsH₃-produced hemolysate was also investigated. The complete hemolysate was toxic and this toxicity was associated with the soluble hemolytic products. AsH₃-induced nephrotoxicity was also studied in the isolated rat kidney. Unmetabolized AsH₃ was more toxic than hemolytic products in this system. Damage was found in the glomeruli, tubular epithelial cells, and vascular peritubular capillaries. Finally, the total amount of arsenicals produced by AsH₃ oxidation in the rat kidney and liver homogenates was determined. As(III) was formed four times as much compared to As(V) in the kidney. By comparison, the liver metabolized less than half of the arsenite formed by the kidney. In summary, in vitro systems were used to model tissue selectivity for AsH₃ toxicity and to investigate AsH₃ renal cytotoxicity. Red blood cells and hepatocytes were susceptible to unmetabolized AsH₃. AsH₃ was required to form As(III) to produce renal toxicity. The soluble hemolytic products produced by AsH₃ also contributed to the in vitro renal toxicity. Renal dysfunction produced by AsH₃ exposure (the cause for mortality), is caused by a combination of AsH₃-produced oxidative-stress toxicity and by cell integrity damage produced by As(III) formed from AsH₃ oxidation, and delivered to the kidney as soluble toxicants in the hemolysate.

Glucocorticoids have diverse physiological functions: they affect central nervous system function, intermediary metabolism and restore homeostasis after stress. Secretion of glucocorticoids is regulated by the hypothalamic-pituitary-adrenal (HPA) axis; negative feedback at the hypothalamus and pituitary suppresses glucocorticoid secretion while the hippocampus exerts additional control over HPA axis activity. Glucocorticoids exert most of their actions, including negative feedback, via the glucocorticoid receptor (GR). The glucocorticoid sensitivity of a given cell/tissue is dependent on the level of GR expression. The regulation of the GR gene is complex; GR levels in adult animals are subject to glucocorticoid regulation and can be permanently "programmed" by early life events, with hippocampal GR permanently increased by neonatal handling (via alterations in serotonin turnover) and decreased by prenatal dexamethasone exposure. Evidence suggests that these effects may be mediated through differential regulation of variant exon-1 containing GR mRNAs; in rats the GR gene contains 8 protein-coding exons (exons 2-9) and at least 11 alternate untranslated exons 1 (exons li-ln) which may reflect transcription regulated by alternate promoters. The aim of this thesis was to further investigate the distribution of these variant GR transcripts and examine whether glucocorticoids themselves differentially regulate GR mRNA and its alternate exons 1 in a tissue and region-specific manner. Tissue and region-specific differences in the expression of variant GR mRNA transcripts were found in rat and mouse. Most GR mRNA variants were ubiquitously expressed, but those containing exon 11 were restricted to rat thymus, liver and hippocampus and mouse spleen, while those containing exon 14 were absent from rat cerebral cortex and mouse lung, heart and abdominal fat. In situ mRNA hybridisation on rat brain showed that all the exons 1 studied showed differences in their regional expression when compared to distribution of the total population of GR mRNAs. In contrast, in rat liver and thymus GR mRNA variants showed the same regional distribution as total GR mRNA with highest expression in periportal region of the liver and the thymic cortex. To investigate whether glucocorticoids differentially regulate variant GR mRNAs, in situ mRNA hybridisation was used to investigate the expression variant exons 1 in the hippocampus of adult rats subjected to 72h (ST) or 3-week (LT) adrenalectomy with glucocorticoid replacement. Variant GR mRNAs containing exons 1₅, 1₇, 1₁₀ and 1₁₁ were not affected by adrenalectomy or supraphysiological glucocorticoid replacement in ST or LT animals. However, both adrenalectomy and supraphysiological glucocorticoid replacement significantly upregulated total GR mRNA in the hippocampus of ST animals while adrenalectomy significantly upregulated total GR mRNA in the hippocampus of LT animals. In situ mRNA hybridisation and RNase protection analysis were used to investigate expression of variant GR mRNAs in the liver. Glucocorticoid manipulations did not significantly affect expression of variant GR mRNAs containing exons 1₅, 1₆, 1₁₀ and 1₁₁. However, in ST adrenalectomised animals glucocorticoid replacement significantly downregulated GR mRNA levels compared to adrenalectomised animals given vehicle alone. Adrenalectomy had no effect on total GR mRNA expression in the LT animals, although in these animals the periportal :perivenous ratio of GR expression was significantly increased by adrenalectomy compared to sham. Preliminary data from DNase I hypersensitive site mapping in control animals showed an area of DNase I sensitive chromatin around the position of exon 1₁₀, present in the majority of GR mRNA in the liver. In the thymus, although adrenalectomy with either high and low dose glucocorticoid replacement in ST animals caused a significant downregulation of GR mRNA in the cortex and medulla compared to sham, the expression of exons 1₁, 1₆ and 1₁₀ of the GR gene was not significantly affected by glucocorticoid manipulations. There was no effect of glucocorticoid manipulation on GR or its variants in the LT animals. These results demonstrate tissue-specific differences in the distribution of GR mRNA variants, suggesting that variations in promoter usage may have a role in determining the "set-point" of GR expression in different tissues. The observed tissue-specific effects of glucocorticoids on GR mRNA expression could not be accounted for by changes in expression of any of the variant GR mRNA transcripts studied. This suggests that either the expression of another variant mRNA (known or novel) is regulated by glucocorticoids or that expression of all or a subset of the variant GR mRNAs changed with a magnitude too small to be detected in this study.

In this thesis I describe my investigations of applying machine learning methods to high throughput experimental and predicted biological data. The importance of such analysis as a means of making inferences about biological functions is widely acknowledged in the bioinformatics community. Specifically, this work makes three novel contributions based on the systematic analysis of publicly archived data of protein sequences, three dimensional structures, gene expression and functional annotations: (a) remote homology detection based on amino acid sequences and secondary structures; (b) the analysis of tissue-specific gene expression for predictive signals in the sequence and secondary structure of the resulting protein product; and (c) a study of ageing in the fruit fly, a commonly used model organism, in which tissue specific and whole-organism gene expression changes are contrasted. In the problem of remote homology detection, a kernel-based method that combines pairwise alignment scores of amino acid sequences and secondary structures is shown to improve the prediction accuracies in a benchmark task defined using the Structural Classification of Proteins (SCOP) database. While the task of predicting SCOP superfamilies should be regarded as an easy one, with not much room for performance improvement, it is still widely accepted as the gold standard due to careful manual annotation by experts in the subject of protein evolution. A similar method is introduced to investigate whether tissue specificity of gene expression is correlated with the sequence and secondary structure of the resulting protein product. An information theoretic approach is adopted for sorting fruit fly and mouse genes according to their tissue specificity based on gene expression data. A classifier is then trained to predict the degree of specificity for these genes. The study concludes that the tissue specificity of gene expression is correlated with the sequence, and to a certain extent, with the secondary structure of the gene’s protein product. The sorted list of genes introduced in the previous chapter is used to investigate the tissue specificity of transcript profiles obtained from a study of ageing in the fruit fly. The same list is utilised to investigate how filtering tissue-restricted genes affects gene set enrichment analysis in the ageing study, and to examine the specificity of age-associated genes identified in the literature. The conclusion drawn in this chapter is that categorisation of genes according to their tissue specificity using Shannon’s information theory is useful for the interpretation of whole-fly gene expression data.

Chlamydia pneumoniae is a wide spread human and animal pathogen, associated with a number of acute and chronic disease states. In this thesis, next-generation sequencing technologies were used to identify genetic markers involved in the diverse presentation of Chlamydia pneumoniae infections. The findings presented in this study describe a phylogenetically distinct human Chlamydia pneumoniae line, as well as a number of minor DNA sequence changes that may account for the biological fitness of particular strains to different diseases and animal hosts. The outcomes of this thesis have considerably expanded our knowledge of the genetic and evolutionary diversity of this common pathogen.

The phenotypic variability in human populations is partly the result of gene polymorphisms and differential gene expression. Studying the variability of gene expression across human populations is essential to understanding the molecular basis for diversity. However, key issues remain unanswered with respect to human expression variability. For example, the role of gene methylation in expression variability is uncertain, nor is it clear what role tissue-specific factors may have. Moreover, the contribution that expression variability has in aging and development is unknown. Here we classified human genes based on their expression variability in normal human breast and brain samples and identified functional aspects associated with high and low expression variability. Interestingly, both high variability and low variability gene sets are enriched for developmentally essential genes. There is limited overlap between the variably expressed genes of different tissues, indicating that tissue-specific rather than individual-specific factors are at work. We also find that methylation likely has a key role in controlling expression variability insofar as genes with low expression variability are likely to be non-methylated. Importantly, we find that genes with high population expression variability are likely to have age-, but not sex-dependent expression. Taken together, our work indicates that gene expression variability is tissue-specific, methylation-dependent, and is an important component of the natural aging process.

El reloj circadiano es un mecanismo celular responsable de la generación de ritmos biológicos con un periodo de 24 horas. La importancia de la función del reloj circadiano es evidente en casi todos los organismos estudiados hasta la fecha, desde bacterias hasta los seres humanos. Dado que las plantas son organismos sésiles, la función circadiana es particularmente relevante para su correcta adaptación al medio y supervivencia. Entender cómo el sistema circadiano de la planta se organiza en el contexto de células, tejidos y órganos surge como una de las preguntas fundamentales para comprender la fisiología y el metabolismo de la planta. Sin embargo, un gran desafío para los estudios de biología vegetal es descifrar cómo los relojes circadianos individuales están interconectados para generar ritmos en toda la planta. En esta Tesis Doctoral, demostramos que el ápice del brote aéreo de la planta Arabidopsis thaliana está compuesto por un conjunto de relojes acoplados que sincronizan los ritmos circadianos en la raíz. Una serie de diversos protocolos desarrollados en este estudio reveló una disparidad de oscilaciones circadianas en hipocotilos, raíces y hojas diseccionadas que exhibían una reducida precisión circadiana. En contraste, los análisis del ápice aéreo de la planta demostraron ritmos altamente sincronizados y precisos. El uso de diferentes mutantes de reloj y líneas de reporteros, así como el análisis global de la transcripción circadiana indicó que tal sincronía y precisión no era debida a una red circadiana molecular específica del ápice del brote. Sin embargo, los estudios in vivo de células individuales, la desincronización de protoplastos dispersos y el análisis matemático usando coordenadas baricéntricas para espacios multi-dimensionales demostraron que la precisión circadiana era debida al acoplamiento o comunicación entre las células del ápice del brote. La mayor sincronía rítmica confería precisión y robustez frente a perturbaciones genéticas y farmacológicas así como capacidades particulares para los reajustes de fase durante experimentos de "jet-lag". Los ritmos en raíces estaban alterados por la ablación del ápice y en estudios de microinjertos, sugiriendo que las señales del ápice pueden sincronizar órganos distales. De una forma similar a la organización circadiana en mamíferos, nuestros estudios demuestran que los ápices juegan un papel dominante dentro del sistema circadiano jerárquico en plantas.
The circadian clock is a timing mechanism that generates 24-hour biological rhythms. The importance of the circadian clock function is evident in almost all organisms examined to date, from bacteria to humans. Since plants as sessile organisms, the circadian function is particularly relevant for adaptation and survival. Understanding how the plant circadian system is organized in the context of cells, tissues and organs raises as one of the fundamental questions to fully understand plant physiology and metabolism. However, a major challenge in plant biology is to decipher how individual clocks are interconnected to sustain rhythms in the whole plant. In this PhD thesis, we show that the Arabidopsis thaliana shoot apex is composed of an ensemble of coupled clocks that influence rhythms in roots. A series of different protocols developed in this study revealed a disparity of circadian oscillations in excised organs, with hypocotyls, roots and leaves displaying reduced circadian precision and robustness. In contrast, analyses of shoot apexes showed highly synchronized and precise rhythms. The use of different clock mutants and reporter lines as well as analyses of the global circadian transcriptional landscape at the shoot apex indicated that such synchrony and precision is not likely due to a molecular circadian network that is specific for the shoot apex. Instead, in vivo live-imaging of rhythmic single cells, desynchronization of dispersed protoplasts and mathematical analysis using barycentric coordinates for high-dimensional space demonstrated that circadian precision relies on a tight circadian coupling (or communication) among the shoot apex clock cells. The increased rhythmic synchrony conferred robustness against genetic and pharmacological perturbations and particular capabilities for phase readjustments during "jet-lag" experiments. Rhythms in roots were altered by shoot apex ablation and micrografting, suggesting that signals from the shoot apex are able to synchronize distal organs. Similar to the circadian organization in mammals, our studies demonstrate that shoot apexes play a dominant role within the hierarchical circadian system in plants.

[Truncated abstract] Cells need to be able to detect changes in their surrounding environment and transduce these signals into the appropriate cellular compartments. One of the major ways that the cell achieves this signal transduction is through the process of phosphorylation. Protein kinases are the enzymes responsible for catalysing this transfer of phosphate groups from ATP to amino acid residues of their specific substrates. A subfamily of serine/threonine kinases known as the Mitogen-Activated Protein Kinases (MAPKs) is essential in a diverse range of cell processes including growth, metabolism, differentiation and death. The first identified MAPKs, the Extracellular Signal-Regulated Kinases (ERKs), were found to be activated in response to mitogenic stimuli such as growth factors. However, since the discovery of the ERKs, other pathways leading to the activation of related kinases have been recognised. These kinases are preferentially activated in response to stress, and are thus termed “Stress-Activated Protein Kinases” or SAPKs. They consist of the c-Jun N-terminal kinase isoforms 1, 2 and 3 (also called SAPK1γ, SAPK1α and SAPKβ respectively) and the p38 MAPKs, p38α, p38β, p38γ and p38δ (also called SAPK2a, SAPK2b, SAPK3 and SAPK4 respectively). A major challenge in this field has been to identify the substrates and functions of the SAPKs. This has been partly achieved by the development of inhibitors for the JNK MAPKs and SAPK2a/b. However, no inhibitors currently exist that specifically inhibit SAPK3 and SAPK4. Therefore, elucidating the function of these SAPKs has proved more difficult. Recent studies suggest that SAPK3 may play a unique role in the cell compared to other members of the p38 MAPK family. For example, several signalling proteins appear to specifically activate SAPK3 in certain circumstances while not activating other members of the p38 MAPK family. In addition, SAPK3 contains a unique sequence motif that allows it to bind to specialised domains known as PDZ domains. The interaction of SAPK3 with proteins containing these domains may regulate its subcellular localisation and interactions with other proteins in the cell. This project was undertaken to expand the knowledge on the expression, localisation, substrate specificity and binding partners of SAPK3. In Chapter 3 of this thesis, a SAPK3 monoclonal antibody was evaluated for its ability to specifically recognise endogenous SAPK3 protein. SAPK3 was found to be expressed in immortalised cell lines and primary cultures of neonatal rat myocytes, and to be colocalised with the mitochondria of these cells. This co-localisation remained unaltered in response to treatment with the nuclear export inhibitor Leptomycin B, and with exposure to osmotic shock, suggesting that SAPK3 substrates may be localised at the mitochondria

La régulation transcriptionelle fine assurée par les Eléments Cis Régulateurs (ECR, eg. promoteurs et «enhancers») et les facteurs protéiques associés, est à la base de la mise en place et le maintien de l'identité tissulaire. Les modifications de la chromatine corrèlent avec l’activité d’ECRs et constituent l’épigénome de la cellule. Au cours de ma thèse, je me suis intéressée aux transitions des modifications des histones (H3K4me1/me2/me3, H3K36me3, H3K27me3 and H3K9me2) accompagnant le développement précoce de la cellule T. Pour cela, j’ai utilisé un modèle murin reproduisant une étape cruciale de la thymopoïèse - la sélection β - et la technique d’Immunoprecipitation de la chromatine couplée à des puces à ADN (ChIP-chip). Au sein des enhancer connus, nos analyses ont mis en évidence une nouvelle signature épigénétique liée à leur activité. De plus, nous montrons que l'étendue d'enrichissement d’H3K4me2 au sein des régions géniques des gènes exprimés, constitue une signature épigénétique des gènes tissus spécifiques. Tout ceci a permis de mieux comprendre le rôle de l’épigénétique dans l'établissement et le maintien de l'identité cellulaire.Le traitement anti-cancer moderne est basé sur les analyses de différents marqueurs d'agressivité (MA) et par la suite, de l’établissement de la thérapie personnalisée. Durant la dernière partie de ma thèse, j’ai participé à un projet collaboratif avec le laboratoire de Thérapie Cellulaire de l’Institut Paoli Calmettes à Marseille, qui visait l’isolation des MA des Leucémies Aiguës Myéloïdes à caryotype normal (LMAcn) grâce aux études de profilage épigénétique (H3K27me3) des blastes des patients atteints de LMAcn
Precise transcriptional regulation underlies the establishment and maintenance of cell type specific identity and is governed by dedicated DNA sequences (i.e., cis regulatory elements (CREs): eg.: promoters, enhancers) and transcription factors. Chromatin modifications (eg.: histone modifications, DNA methylation) impinge on CREs activity and constitute the epigenome of the cell.During my PhD, I was interested in the transitions of a set of histone modifications (H3K4me1/me2/me3, H3K36me3, H3K27me3 and H3K9me2), during one of the major checkpoints of thymopoiesis - the β-selection. I used a dedicated mouse model and Chromatin Immunoprecipitation coupled with microarrays (ChIP-chip) technique. Our data evidenced a previously unappreciated epigenetic signature linked to enhancer activity. In parallel, computational analyses of the patterns of gene body enrichment of H3K4me2 highlighted an epigenetic signature linked to the regulation of the tissue specific gene expression. Altogether, this enabled to deepen the relationship between chromatin states and regulation of cell type specific identity.Modern anticancer treatment is based on the analyses of a number of cancer aggressiveness markers (CAM) and results in a highly personalized therapy. Epigenetic profiling can constitute a powerful tool for CAM’s isolation. In the second part of the presented work, I participate in a collaborative project (with Cellular Therapy Centre at the Paoli Calmettes Institut, Marseille) aiming to isolate new CAM for Acute Myeloid Leukemia with normal karyotype (AMLnc) patients. For this purpose I performed epigenetic (H3K27me3) profiling of blasts of AMLnc

Thesis (Ph.D)--Biomedical Engineering, Georgia Institute of Technology, 2010.
Committee Chair: Andres Garcia; Committee Member: Andrew Lyon; Committee Member: Barbara Boyan; Committee Member: Johnna Temenoff; Committee Member: Todd McDevitt. Part of the SMARTech Electronic Thesis and Dissertation Collection.

The majority of genetic differences between species and individuals have been hypothesised to impact on the regulation, rather than the structure, of genes. As the details of genetic variation are uncovered by the various genome sequencing projects, understanding the functional effects on gene regulation will be key to uncovering the molecular mechanisms underying the genesis and inheritance of common phenotypes, such as complex human disease and commercially important traits in plants and animals. Unlike coding sequence polymorphisms, genetic variants affecting gene expression will reside in the transcriptional machinery and its regulatory inputs. As these are largely specific to cell- or tissue-types, we would expect that regulatory variants will also affect final mRNA levels in a tissue specific manner. Genetic variation between individuals may therefore be more complex than the sum total of sequence differences between them. Demonstrating this hypothesis is the main focus of this thesis. We use microarrays to measure mRNA levels of approximately 22,000 transcripts in inbred and recombinant inbred strains of mice, and present compelling evidence that the genetic influences on these levels are tissue-specific in at least 85% of cases. We uncover two loci which apparently influence transcript levels of multiple genes in a tissue-specific manner. We also present evidence that failure of microarray data normalisation may cause spurious linkage of expression phenotypes leading to erroneous biological conclusions, and detail a novel, extensible mathematical framework for performing tailored normalisation which can remove such systematic bias. The wider context of these results is then discussed.

Cell transplantation to the brain and spinal cord is a well-established tool for studies on cellular and molecular mechanisms involved in development and plasticity. Experimental data from animals has suggested that restoration or preservation of function through cell transplantation has potential as a useful therapeutic approach in neurodegenerative disorders. Experimental evidence of graft-induced behavioral ameliorations has raised the question of the applicability of this technique to the cognitive neurodegenerative disorder, Alzheimer's disease (AD).
This present study is based on previous work from our lab that has indicated a potential for cortical grafts of chromaffin cells of the adrenal medula to alleviate nucleus basalis magnocellularis (nbm) lesion-induced cognitive and neurochemical deficits; such lesions produce deficits which mimic certain of the deficits seen in AD. This study has found that chromaffin cell grafts ameliorate behavioral deficits and that a neurochemical recovery occurs in the cortex consequential to it. This particular behavioral improvement was evidenced 6 weeks but not 5 days post-graft, indicating a gradual effect by the graft. Additionally, in this behavioral test, control grafts of kidney cells were found to have no ameliorating effects. A final objective to establish immunocytochemical techniques for detection of graft constituents was also actualized.

Abstract The collagens are a family of extracellular matrix proteins with a widespread tissue distribution. Collagen biosynthesis requires the hydroxylation of a number of proline residues by prolyl 4-hydroxylase. This posttranslational modification is essential for the synthesis of all collagens, as 4-hydroxyproline deficient collagens cannot form stable triple helices at body temperature. The genes for the human and mouse prolyl 4-hydroxylase α(II) subunits were cloned and characterized in this study. The human and mouse genes are 34.6 and 30.3 kb in size, respectively, consisting of 16 exons and 15 introns. The intron sizes vary from 48-49 bp to over 8 kb in both genes. The 5' flanking regions contain no TATA box, but there are several motifs that may act as transcription factor binding sites. A novel mutually exclusively spliced exon 12a was identified in both genes. Both variants of the α(II) subunit were found to be expressed in a variety of tissues and both formed a fully active recombinant tetramer with the β subunit when expressed in insect cells. Tissue distribution of the type I and type II prolyl 4-hydroxylase isoenzymes was studied in developing, mature, and malignant cells and tissues by immunofluorescence and Western blotting. The results indicate that the type I isoenzyme is the main form in many cell types. Skeletal myocytes and smooth muscle cells appeared to have the type I isoenzyme as their only prolyl 4-hydroxylase form, whereas the type II isoenzyme was clearly the main form in chondrocytes. A strong signal for the type II enzyme was detected in cultured umbilical and capillary endothelial cells, whereas the type I isoenzyme could not be detected in these cells by immunostaining or Western blotting. Similar studies on primary chondro- and osteosarcomas and benign bone tumours indicated that the type I isoenzyme is the predominant form in both types of bone sarcoma, whereas the type II isoenzyme was more abundantly expressed in benign tumours. In chondrosarcomas, the type II isoenzyme was expressed in the nonmalignant chondrocytes, whereas their malignant counterparts switched their expression pattern to that of the type I isoenzyme. Two isoforms of the catalytic prolyl 4-hydroxylase α subunit, PHY-1 and PHY-2, have previously been characterized from Caenorhabditis elegans. This study reports the cloning and characterization of a third C. elegans α subunit isoform, PHY-3, which is much shorter than the previously characterized vertebrate and C. elegans α subunits. Nematodes homozygous for a phy-3 deletion were phenotypically wild type and fertile, but the 4-hydroxyproline content of their early embryos was reduced by about 90%. The expression of PHY-3 was found to be restricted to spermatheca of late larvae and adult nematode, indicating that PHY-3 is likely to be involved in the synthesis of collagens of the early embryo egg shells.

In this study, we assessed pirAB toxin transcription in Photorhabdus luminescens laumondii (strain TT01) (Enterobacteriaceae) by comparing mRNA abundance under in vivo and in vitro conditions. In vivo assays considered both natural and forced infections with two lepidopteran hosts: Galleria mellonella and Manduca sexta. Three portals of entry were utilized for the forced infection assays: (a) integument; (b) the digestive route (via mouth and anus); and (c) the tracheal route (via spiracles). We also assessed plu4093-2 transcription during the course of a natural infection; this is when the bacteria are delivered by Heterorhabditis bacteriophora nematodes. Transcript abundance in G. mellonella was higher than in M. sexta at two of the observed time points: 15 and 18 h. Expression of pirAB plu4093-2 reached above endogenous control levels at 22 h in G. mellonella but not in M. sexta. Overall, pirAB plu4093-2 transcripts were not as highly expressed in M. sexta as in G. mellonella, from 15 to 22 h. This is the first study to directly compare pirAB plu4093-2 toxin transcript production considering different portals of entry.

Dans l’histoire évolutive, deux gènes paralogues sont issus d’un évènement de duplication de leur ancêtre commun. Les gènes paralogues sont caractérisés par des duplications globales de génome (WGD) ou à petite échelle (SSD) et par leur datation. Les WGDs ont lieu à deux reprises à la base de la lignée des vertébrés. Les évènements de SSD ont lieu à plusieurs moments pouvant être plus récents, plus anciens ou contemporain de la période des évènements de WGD. La rétention des paralogues dans le génome, associée à une divergence de l’expression spatiale est une contribution importante pour l’augmentation de la complexité de l’organisme au cours de l’évolution. Certaines études ont montré que les duplications anciennes seraient plus associées aux maladies. L’objectif de la première partie de la thèse est de créer une ressource sur les paralogues en collectant et en analysant différentes annotations. Nous avons construit une ressource robuste de paralogues humains à partir de listes publiées mais aussi à partir d’annotations externes. L’exploration de différentes annotations nous a permis d’identifier une identité de séquence élevée entre gènes paralogues pouvant biaiser la mesure d’expression des gènes et diminuer leur expression. L’objectif de la seconde partie, est d’explorer l’expression spatiale et la co- expression des paralogues au sein du cerveau humain, à partir des données RNA-seq du consortium GTEx. Les données d’expression GTEx de 13 tissus cérébraux, nous ont permis de montrer que la datation récente mais aussi que le type SSD contribuaient à une expression plus tissu-spécifique. Nous avons utilisé l’analyse de la co-expression (WGCNA) afin de regrouper les paralogues possédant une expression similaire au travers des tissus et nous avons pu suggérer une co-expression des SSD récents. Nos études sur les maladies ont montré que les SSD récents accumulaient des mutations associées à des maladies cérébrales. Finalement, nous avons trouvé que la co-expression des paralogues et leur tissu-spécificité au travers des régions cérébrales pouvaient enrichir nos connaissances sur les gènes associés à des maladies cérébrales
In evolution history, two paralogous genes originate from the duplication event of a common ancestor gene. Paralogous genes are characterized by whole genome (WGD) or small-scale (SSD) duplications and their duplication date. The WGDs happened twice in the early vertebrate lineage. SSD events take place at any moment in evolutionary history and can be younger, older or dating to the same period than WGD events. Retention of paralogs in the genome associated with divergence of spatial expression is an important contributor to the increase of organism complexity through evolution. Different studies found that old duplications are more associated with diseases. The objective of the first part of the thesis is to create a resource on paralogs by collecting and analyzing annotations. We built a robust resource of human paralogs from published lists of paralogous genes and also from external annotations. Annotation exploration allowed us to identify a high sequence identity between paralogous genes impacting the gene expression measurement from RNA-seq data and decreasing the gene expression. The objective of the second part is to explore spatial expression and co-expression of paralogs in the human brain, from the GTEx consortium RNA-seq data. The GTEx expression data of 13 brain tissues allowed us to show that duplication youth and SSD type contributed to a more tissue-specific expression. We used co-expression analyses (WGCNA) to group paralogs with similar expression across tissues and we suggested the co-expression of younger SSDs. Our disease studies showed the younger SSD accumulation of mutations associated with brain diseases. We finally found that paralog co-expression and their tissue-specificity across brain regions could enrich information of known brain disease-associated genes

Durch ihren Einfluß auf die Genexpression reguliert die zirkadiane Uhr physiologische Funktionen vieler Organe. Obwohl der zugrundeliegende allgemeine Uhrmechanismus gut untersucht ist, bestehen noch viele Unklarheiten über die gewebespezifische Regulation zirkadianer Gene. Neben ihrer gemeinsamen 24-h-Periode im Expressionsmuster unterscheiden diese sich darin, zu welcher Tageszeit sie am höchsten exprimiert sind und in welchem Gewebe sie oszillieren. Mittels Überrepräsentationsanalyse lassen sich Bindungsstellen von Transkriptionsfaktoren identifizieren, die an der Regulation ähnlich exprimierter Gene beteiligt sind. Um diese Methode auf zirkadiane Gene anzuwenden, ist es nötig, Untergruppen ähnlich exprimierter Gene genau zu definieren und Vergleichsgene passend auszuwählen. Eine hierarchische Methode zur Kontrolle der FDR hilft, aus der daraus entstehenden Menge vieler Untergruppenvergleiche signifikante Ergebnisse zu filtern. Basierend auf mit Microarrays gemessenen Zeitreihen wurde durch Promotoranalyse die gewebespezifische Regulation von zirkadianen Genen zweier Zelltypen in Mäusen untersucht. Bindungsstellen der Transkriptionsfaktoren CLOCK:BMAL1, NF-Y und CREB fanden sich in beiden überrepräsentiert. Diesen verwandte Transkriptionsfaktoren mit spezifischen Komplexierungsdomänen binden mit unterschiedlicher Stärke an Motivvarianten und arrangieren dabei Interaktionen mit gewebespezifischeren Regulatoren (z.B. HOX, GATA, FORKHEAD, REL, IRF, ETS Regulatoren und nukleare Rezeptoren). Vermutlich beeinflußt dies den Zeitablauf der Komplexbildung am Promotor zum Transkriptionsstart und daher auch gewebespezifische Transkriptionsmuster. In dieser Hinsicht sind der Gehalt an Guanin (G) und Cytosin (C) sowie deren CpG-Dinukleotiden wichtige Promotoreigenschaften, welche die Interaktionswahrscheinlichkeit von Transkriptionsfaktoren steuern. Grund ist, daß die Affinitäten, mit denen Regulatoren zu Promotoren hingezogen werden, von diesen Sequenzeigenschaften abhängen.
A circadian clock in peripheral tissues regulates physiological functions through gene expression timing. However, despite the common and well studied core clock mechanism, understanding of tissue-specific regulation of circadian genes is marginal. Overrepresentation analysis is a tool to detect transcription factor binding sites that might play a role in the regulation of co-expressed genes. To apply it to circadian genes that do share a period of about 24 hours, but differ otherwise in peak phase timing and tissue-specificity of their oscillation, clear definition of co-expressed gene subgroups as well as the appropriate choice of background genes are important prerequisites. In this setting of multiple subgroup comparisons, a hierarchical method for false discovery control reveals significant findings. Based on two microarray time series in mouse macrophages and liver cells, tissue-specific regulation of circadian genes in these cell types is investigated by promoter analysis. Binding sites for CLOCK:BMAL1, NF-Y and CREB transcription factors are among the common top candidates of overrepresented motifs. Related transcription factors of BHLH and BZIP families with specific complexation domains bind to motif variants with differing strengths, thereby arranging interactions with more tissue-specific regulators (e.g. HOX, GATA, FORKHEAD, REL, IRF, ETS regulators and nuclear receptors). Presumably, this influences the timing of pre-initiation complexes and hence tissue-specific transcription patterns. In this respect, the content of guanine (G) and cytosine (C) bases as well as CpG dinucleotides are important promoter properties directing the interaction probability of regulators, because affinities with which transcription factors are attracted to promoters depend on these sequence characteristics.

L'analyse de la repartition des transcrits de l'arnr16s de plastes d'epinard a permis d'observer une accumulation preferentielle de ces transcrits dans les cotyledons (organes photosynthetiques) par rapport aux racines (organes non photosynthetiques). Cette expression variable suivant les organes de la plante a pu etre associee a la presence de transcrits alternatifs inities au niveau des sites d'initiation de transcription pc et parnt-16s. Alors que parnt-16s est constitutivement represente, le transcrit pc est associe aux organes photosynthetiques. L'expression de l'arnr16s au niveau du site pc ne demarre qu'au 5#e#m#e jours de germination, juste avant l'apparition des cotyledons. Le site d'initiation de transcription pc est localise entre deux sites promoteurs p1 et p2 qui presentent de fortes homolgies avec les promoteurs procaryotiques. L'arn polymerase de e. Coli initie correctement la transcription in vitro au niveau de ces 2 sites. In vivo, seul le site pc est utilise ; p1 et p2 ne sont pas utilises en depit de l'existence de l'arn polymerase chloroplastique de type e. Coli. Dans la deuxieme partie de ce travail, nous avons donc etudie les mecanismes regulant l'expression du 16s au niveau du site pc. Deux complexes nucleoproteiques cl et cs se formant entre la region promotrice du 16s et des proteines chloroplastiques ont ete identifies. Le complexe cs resulte de la fixation des proteines cdf2 (baeza et coll. , 1991). Le complexe cl resulte de la fixation concomitante de l'arn polymerase plastidiale de type e. Coli et des proteines cdf2 sur le promoteur de l'arnr16s. Complexee aux proteines cdf2, cette polymerase ne transcrit pas le 16s au niveau des promoteurs p1 et p2. La transcription de l'arnr 16s au niveau du site pc serait probablement assuree par la polymerase plastidiale monomerique d'origine nucleaire. Le role probable de facteurs d'initiation ou d'activateurs de transcription par cette polymerase, pour les proteines cdf2 est discute

La decouverte de l'homeobox: sequence d'adn, presente dans plusieurs genes jouant un role decisif dans la formation des structures de la drosophile et conservee dans des organismes aussi differents que les annelides, la souris et l'homme. Isolement de gene de souris contenant une homebox, en criblant une banque d'adn genomique. Six homeoboxes groupees sur une distance de 75 kilobases ont ete isolees, demontrant que la structure en complexes rencontree chez la drosophile et l'homme est conservee chez la souris. L'analyse de l'expression des homeo-genes a montre qu'elle etait restreinte a certains jours du developpement embryonnaire et a quelques tissus de l'adulte, specifiques pour chaque homeo-gene. Ceci suggere que les homeogenes pourraient avoir un role de regulation des mecanismes impliques dans le developpement embryonnaire

Le controle de l'initiation de la traduction est une etape importante de l'expression des genes chloroplastiques et fait intervenir, chez e. Coli, la proteine ribosomique (proteine-r) s1. Afin d'etudier l'etape d'initiation de la traduction chez le chloroplaste, nous avons caracterise chez spinacia oleracea une proteine (cs1) apparentee a la proteine s1. Nous avons isole un adnc codant pour le precurseur de la proteine-r cs1. Cs1 est considerablement plus courte que sa contrepartie bacterienne. Un noyau central homologue se compose de trois repetitions degenerees qui presentent de l'homologie principalement avec le domaine de liaison a l'arn de la proteine s1 d'e. Coli. Cs1 a ete surproduite dans e. Coli et purifiee. Nous avons etudie les proprietes de liaison a l'arn de la proteine cs1 au sein de la sous-unite ribosomique 30s et de la proteine isolee. Nous montrons que cs1 joue un role actif dans la liaison des arnm durant l'initiation de la traduction. Les implications de ces resultats pour la comprehension du systeme traductionnel du chloroplaste sont discutees. Dans la seconde partie de ce travail, nous avons montre que l'arnm codant pour la proteine-r cs1 est synthetise tres precocement durant les premieres etapes du developpement des plantes et est accumule meme en absence de lumiere. Nous avons clone et sequence la totalite de la region codante ainsi que la region 5 regulatrice du gene nucleaire unique (rps1) codant pour cs1. Nous avons observe la presence de deux departs de transcription. L'arnm le plus long code par le gene rps1 est specifiquement transcrit dans les feuilles. Ces resultats sont a relier a une accumulation differentielle de l'arnm cs1 dans les racines et dans les feuilles. Ces observations nous amenent a conclure que le gene de menage rps1 est regule transcriptionnellement de maniere tissu-specifique

Le mutant d'arabidopsis thaliana dal1-2, etiquete par un element transposable ac, est bloque precocement dans le developpement du chloroplaste. Son etude phenotypique a permit de mettre en evidence une couleur des feuilles variant du jaune au vert clair, au cours de son developpement. Morphologiquement, des coupes histologiques de feuilles montrent une desorganisation de la couche palissadique mesophyllienne. Le nombre de chloroplastes par cellule est plus important chez le mutant compare avec le sauvage mais leur taille est plus petite. La structure des chloroplastes par microscopie electronique a transmission montre une desorganisation et une quantite reduite des membranes thylacoidiennes. Les dosages realises sur les chlorophylles, carotenoides et lipides indiquent une forte reduction quantitative de ces molecules chez le mutant. La respiration chez le mutant est normale mais la photosynthese est residuelle pour un stade de developpement avance du mutant. Dal1-2 n'est pas affecte dans la skotomorphogenese. Au niveau genetique, l'isolement de l'adnc et du gene ont ete realises. L'expression du gene dal est absente chez le mutant. Une seule copie du gene dal est presente dans le genome nucleaire d'arabidopsis thaliana et celui-ci appartient a une famille de genes. L'expression de dal est elevee dans les boutons floraux et les fleurs. La proteine dal contient une sequence d'adressage chloroplastique et est importee dans le chloroplaste. L'analyse de l'expression de genes impliques dans le developpement des organites a permit de montrer clairement que la coordination de l'expression de genes nucleaires et chloroplastiques est toujours assuree chez le mutant dal1-2. L'expression des genes nucleaires de proteines ribosomiques est inchangee dans le mutant alors que l'expression des genes photosynthetiques cab et rbcs est reduite. Globalement, l'expression des genes mitochondriaux montre un niveau d'expression normal. Par contre, l'expression de tous les genes chloroplastiques testes est fortement reduite. L'analyse de l'expression de l'adnr 16s chloroplastique revele la presence de 50% d'arnr 16s non matures. Une explication possible etant que la proteine dal soit impliquee dans la maturation post-transcriptionnelle des arnr 16s. Ainsi, l'absence de la proteine dal conduirait a une chute de l'assemblage des ribosomes 70s, et par consequent a une perte de l'integrite chloroplastique.

La maladie de Huntington (MH) est une maladie neurodégénérative fatale, causée par l'expansion des répétitions CAG du gène de Huntingtine. La longueur de l'expansion est instable et proportionnelle à la gravité de la maladie. L'instabilité varie selon les tissus, p.ex. le striatum est très instable et dégénère, alors que le cervelet a une instabilité limitée et est épargné par la maladie. Nous avons étudié le rôle des lésions oxydantes et du mécanisme de réparation par excision de base (BER) dans la sélectivité tissulaire de l'instabilité dans ces deux tissus de souris R6/1. Le niveau des lésions était similaire dans ces tissus, alors que les niveaux et les activités des principales protéines BER étaient globalement diminués dans le striatum. L'efficacité de réparation dépendait de la stoechiométrie de BER, la position de la lésion et la séquence d'ADN. Nos résultats suggèrent une faible coopération entre les activités BER associée à la spécificité tissulaire de l'instabilité de la MH.

Au cours de ce travail de these nous avons caracterise un adnc correspondant a la proteine t (1560 pb) du complexe de la glycine decarboxylase. Il possede un cadre de lecture ouvert de 1220 pb codant pour un peptide mature de 378 acides amines. La sequence primaire ainsi que la masse deduite (40 961 da) ont ete confirme respectivement par microsequencage et par mesure au spectrometre de masse. Dans la perspective d'une etude structurale, nous avons reussi a surexprimer chez e. Coli la proteine t en construisant un vecteur d'expression produisant un arnt rare chez les bacteries. L'etude de l'expression de la proteine t ainsi que de ses transcrits a montre qu'ils sont principalement presents dans les tissus folaires et semblent subir une forte induction a la lumiere. De plus, nous avons demontre que les transcrits correspondant aux proteines de la gdc (p, h et t) sont exprimes des le 4#i#e#m#e jours de developpement de la plante avec un pic au 7#i#e#m#e jour. . A ce stade, la faible representation des proteines de la gdc (p, h et t), suggere l'existence d'un controle post-transcriptionnel de l'expression de leurs genes. Nous avons egalement isole et sequence le gene codant pour la proteine t. Ce gene, compose de quatre exons, possede deux sites d'initiations dont l'un presente une sequence riche en pyrimidine proche de la sequence inr (initiator element). Enfin, l'analyse de la region promotrice a permis de caracteriser trois regions consensus qui semblent etre impliquees dans la regulation a la lumiere et dans la specificite tissulaire

Deux lectines endogenes, l'une soluble (appelee csl), l'autre membranaire (appelee r1), ont ete isolees du cervelet de jeunes rats, par un cycle d'extractions sequentielles et purifiees par chromatographie d'immunoaffinite, sous une forme active. Cette purification a permis d'aborder l'etude de leur specificite glycannique fine par des tests d'inhibition de l'agglutination des erythrocytes induite par ces lectines ou par l'utilisation d'une technique similaire a l'elisa. Ces tests, qui ont ete realises avec des mono-, oligo- et polysaccharides, ainsi qu'avec des glycoconjugues de structure connue, ont permis de montrer que ces deux lectines different par leur specificite glycannique des autres lectines decrites tant dans le cerveau que dans d'autres tissus. La csl et r1 ont une affinite tres forte pour une classe particuliere de glycannes endogenes du cervelet, riches en mannose qui portent des epitopes reconnues par certains anticorps du type onco-ftal hnk-1. L'utilisation de deux lectines iodees pour reveler leurs ligands glycoproteiques endogenes apres electrophorese et electrotransfert a permis de mettre en evidence que nombre de ces ligands sont des molecules par ailleurs considerees comme des cams. C'est le cas des glycoproteines myeliniques po et mag. Etant donne l'interet des etudes sur la regulation de l'expression de la csl, le clonage de cette lectine a ete entrepris. Des banques d'adnc de cervelet de rat ont ete preparees dans le vecteur lambda zap ii et cribles avec differents anticorps anti-cls. Trois clones ont ete isoles et caracterises. Des travaux recents de notre groupe ont montre que la csl etait une cible immunologique dans la sclerose en plaques (sep). Nous avons pu verifier que les proteines de fusion reconnues par les anticorps anti-csl produits chez le lapin sont egalement reconnues par des anticorps presents dans le liquide cephalo-rachidien de patients atteints de s

Les androgens(ADs) et les glucocorticoïdes (GCs) sont des hormones stéroïdiennes qui exercent des effets pléiotropes chez les mammifères. Leurs effets sont relayés par deux récepteurs nucléaires, le récepteur des androgènes (AR) et le récepteur des glucocorticoïdes (GR), respectivement. Même si les GCs sont fréquemment utilisés pour traiter les maladies inflammatoires et les antiandrogènes pour le cancer de la prostate, les traitements à long terme induisent des effets secondaires majeurs, notamment l'atrophie musculaire.Afin de préciser les mécanismes d’action de ces hormones, nous avons réalisé des analyses phénotypiques, transcriptomiques et cistromiques. La première partie de ce travail démontre que GR des myofibres contrôle négativement la masse et la force musculaire aux niveaux physiologiques de GCs. La perte de GR dans les muscles squelettiques n'affecte pas les voies cataboliques, mais augmente l’expression de facteurs anaboliques et réduit celle de facteurs anti-anaboliques. Nous avons également montré que GR se lie à des éléments de réponse du GR (GREs) situés aux enhancers, en association avec Myod1 et Foxf2, et interagit avec des facteurs liés aux promoteurs, tels que Nrf1, pour favoriser la transcription des gènes.Dans la deuxième partie de ce travail, nous avons comparé le cistrome et le transcriptome du GR dans la prostate et le muscle squelettique, et identifié des sites de liaison pour d'autres facteurs de transcription proche des GREs, indiquant que ces facteurs contribuent à la spécificité tissulaire. De plus, en comparant les cistromes et transcriptomes d’AR et de GR dans la prostate, nous montrons que les éléments de réponse liés par les deux récepteurs sont distincts de ceux liés uniquement par AR ou GR, et que la sélectivité du récepteur dépend de la liaison d’autres facteurs de transcription.Enfin, nous avons comparé les données transcriptomiques et épigénétiques du tissu musculaire squelettique et de myoblastes et myotubes C2C12, et nous fournissons une description détaillée de gènes, voies de signalisation et facteurs de transcription exprimés de façon différentielle pendant la différenciation myogénique.En conclusion, nos travaux ont permis de clarifier les mécanismes moléculaires régulant l'homéostasie musculaire et ont établi la base d'une compréhension moléculaire des effets spécifiques des ADs et des GCs dans divers types cellulaires
Androgens (ADs) and glucocorticoids (GCs) are steroid hormones exerting pleiotropic effects in mammals. Their effects are mediated by two nuclear receptors, the androgen (AR) and the glucocorticoid (GR) receptor, respectively. Although GCs are extensively used to treat inflammatory diseases and antiandrogens for prostate cancer, long-term treatments induce major side effects such as muscle atrophy.To determine the mechanisms underlying their effects in muscle, we performed phenotypic, transcriptomic and cistromic analyses. The first part of this work demonstrates that myofiber GR negatively controls muscle mass and strength under physiological GCs levels. GR loss in skeletal muscle did not affect catabolic pathways, but enhanced the expression of anabolic factors and reduced that of anti-anabolic ones. We also showed that myofiber GR binds DNA to GR response elements (GREs) located at enhancers, in association with Myod1 and Foxf2, and interact with promoter-bound factors such as Nrf1 to promote gene transcription.In the second part of this work, we compared GR cistromes and transcriptomes in prostate and skeletal muscle, and identified binding sites for additional transcription factors in the vicinity of GREs, indicating that they contribute to the tissue specificity. In addition, by comparing the AR and GR cistromes and transcriptomes in prostate, we show that the response elements bound by both receptors are distinct from those bound by either AR or GR, and that the receptor-selectivity depends mostly on the surrounding factors.Finally, we compared transcriptomic and epigenetic data of skeletal muscle tissue and C2C12 myoblasts and myotubes and provide a detailed description of genes, signaling pathways and transcription factors that are differentially expressed during myogenic differentiation.In conclusion, our work allowed to clarify the molecular mechanisms regulating muscle homeostasis and provides the basis of a molecular understanding of tissue- and/or promoter-specific activity of ADs and GCs

Macroautophagy is a degradative system that cells employ to degrade proteins, lipids, pathogens or whole organelles. Dysfunctional autophagy has been implicated in diseases ranging from cancer to neurodegeneration. Animal models lacking macroautophagy fail to preserve a functional liver or central nervous system, supporting the importance of autophagy in maintaining the health of these tissues. However, it is unclear why this degradative pathway is critical in maintaining homeostasis. All macroautophagic cargo are sequestered by the multilamellar organelle called the autophagosome. The formation of the autophagosome depends on the lipidation of a cytosolic protein LC3, so that it associates with the autophagosomal membrane throughout the autophagic process. Using a mouse model expressing GFP-LC3, we have developed an approach to immunopurify autophagosomes from different tissue, then identified their autophagosomal content using tandem-mass-tag (TMT) quantitative proteomics. We have found that the tissues rely on autophagy differently based on the turnover of their organelles as liver depended more on autophagy for ER turnover and brain relied on autophagy more for mitochondrial turnover and its synaptic vesicle homeostasis. Starvation can activate macroautophagy, and is the most studied means through which this pathway has been studied. The importance of autophagy activation in the liver during starvation has been well characterized whereas its importance in the brain has been debated. In this study, we have found that both the liver and brain rely on autophagic degradation of mitochondria differently during starvation. As expected, liver increases its autophagic response upon 24 hr nutrient deprivation, but surprisingly cargo capture transitions from whole mitochondrial turnover to piecemeal mitochondrial turnover. In contrast, in brain, mitochondria-turnover remains largely unchanged. Moreover, although neuronal cargo proteins also remained largely unchanged in response to nutrient deprivation, there was a robust response driven by the non-neuronal cells of the CNS including glial cells and brain endothelial cells, indicating how the discrete cell types of the CNS respond to this physiologic stressor differently. Taken together, this work reveals the tissue-specificity and cell-specificity in the physiological role of autophagy, providing insight in how vertebrates use autophagy to maintain health and react to stress.

碩士
國立清華大學
生命科學系
90
Ataxia-telangiectasia (A-T) is a recessive genetic disease that progressively and degenerately affects a startling variety of body systems. Several epidemiological studies have found that patients with A-T heterozygotes tend to have higher risk to develop epithelial carcinoma, especially breast carcinomas. Although tumorigenic mechanisms to explainthe occurance of tissue specificity is not clear, the gene responsible for A-T (ataxia-telangiectasia mutated;ATM) has been identified recently, and it is believed that ATM plays a key role In tumor suppression this disease. In this study, we aimed at investigating how ATM down-stream ptoteins react againt ioizingradition when the ATM protein were down-regulated,and we focus on the differences among cells of tissue origins. The Tet-on gene expression system, using Doxycycline as a cofactor to control the gene expression, was used to generate an ATM antisense mRNA to suppress the expression of ATM protein. Immunoblotting was used to monitor possible functional alteration of ATM down-stream genes (p53, chk2, p95/NBS1). Our data shows that the ATM protein was successfully down regulated by adding increasing concentration of Doxycyclin in Tet-on gene expression system. however, no significantly functional differences of ATM down-stream genes was found in MCF-7 (Breast Carcinoma), HepG2 (Human Hepatocellular carcinoma), 293T (Human embryonic kidney cell) and Jurkat (human acute T-cell Leukemia). Only when the ATM protein was totally absent, the function of ATM down-stream protein was abolished. In conclusion, the outcome of using Tet-on system expressing antisense mRNA to inhibit the expression of ATM within the cells is good. When the ATM expression was down regulated, the expression of the downstream regulated proteins did not decrease significantly. This phenomenon could be explained as even low amount of ATM was expressed, but all function normally, therefore, the effect of down regulated ATM downstream proteins is slightly weaker than those not expressing antisense mRNA

Alongside with the drought, soil salinity is one the major environmental constraints that reduces crop growth and yield worldwide. According to recent reports, salinity stress is costing agricultural sector over US $27.3 billion per annum in lost revenues; which also aggravates the global food security. Improving salinity tolerance is a challenging task that requires understanding key physiological traits in naturally salt tolerant plant species. Halophytes are salt loving plants and they have very diverse array of physiological and biochemical mechanisms to encounter salinity stress. Therefore, understanding the fine-print of salinity-induced constraints on plant growth and/or salinity tolerance mechanisms in salt-tolerant halophytes is of a key importance for enhancing salinity stress tolerance in salt sensitive crop plants. The extent of salinity tolerance depends considerably on crop species and families. Thus, comparing different plant species from the same family or genus can provide much better understanding of physiological mechanisms conferring differential salinity tolerance. In this work, we have selected Chenopodiaceae family, one of most important subfamilies of Amaranthaceae family that contains numerous plant species, including halophtytic (e.g. Chenopodium quinoa, Chenopodium album, Atriplex lentiformis) and glycophytic species (e.g. Spinacia oleracea (spinach). Amongst different plant species, we have selected Chenopodium quinoa (quinoa hereafter) as a dicotyledonous halophytic plant species and Spinacia oleracea (spinach hereafter) as glycophytic plants species. Some previous and basic reports showed that Chenopodiaceae family showed a considerable intra- and inter-specific variation at the whole plant level under saline conditions. However, to the best of our knowledge, no much specific details on the cellular mechanisms conferring this intra- and inter-specific variability in the context of salinity tolerance are available in the literature. Salinity stress is very complex abiotic stress that induces cytosolic toxicity and oxidative damage by causing ROS production. Under saline conditions, the production of different ROS such as hydrogen peroxide (H\(_2\)O\(_2\)), superoxide radical (O\(_2\)\(^-\)) and hydroxyl radical (•OH) at different sites in cells causes significant damages to nucleic acids, proteins, and lipids. Elevated ROS levels also cause major disturbance to plant ionic homeostasis. At the same time, at low concentrations ROS can act as signalling molecules to control various physiological processes such as cell growth, pollen development, hormonal control, stress signalling and transduction, and ion transport across the plasma membrane. Until now very few studies have been published showing the role of different types of ROS in regulating ion transport at tissue levels. Therefore, in order to understand above tissue specific and intra- and inter-specific variability in salinity tolerance, set of physiological, electrophysiological and confocal imaging experiments were conducted to answer some of these specific questions: I. How does K\(^+\) retention pattern (a key determinant of the salinity tissue tolerance mechanism) in different tissues (root and leaf mesophyll) differ between halophyte and glycophyte species? II. Can plants (especially halophytic plants) employ other ROS such as •OH and O\(_2\)\(^-\) to shape Ca\(^{2+}\) flux signatures? III. Is there any specific Ca\(^{2+}\) or ROS signatures involved in early salt sensing in halophytes? IV. How do halophytes avoid Na\(^+\) cytosolic toxicity and enhance K\(^+\) retention ability? V. How can halophytes retain more K\(^+\)? Do they spend much energy (H\(^+\)-ATPase activity) to retain K\(^+\) as compared with spinach? VI. What could be possible players behind reduced K\(^+\)-efflux from leaf mesophyll and roots in quinoa in relation to acclimation? This work showed that salinity application arrested plant growth in a highly tissue- and treatment-specific manner and was more severe in glycophytic spinach plants however quinoa was able to withstand salinity stress and produced relative higher plant biomass even at sea level saline conditions (500mM NaCl). Analysis of shoot and xylem sap Na\(^+\) and K\(^+\) contents have revealed the key factor determining differential salinity tolerance between quinoa and spinach species was shoot K\(^+\) (not Na\(^+\)) content and kinetics of xylem ion loading suggested that quinoa species actively load and used Na\(^+\) for osmotic adjustment in shoot to avoid energy expensive synthesis of organic osmolytes. To further gain insight into such whole-plant observations, kinetics of K\(^+\), H\(^+\), Ca\(^{2+}\) flux responses from leaf mesophyll in were measured using non-invasive ion flux measuring MIFE technique in response to salinity stress and H\(_2\)O\(_2\) stress. Moreover, laser microscope confocal imaging technique was used to measure the cytosolic and vacuolar intensities of the fluorescent signals of K\(^+\), Na\(^+\) and Ca\(^{2+}\) from different root zones in response to salinity stress and H\(_2\)O\(_2\)2 stress. It was also observed that mesophyll cells in glycophytic spinach lost 2 to 6-fold more K\(^+\) compared with its halophytic quinoa counterpart. Treatment with NaCl resulted in significant increase in a transient H\(^+\)-efflux in the leaf mesophyll in spinach, suggesting that spinach spent more ATP to up-regulate H\(^+\)-ATPase activity while quinoa avoid this mechanism to use same energy in defence system, consistent with recently suggested concept of the 'metabolic switch‘. Among root zones, NaCl- and ROS- induced K\(^+\)-efflux was more pronounced in the root apex while mature zone showed relatively higher K\(^+\) retention, especially in quinoa. This differential sensitivity between different root zones was specifically originated from a 10-folds difference in K\(^+\)-efflux between the mature zone and the apical region (much poorer in the root apex) of the root. Three factors behind this poor K\(^+\) retention ability were: (1) an intrinsically lower H\(^+\)-ATPase activity in the root apex; (2) greater salt-induced membrane depolarization and (3) a higher ROS production under NaCl and a larger density of ROS-activated cation currents in root. Moreover ROS (H\(_2\)O\(_2\)) production was increased with time in all root zones in both species and accompanied with cytosolic Ca\(^{2+}\) elevation in quinoa, suggesting that quinoa (halophytic species) used ROS as a signalling moiety in stress adaptation. Elevation in cytosolic Ca\(^{2+}\) reduced cytosolic Na\(^+\), possibly by SOS1 pathway. Both species showed tremendous K\(^+\)-efflux in response to •OH and O\(_2\)\(^-\) radical but Ca\(^{2+}\)\) flux patterns revealed different results. In response to O\(_2\)\(^-\), a net Ca\(^{2+}\)-efflux was observed while in response to •OH, a net Ca\(^{2+}\)-influx was noted, suggesting halophytes may use ROS specific Ca\(^{2+}\) signatures to activate stress adaptation process. Moreover, halophytes (or at least quinoa) may use Ca\(^{2+}\)-efflux system to restore basal cytosolic Ca\(^{2+}\) level upon O\(_2\)\(^-\) treatment. Under long term salinity conditions, quinoa grown under higher NaCl level (300mM NaCl) showed much reduced responses to external H\(_2\)O\(_2\), suggesting desensitization of K\(^+\)-permeable ion channels to ROS. Moreover, quinoa showed strongest Ca\(^{2+}\) flux response to H\(_2\)O\(_2\) during acclimation, suggesting the important role of ROS-induced cytosolic Ca\(^{2+}\) elevation in stress signalling and adaptive cascade. Spinach was less efficient in doing so, thus showed massive K\(^+\)-efflux and reduced K\(^+\) retention ability. In conclusion, results from current work showed that NaCl and ROS stress induced massive K\(^+\)\(^+\) loss. This loss was highly tissue-specific and more pronounced in glycophytic spinach plants. Several mechanisms were highlighted in this work behind such response in quinoa (i) higher vacuolar Na\(^+\) sequestration ability in roots, thus reduced Na\(^+\) cytosolic toxicity. (ii) It can employ H\(_2\)O\(_2\) to activate stress signalling cascades, (iii) higher K\(^+\) retention in leaf mesophyll was strongly correlated with plant biomass, SPAD and stomatal conductance, (iv) The results obtained indicated a major difference in distribution of energy between "metabolic" and "defence" pools. Future research should focus on the difference and mechanisms of the regulation of H\(^+\)-ATPase activity between halophytes and glycophytes, and the role of ROS in this process, (V) during acclimation, quinoa showed relatively more Na\(^+\) accumulation (based on coroNa green fluorescence signal) than spinach. Thus, it will be of a significant importance to reveal the contribution of numerous components (e.g. tonoplastic NHX, or FV/SV channels) towards vacuolar Na\(^+\) sequestration. One of most important discoveries in this study was the identification of the electrophysiological role of ROS specific Ca\(^{2+}\) signatures in the regulation of K\(^+\) homeostasis and stress adaptation. The use of some techniques such patch clamp and CRISPR/CAS 9 will help to reveal the molecular identity of different ion transporters in quinoa and spinach in response to different ROS especially to •OH and O\(_2\)\(^-\) radical at transcriptional and post transcriptional levels, to further understand the molecular basis of the observed physiological salinity tolerance mechanisms.

Soil salinity is predicted to become more severe and widespread adding more challenges for sustainable crops production worldwide. Salinity stress tolerance is a complex trait, both physiologically and genetically, and halophytes represent a rich resource for understanding different strategies used by plants to cope with saline condition. While many previous studies explored various physiological and genetic aspects of salinity tolerance in halophytes, most of them were focused on annual species. However, this knowledge is hardly applicable to perennial crops that need to maintain capability of growth and development for many successive seasons and, therefore, cannot rely on the strategy associated with salt exclusion from uptake. Succulence is one of that important strategies by which perennial halophytes conserve water and dilute immense salt concentration in their susceptible tissues. Succulent halophytes can be used as convenient models for understanding the mechanistic basis of plant adaptation to salt stress. This understanding is important for improving water use efficiency (WUE) in crop plants that undergo a salt-induced physiological drought under saline condition. How a perennial succulent halophyte then will manage this issue, morphologically, anatomically, and physiologically? The major aim of this study was understanding the mechanistic basis of long-term salt tolerance strategy employed by the succulent perennial halophyte, Sarcocornia quinqueflora. The following specific objectives were addressed: • To quantify the relative contribution of organic and inorganic osmolytes towards osmotic adjustment and turgor-induced growth in succulent shoots. • To link osmotic adjustment and stomatal characteristics with salinity stress tolerance and water use efficiency (WUE) of succulent stems. • To dissect specific morphological and anatomical features of succulent shoots and investigate their potential contribution toward their salt tolerance strategy. • To investigate the causal relationship between salinity and oxidative stress tolerance in succulent stems of this perennial halophyte. • To understand the role of ROS signalling in regulating activity of membrane transporters mediating ion homeostasis in this plant. The whole-plant responses of S. quinqueflora to soil salinity was investigated using a broad range of salinity (0 – 1000 mM NaCl). S. quinqueflora showed the typical growth response of a succulent halophyte, with maximum growth obtained at 200 mM NaCl, while growth was reduced at concentrations exceeding 600 mM NaCl. Elevated salinity levels up to 400 mM NaCl largely promoted dry matter yield, succulence, shoot surface area and stomatal characteristics. The osmolality of shoot sap increased as salinity increased from 0 to 1000 mM NaCl. Osmotic adjustment in succulent shoot was achieved, even at the highest salinity levels, by a massive accumulation of inorganic ions, with Na\(^+\) and Cl\(_-\) contributing ~85 % of its osmolality, while organic compatible solutes and K\(^+\) were responsible for only ~15%. These facts suggest that cell expansion growth in this species is relying heavily upon the coordination between the cell vacuolar sequestration capacity (VSC) of Na\(^+\) and Cl\(_-\) and the extent of the cell wall extensibility (CWE). The maximum VSC of Na\(^+\) and Cl\(_-\) is required to keep the cytosol toxic-free and to lower the cell osmotic potential which in turn elevates turgor pressure (hence, succulence). Carbohydrates were not reduced at high salinity compared to plants at optimal conditions, implying that growth retardation at severe salt dosages was attributed to limitations in VSC rather than inadequate photosynthesis and substrate limitation. The control of stomatal operation seems to be critical for S. quinqueflora performance under saline conditions. The fact that transpiration rate was maintained unchanged over the very broad (200 to 1000 mM NaCl) range of salinities, despite the large difference in stomatal density and aperture size, suggest superior plant’s ability to optimize WUE and balance water loss with CO\(^2\) assimilation. Importantly, shoot K\(^+\) was unchanged across the entire range of salinity treatments (200 – 1000 mM NaCl) and positively correlated with transpiration rate (R=0.98), indicative of the likely role of K\(^+\) in controlling stomatal transpiration. Therefore, the superior salt tolerance of succulent shoots is achieved by effective reliance on Na\(^+\) and Cl\(_-\) accumulation for osmoregulation and turgor-induced growth and maintaining K\(^+\) threshold levels for efficient stomatal operation. The leafy stems of S. quinqueflora shoot are composed of assimilating oblong internodes (beads), representing the main photosynthetic organ. Anatomically, the plant develops two distinct layers: an endodermis-like layer (ED), and an additional internal photosynthetic layer (IP). We followed the morpho-anatomical changes in S. quinqueflora leafy stem under varied salt levels in beads of different ages to assess this biological barrier (ED) from non-senescent to senescent stages. Our findings revealed that S. quinqueflora utilizes senescence process to discard excess salt being accumulated in outer tissues of their leafy stems (salt shedding). The development of ED and IP appears to be important to enable this process and determines the whole salt-coping strategy for the plant. Elevated salinity leads to an accelerated development of the ED. In addition, its development strongly affected ion distribution between outer (senescent) and inner (non-senescent) tissues. A positive correlation between the ratio of ED to a bead diameter and the outer to inner concentration of Na\(^+\) was observed. These ratios were highest in older (basipetally-located) beads and progressively decreased towards the tip. The Na\(^+\)/K\(^+\) ratio was substantially higher in outer region compared to the inner one. In addition, different leafy stem regions had showed that a ratio for any given NaCl concentration was the lowest for the top stem region with maximum impact being observed in the bottom stem region. High K\(^+\) content in the tip of the leafy stem also drives the expansion growth of the plant. Accordingly, the top leafy stem region kept Na\(^+\)/K\(^+\) ratio at the constant level regardless of external salt concentration. This correlates with the plant’s ability to grow/survival even at the highest (1000 mM) NaCl concentration tested. Furthermore, the Na\(^+\)/K\(^+\) ratio in inner tissues of bottom beads at highest salinity treatments (800 and 1000 mM NaCl) that showed clear senescence symptoms was ~1.0, indicative of complete separation of the outer and inner tissues at late developmental stage due to the presence of the fully suberized endodermis multilayer (ED). Accordingly, a model was suggested for bead tolerance strategy at early, middle, and late stages under saline conditions involving a suggested role for the internal photosynthetic layer (IP). Two main features are envisaged. The first one is an accelerated development of a biological barrier (ED) which, in its earlier developmental stage, control water and solute movements to the water storage tissue (WS) and determine Na\(^+\)/K\(^+\) ratio in both senescing and non-senescing tissues. At a later stage, this tissue becomes multilayered and highly suberized, thus protecting the internal tissues for many successive seasons. The second feature is a difference in the energy supply (source) for the outer and the inner tissues. The outer layer (palisade tissue) will fuel the water storage cells to mediate Na\(^+\) and Cl\(_-\) sequestration in their vacuoles while the internal photosynthetic layer operates as an energy provider for young beads and roots. The ability of S. quinqueflora to extend growth for many successive seasons under saline condition also implies an efficient and well-regulated ROS-scavenging and signalling systems at both organ and tissue levels. The causal relationship between activity of key membrane transporters involved in maintaining plant ionic homeostasis and oxidative stress tolerance in succulent stems was investigated. S. quinqueflora possess a well-developed antioxidant system, including betalains, ascorbic acid, α-tocopherol, polyphenols, and flavonoids. The optimal growth level (200 mM NaCl) had the lowest antioxidants concentrations, indicating its capability to maintain favorable ROS levels at moderate salinity. Moreover, both sugars and ROS were positively correlated suggesting that growth/biomass reduction at these conditions probably mediated by re-allocation of the energy pool towards sugar production to operate as non-enzymatic antioxidant scavengers. Also, a negative correlation was recorded between plant biomass and antioxidant activity, implying that the latter should be treated as a damage control mechanism rather than a trait that confers salinity tolerance. In addition, ROS-induced net K\(^+\) and Ca\(^{2+}\) fluxes were measured from various bead tissues being located either in outer (palisade tissue, Pa; and water storage tissue, WS), or inner leafy stem part (internal photosynthetic layer, IP; and stele (vascular cylinder) parenchyma, SP) in addition to the in-between barrier (ED). Two types of ROS were used to induce ion fluxes, hydroxyl radical (OH\(^•\)) and hydrogen peroxide (H\(_2\)O\(_2\)). The flux responses to oxidative stresses were governed largely by (1) the type of ROS applied (OH\(^•\) or H\(_2\)O\(_2\)); (2) the tissue-specific origin and function (parenchymatic or chlorenchymatic); and (3) the tissue location in either outer (senescent) or inner (non-senescent) bead part. ROS-induced K\(^+\) effluxes that were highly tissue-specific, with inner tissues of the plant beads being more sensitive to ROS applied, as compared to the plant outer parts. The magnitude of ion flux response to OH\(^•\) was higher compared to response to H\(_2\)O\(_2\). The ability to retain cellular K\(^+\) under OH\(^•\) stress varied between different bead tissues and was ranked in the following descending order: WS > Pa > IP > SP. Hydroxyl radicals (OH\(^•\)) always led to Ca\(^{2+}\) influx with all bead tissues, while treatment with H\(_2\)O\(_2\) induced opposite Ca\(^{2+}\) flux responses: the Ca\(^{2+}\) influx was recorded only from the photosynthetically active tissues (Pa and IP), while the parenchyma tissues (WS and SP) had transient Ca\(^{2+}\) efflux. These results indicate high tissue-specific ability of S. quinqueflora to maintain their ion homeostasis upon exposure to ROS; a feature that is mainly determined by the developed suberized barrier (ED). In conclusion, the salt tolerance strategy of perennial S. quinqueflora halophyte relies on a set of morphological, anatomical, and physiological traits, enabling the ability to extend growth for many successive seasons. This work emphasized the importance and a requirement to engage specific anatomical features in studying salinity tolerance mechanisms of halophytes, in addition to the traditional whole-plant phenotyping. To the best of our knowledge, none of the previous works provided a causal link between salinity-stress tolerance and ROS activation of ion transporters mediating ionic homeostasis in S. quinqueflora succulent tissues. This gap in our knowledge was filled by the current study. The future work should be focused on comparing tissue-specificity of long-term salinity tolerance mechanisms in other perennial succulent and non-succulent halophytes.

碩士
國立陽明大學
生物化學研究所
89
Abstract The covalent modification of DNA provides a direct and powerful mechanism to regulate gene expression. DNA methylation in eukaryotes involves addition of a methyl group to the carbon 5 position of the cytosine ring. This reaction is catalyzed by DNA methyltransferase in the context of the sequence 5’-CG-3’, which is also referred to as a CpG dinucleotide. It’s the most common eukaryotic DNA modification and is one of the many epigenetic mechanism. This special DNA marking has been closely associated with controlling the expression of housekeeping genes and possibly also tissue-specific genes, as well as several important cellular functions such as X chromosome inactivation and genomic imprinting , as well as mutagenesis and tumorigenesis. Unfortunately, the ongoing genomic sequencing projects are unable to provide information on this important area of gene regulation and all current investigations on changes in methylation status have been limited to known genes. To initiate a genome-wide search for differentially methylation pattern in a tissue- or development-specific manner, we developed a methylation-sensitive Alu-PCR technique to examine the mathylated DNA sequence in different mouse tissues at different stage of development. Using this technique we have cloned and sequenced several methylated sequences, some tissue-specific. We have then examined in detail the methylation status of each CpG sites in a 6 kb region surrounding the s27-p53 gene using bisulfite DNA sequencing technique. The results revealed an interesting methylation transition boundary that demarcated the full methylated region from the unmethylation region and a tissue-specific methylated site in the transition boundary. Currently, we are developing a more global methodology for fishing out tissue- and developmental stage-specific methylated sites.

碩士
國立海洋大學
食品科學系
87
Zinc concentrations in most tissues of animals and fishes were around 10~50 ppm; however, those in the digestive tract tissue of common carp were found to be extraordinarily high, being 300-500 ppm. It is also known that the nuclei/cell debris fraction was the major fraction responsible for these high concentration of zinc. In order to survey the specificity and affinity of zinc binding substance(s) in the digestive tissue of common carp, 65Zn was added to the Debris Fraction. Binding increased linearly with increase amounts of 65Zn. The Debris Fraction in the digestive tract tissue of common carp showed specific, saturable binding with 65Zn. Binding of 65Zn to Debris Fraction is fast and reversible. Other metal ions (including Fe, Cu, Mn, Mg, Ca, Ni, Hg, Cd and Co) all fail to displace zinc at 0.06 mM concentration. These attributes are consistent with the presence of “specific zinc binding substance(s)” in the digestive tract tissue of common carp. The specific binding of 65Zn and Debris Fraction is eliminated by protease treatment. Saturation analyses of the binding of 65Zn to digestive tract tissue Debris Fraction of common carp, and Scatchard plot for each saturation analysis were presented. The Kd and Nmax of zinc specific binding protein of common carp derived from these plot were 0.55~8.06 M and 0.14~ 2.35 nmol/g fresh tissue respectively. Therefore, it is concluded that there is a zinc specific binding protein existed in the Debris Fraction of digestive tract tissue of common carp. The saturation analyses of the binding of 65Zn to the digestive tract tissue Debris Fraction of of grass carp, silver carp and tilapia were also performed. It was found that in these fishes, there were also existed the zinc specific binding protein with Kd of 0.12~ 0.78 M. But Nmax of these fishes were 3 to 16 times lower than that of common carp. Obviously the reason why common carp has higher zinc concentration in its digestive tract tissue is due its higher concentration of the zinc specific binding protein.

Wheat and barley are ranked the second and fifth most important crops in terms of dry matter production. Both of them are classified as glycophytes, and their production is strongly affected by soil salinity. Thus, given the extent of land salinization in the world and predicted population growth to 9.3 billion by 2050, creating salt tolerant wheat and barley germplasm remains one of highest priorities for breeders. Salinity tolerance is a complex physiological trait composed of numerous sub-traits controlled by multiple regulatory pathways. Until now, most studies were focused on traits related to sodium, such as `Na^+` exclusion from uptake, control of xylem `Na^+` loading, `Na^+` retrieval from the shoot or vacuolar Na+ sequestration. However, it is not `Na^+` but the `K^+``/Na^+` ratio in the cytosol that ultimately determines plant performance under saline conditions. In recent years, `K^+` retention in root mature zone has emerged as an important component of salt tolerance mechanisms in many plant species. However, whether the importance of cell’s ability to maintain `K^+` in plant overall salt tolerance can be extrapolated to other root zones or tissues (e.g. leaves) remained obscure prior to this work. Also elusive remained the essentiality of root `Na^+` exclusion and vacuolar `Na^+` sequestration in various root tissues. Furthermore, the relative contribution of each of the above salt tolerant mechanisms towards the overall salinity tolerance remained unclear, especially at the tissue specific level.

Mgr. Daniela Fornuskova PhD thesis Biochemical and molecular studies of cytochrome c oxidase and ATP synthase deficiencies ABSTRACT The mammalian organism fully depends on the oxidative phosphorylation system (OXPHOS) as the major energy (ATP) producer of the cell. Disturbances of OXPHOS may be caused by mutations in either mitochondrial DNA (mtDNA) or nuclear DNA (nDNA). One part of the thesis is focused on the role of early and late assembled nuclear-encoded structural subunits of cytochrome c oxidase (CcO) as well as Oxa1l, the human homologue of the yeast mitochondrial Oxa1 translocase, in the biogenesis and function of the human CcO complex using stable RNA interference of COX4, COX5A, COX6A1 and OXA1L, as well as expression of epitope-tagged Cox6a, Cox7a and Cox7b, in HEK (human embryonic kidney)- 293 cells. Our results indicate that, whereas nuclear- encoded CcO subunits Cox4 and Cox5a are required for the assembly of the functional CcO complex, the Cox6a subunit is required for the overall stability of the holoenzyme. In OXA1L knockdown HEK-293 cells, intriguingly, CcO activity and holoenzyme content were unaffected, although the inactivation of OXA1 in yeast was shown to cause complete absence of CcO activity. In addition, we compared OXPHOS protein deficiency patterns in mitochondria from skeletal...

A doubly resonant cavity was used to search for nonlinear radiofrequency (RF) energy conversion in a range of biological preparations, thereby testing the hypothesis that living tissue can demodulate RF carriers and generate baseband signals. The samples comprised high-density cell suspensions (human lymphocytes and mouse bone marrow cells); adherent cells (IMR-32 human neuroblastoma, G361 human melanoma, HF-19 human fibroblasts, N2a murine neuroblastoma (differentiated and non-differentiated) and Chinese hamster ovary (CHO) cells) and thin sections or slices of mouse tissues (brain, kidney, muscle, liver, spleen, testis, heart and diaphragm). Viable and non-viable (heat killed or metabolically impaired) samples were tested. Over 500 cell and tissue samples were placed within the cavity, exposed to continuous wave (CW) fields at the resonant frequency (f) of the loaded cavity (near 883 MHz) using input powers of 0.1 or 1 mW, and monitored for second harmonic generation by inspection of the output at 2f. Unwanted signals were minimised using low pass filters (/= 1 GHz) at the output from, the cavity. A tuned low noise amplifier allowed detection of second harmonic signals above a noise floor as low as -169 dBm. No consistent second harmonic of the incident CW signals was detected. Therefore, these results do not support the hypothesis that living cells can demodulate RF energy, since second harmonic generation is the necessary and sufficient condition for demodulation.

碩士
元智大學
資訊工程學系
104
S-palmitoylation, the covalent attachment of 16-carbon palmitic acids to a cysteine residue via a thioester linkage, is an important reversible lipid modification (PTM) that regulates protein trafficking, protein-protein interaction. It also related to diversity of physiological and biological process, etc… However, the substrate specificity of cysteine S-palmitoylation remains unknown. Thus, finding the effective computational method to predict S-palmitoylation sites is urgent demand in bioinformatics. Based on total of 710 experimentally verified S-palmitoylation sites in the liver tissues were collected from UniProtKB, Forrester MT et al and Yang W et al papers. This study presents a recursively statistical method to identify conserved substrate motifs for S-palmitoylation in the liver tissues. Statistical significance Support vector machine (SVM) was applied to construct predictive model learned from verified substrate motifs. The evaluation of five-fold cross-validation indicated that the model trained with identified motifs were effective in identification of S-palmitoylation sites with an enhanced sensitivity, specificity, and accuracy. It also provided a promising performance in an independent testing set. The correct identification of previous report S-palmitoylation sites of mouse protein demonstrated the effectiveness of the proposed method and it indicated that the proposed method could be a practicable ways of conducting primary analyses of proteins S-palmitoylation in the liver tissues. Finally, the constructed models have been implemented as a web-based system freely available at http://csb.cse.yzu.edu.tw/MDDPalm/ for identifying uncharacterized S-palmitoylation sites on the protein sequences.