Dissertations / Theses on the topic 'TRPS1 gene'

The release of vasopressin (VP) from magnocellular neurosecretory cells (MNCs) of the supraoptic (SON) and paraventricular (PVN) nuclei is essential to hydromineral homeostasis. This release is controlled by several physiological stimuli, including changes in the osmotic pressure of the extracellular fluid, and in core body temperature. The osmotic control of VP release is mediated by specific and highly sensitive 'osmoreceptors'. Indeed, VP-releasing neurons in the SON are directly osmosensitive, and this osmosensitivity is mediated by stretch-inhibited cation channels. The molecular identity of these channels, however, remains unknown. The thermal control of VP release, on the other hand, is largely unexplained. In this thesis, we demonstrate that the mouse SON is a valid model for investigating the molecular basis of osmotransduction. We show that hyperosmotically-induced increases in membrane conductance are blocked by ruthenium red (RR), a non selective blocker of TRPV channels. In addition, SON neurons were found to express an N-terminal splice variant of TRPV1, but not full-length TRPV1. Unlike their wild-type counterparts, SON neurons in Trpv1 knockout (Trpv1-/-) mice could not generate RR-sensitive increases in membrane conductance and depolarizing potentials in response to hyperosmotic stimulation. Moreover, Trpv1-/-mice showed a pronounced serum hyperosmolality under basal conditions and severely compromised VP responses to osmotic stimulation in vivo. These results suggest that the Trpv1 gene may encode a central component of the osmoreceptor. Furthermore, we demonstrate that VP neurons are intrinsically thermosensitive. In these neurons, thermal stimuli spanning core body temperatures activate a RR-sensitive non selective cation current. Interestingly, VP neurons isolated from Trpv1 -/-mice are significantly less thermosensitive. These results suggest that channels encoded by the Trpv1 gene can confer thermosensitivity in the physiological range. Overall, these data suggest that products of the Trpv1 gene in VP neurons may represent a molecular point of convergence for the detection of osmotic and thermal stimuli.

La cathepsine D est une aspartyl protéase lysosomale surexprimée et hypersécrétée par les cellules épithéliales cancéreuses mammaires. C'est un marqueur de mauvais pronostic du cancer du sein. Elle stimule la prolifération des cellules cancéreuses, la croissance invasive des fibroblastes et la formation des métastases. Les travaux de l'équipe ont montré qu'elle peut agir indépendamment de son activité catalytique par interaction protéique. Le répresseur transcriptionnel Tricho-Rhino-Phalangeal Syndrome type 1, TRPS1, a été identifié comme un partenaire potentiel de la cathepsine D. Différentes études indiquent que des cystéines cathepsines peuvent être localisées au noyau et être protéolytiquement actives. Par exemple, la cystéine cathepsine L agit par protéolyse limitée sur le facteur de transcription CDP/Cux et sur l'histone H3 lorsqu'elle est localisée au noyau.Dans cette thèse nous avons étudié le rôle de la cathepsine D nucléaire dans des cellules cancéreuses mammaires. Nos résultats indiquent que la cathepsine D, comme TRPS1, est localisée au noyau et est associée à la chromatine dans les cellules positives aux récepteurs aux œstrogènes. De plus elle interagit de manière directe et endogène avec TRPS1 et participe à la régulation transcriptionnelle de PTHrP (parathyroïd hormone-related protein) un gène cible de TRPS1. Finalement nous avons identifié de nouveaux gènes co-régulés par TRPS1 et la cathepsine D dans le cancer du sein montrant que leur action n'est pas limitée à PTHrP. L'ensemble de ces résultats suggère que la cathepsine D est la première cathepsine identifiée comme un co-facteur transcriptionnel et que son rôle dans le cancer pourrait impliquer, en plus de ses activités extracellulaires, ses activités nucléaires
Cathepsin D is a lysosomal aspartyl protease which is overexpressed and hyper-secreted by epithelial breast cancer cells. This is a poor prognosis factor in breast cancer. It stimulates cancer cell proliferation and metastasis formation. Team works have shown it can acts in an independent manner of its catalytic activity by protein interactions. The transcriptional repressor trichorhinophalangeal syndrome type 1 protein, TRPS1, has been identified as a new potential partner of Cathepsin D. Several studies indicate that cystein cathepsins can be localized in nucleus and are proteolytically actives. For example, the cystein Cathepsin L acts by limited proteolysis of the CDP/Cux transcription factor and histone H3 when located to the nucleus.During this thesis, we studied the role of nuclear Cathepsin D in breast cancer cells. Our results indicate that Cathepsin D, as TRPS1, is localized in nucleus and is associated with chromatin in estrogen-receptor positive breast cancer cells. Furthermore it interacts in a direct and endogenous manner with TRPS1 and participates to the transcriptional repression of PTHrP, parathyroïd hormone-related protein, a TRPS1 target gene. Finally, we identified new co-regulated genes by TRPS1 and Cathepsin D in breast cancer showing their action is not limited to PTHrP.Together, our results suggest that Cathepsin D is the first cathepsin identified as a transcriptional co-repressor and that its role in cancer may involve, in addition to its extracellular activities, its nuclear activities

A adaptação temporal é fundamental para a sobrevivência de espécies que precisam coordenar sua fisiologia e comportamentos ajustando-se a sinais externos. Ritmos biológicos não são simplesmente uma resposta às mudanças de 24 horas no ambiente físico impostas pela rotação da Terra sobre o seu próprio eixo, ao contrário, surgem a partir de um sistema de cronometragem endógeno. No teleósteo Danio rerio, ainda não foi identificada a presença de uma região que atue como relógio central; alguns estudos têm evidenciado a existência de células e tecidos que contêm relógios circadianos autônomos, fotossensíveis, comprovando um outro tipo de regulação dos ritmos circadianos onde a percepção do ambiente e o ajuste do período circadiano são efetivados diretamente em nível celular. As consequências deletérias do aumento da temperatura são impedidas, em certa medida, por uma resposta adaptativa que assegura a sobrevivência celular na presença de calor. Esta via de sobrevivência ativada por calor, conhecida como resposta ao choque térmico, é composta por uma cascata de eventos que conduzem à indução de proteínas de choque térmico (HSPs) que minimizam a lesão celular aguda. Acredita-se que os sistemas de percepção dos ciclos diários de temperatura e luminosidade sofreram as mesmas pressões seletivas em sua co-evolução, resultando em sua associação. As bases da sensação térmica estão em um grupo de canais altamente conservados, presente em todos os metazoários estudados até o momento e envolvidos em uma série de modalidades sensoriais, os canais de potencial receptor transiente (TRP); os que respondem a estímulos térmicos foram agrupados em uma subfamília e denominados termoTRPs. O objetivo deste trabalho foi investigar a influência do pulso de temperatura (33 ºC) na expressão de genes de relógio e de proteínas de choque térmico, bem como o papel do canal TRPV1, em células embrionárias de blástula de Danio rerio, denominadas ZEM-2S, submetidas a escuro constante (DD) ou ciclos claro-escuro (LD 12:12). Através de PCR em tempo real (quantitativo) demonstrou-se que as células ZEM-2S expressam os genes dos seguintes canais TRP: trpA1a, trpA1b, trpV1/2, trpV4, trpC6, trpM2, trpM4a, trpM4b/c e trpM5. Após um pulso de temperatura, observou-se um aumento no transcrito de hsp90 aa1 em células mantidas tanto em DD como em LD, sendo a expressão de hsp90 aa1 em LD, no ponto uma hora, duas vezes menor quando comparada a sua expressão no mesmo ponto temporal em DD. O pulso de temperatura não promoveu efeito em nenhum dos genes do relógio estudados (bmal1a, bmal1, bmal2, cry1a, cry1b, per1, per2) quando as células foram mantidas em DD. Porém, o transcrito de per2 aumentou em resposta ao pulso de temperatura quando as células foram sincronizadas pelos ciclos claro-escuro. A inibição do canal TRPV1 não alterou o efeito induzido pelo pulso de temperatura na expressão do gene hsp90 aa1 em células ZEM-2S mantidas em DD. Por outro lado, nossos dados permitem afirmar que o mesmo participa parcialmente na indução do aumento da expressão do gene per2 pelo estímulo térmico em células mantidas em LD, tendo em vista um decaimento significativo na resposta deste gene. Os dados obtidos neste trabalho abrem uma nova perspectiva sobre a investigação da relação temperatura e genes de relógio, colocando um novo “ator” na regulação deste fenômeno: o canal TRPV1
Temporal adaptation is essential for the survival of species which need to coordinately adjust their physiology and behavior to external signals. Biological rhythms are not just a response to the 24 hour changes in the physical environment imposed by the rotation of the Earth around its own axis, but they arise from an endogenous timing system. In the teleost Danio rerio, there has not been identified so far a region in the nervous system that could act as a central clock; some studies have reported the existence of cells and tissues which contain photosensitive, autonomous circadian clocks, demonstrating the existence of another type of circadian rhythm regulation in which environment perception and entrainment of the circadian period are directly effected at cell level. The deleterious consequences of temperature increase are prevented by an adaptive response which assures cell survival in the presence of heat. This survival pathway activated by heat, known as response to temperature shock, is signaled by a cascade of events leading to the induction of thermal shock proteins (HSPs) which attenuate the acute cell lesion. It is believed that the systems perceiving temperature and light daily cycles were subject to the same selective pressures during their co-evolution, resulting in their association. The base of thermal sensation is a family of highly conserved channels, present in all metazoans studied to date, and involved in a variety of sensorial modalities, the transient receptor potential channels (TRP); those responding to thermal stimuli were grouped in a sub-family named thermo-TRPs. The aim of this work was to investigate the influence of a temperature pulse (33 ºC) on the expression of clock and heat shock protein genes, as well as the role of TRPV1 channel, in blastula embryonic cells of Danio rerio, named ZEM-2S, subject to constant dark (DD) or light-dark cycles (LD). Using quantitative PCR, we demonstrated that ZEM-2S cells express genes for the following TRP channels: trpA1a, trpA1b, trpV1/2, trpV4, trpC6, trpM2, trpM4a, trpM4b/c and trpM5. After the pulse of temperature, we observed an increase of hsp90 aa1 transcripts in DD as well as in LD; hsp90 aa1 expression 1 hour after the stimulus was two-fold lower in LD than in DD. Temperature pulse did not affect the expression of any of the studied clock genes (bmal1a, bmal1, bmal2, cry1a, cry1b, per1, per2), when the cells were kept in DD. However, per2 transcript increased in response to the temperature pulse when the cells were synchronized by light-dark cycles. Inhibition of TRPV1 channel did not change the effect induced by the temperature pulse on hsp90 aa1 in ZEM-2S cells kept in DD. On the other hand, our data suggest that this channel participates, at least partially, in the temperature-induced increase of per2 in cells maintained in LD, as indicated by the significant decay observed in the gene response in the presence of the inhibitor. Our results open new investigative perspective about the relationship between temperature and clock genes, placing a new “actor” in the regulation of the phenomenon: the TRPV1 channel

Trichorhinophalangeal syndrome is a malformation syndrome characterized by craniofacial and skeletal abnormalities and is inherited in an autosomal dominant manner. We distinguish free subtypes on clinical and molecular level - TRPS I, TRPS II, TRPS III. All TRPS patients have sparse hair, a pear-shaped nose, a long flat philtrum, a thin upper lip and protruding ears. Skeletal abnormalities include cone-shaped epiphyses at the phalanges, hip malformations and short stature are present. The subgroups TRPS I and TRPS III are result of the mutated TRPS1 gene, which is maped into the 8q24 region. This gene is situated proximal of the EXT1 gene, both genes are affected in a subgroup of patients with TRPS II. These patients suffer more from multiple (cartilaginous) exostoses and mental retardation. In this work we performed molecular genetic analysis of a sample of 16 patients, 8 probands showed a TRPS phenotype and 8 probands had only isolated exostoses. The peripheral venous blood of patients was used to gain purified DNA, which was subsequently used to investigate the chromosome 8q24 region using MLPA ("multiplex ligation-dependent probe amplification"). This analysis revealed a deletion in 1 TRPS patient and 1 patient with exostoses. Sequencing of the TRPS1 gene coding exons in remaining 7 TRPS...

博士
國立陽明大學
藥理學研究所
105
Notch1 signaling involves in several physiological and pathological cellular processes, including proliferation, apoptosis, stem cell maintenance and regulation of erythroid and megakaryocyte differentiation. Notch1 intracellular domain (N1IC), the activated form of Notch1, induced TRPA1 expression. TRPA1 is a non-selective calcium channel. Inflammatory cytokines enhance TRPA1 expression, and TRPA1 activation induces neurotransmitter release. Inflammatory cytokines suppress erythroid differentiation and result in anemia. The roles of TRPA1 in erythroid/megakaryocyte differentiation are poorly understood. Herein, the data indicated that N1IC activated TRPA1 promoter in a CBF1-independent manner. N1IC enhanced TRPA1 promoter activity via Ets-1, and both of them bound to TRPA1 promoter. N1IC modulated TRPA1 promoter depend on promoter methylation, and N1IC and Ets-1 inhibited DNA methyltransferase 3B (DNMT3B) expression synergistically. TRPA1 decreased hemin-induced erythroid differentiation of K562 and HEL cells. TRPA1 agonist AITC suppressed erythroid differentiation and increased phosphorylation of ERK in K562 and HEL cells, which were reversed by TRPA1 antagonist or EGTA pretreatment. TRPA1 mediated N1IC- or Ets-1- restrained erythroid differentiation. TRPA1 improved PMA-induced megakaryocyte differentiation, and the levels of megakaryocytic markers were increased. Notch1 receptor or Ets-1 knockdown reduced me megakaryocyte differentiation, which could be restored by TRPA1 expression. Knockdown of DNMT3B increased TRPA1 level, inhibited erythroid differentiation as well as promoted megakaryocyte differentiation. Moreover, TRPA1 inhibition enhanced migration, invasion and colony forming abilities of K562 cells. These results demonstrate that N1IC-induced TRPA1 play a critical role in the regulation of erythroid and megakaryocyte differentiation.

碩士
國立中央大學
生命科學系
103
Autoimmune diseases (ADs) are chronic conditions initiated by the loss of immunological tolerance to self-antigens and represent a heterogeneous group of disorders that afflict specific target organs or multiple organ systems. Rheumatoid arthritis (RA), one of ADs, can be defined as a chronic, progressive, systemic, inflammatory disease of connective tissues characterized by the severe destruction in synovial joints. RA involves primary tissue inflammation rather than joint degeneration. Although inflammation contributes to pain in RA, however, it may not be the only factor. For some patients, pain is not improved despite of the treatment with anti-inflammatory disease-modifying anti-rheumatic drugs. The aim of this thesis is to identify novel genes that are involved in RA-induced pain. Given that joint inflammation is usually accompanied by tissue acidosis, proton-sensing receptors could be the candidates for RA. Two knockout mice (ASIC3-/- & TRPV1-/-) were generated for the RA study. After intra-articular injection into a stifle joint with complete Freund’s adjuvant (CFA) once per week for 4 weeks, long-term mechanical hyperalgesia was developed in wildtype and knockout mice. Both of ASIC3-/- & TRPV1-/- reduced RA-induced mechanical hyperalgesia in the late phase.

碩士
國立中央大學
生命科學系
103
When tissues are injured, damaged cells release multiple chemical mediators, such as protons, PGE2, growth factors, cytokines, kinins and so on. These inflammatory mediators activate protein kinase A (PKA) or protein kinase Cε (PKCε) to sensitize pain-related nociceptor afferents, leading to inflammatory pain. Previous studies have been demonstrated that the transition from acute to chronic inflammatory pain requires the switch of PKA and PKCε-dependency. However, it remains unclear which genes regulate the switch of kinase dependence. Since proton is the decisive factor to induce inflammatory pain, proton-sensing receptors could play an important role in the switch of PKA and PKCε dependency. To address this question, we generated knockout or overexpression mice for proton-sensing receptors. I found that both ASIC3 and TRPV1 knockout mice had shortened CFA-induced mechanical hyperalgesia. Moreover, blocking of TRPV1 by TRPV1 antagonist also shortened CFA-induced chronic mechanical hyperalgesia. Overexpression of G2A reduced CFA-induced mechanical hyperalgesia in early phase through Gαi pathway.

碩士
國立中央大學
生命科學系
102
Chest pain is the hallmark of myocardial ischemia, that often occurs in heart when sufficient oxygen for the needs is not acquired. Myocardial ischemia induces production of proton or bradykinin, which activates cardiac primary afferents, specifically Aδ- and C-fibre afferents, to transduce cardiac nociception to the central nervous system, leading to pain. In previous studies, acid-sensing ion channel 3 (ASIC3) on cardiac sensory afferents to sense protons and some regulations, participate in the regulation of myocardial ischemia. Transient myocardial ischemia induces prolonged ST-segment depression and more severe cardiac fibrosis in ASIC3-/-, but not in ASIC3+/+ or TRPV1-/-. However, molecular involved in ischemia-induced pain is not clear. In this study, I used low-dose isoproterenol to induce transient myocardial ischemia in ASIC3 or TRPV1 knockout mice. Isoproterenol injection caused transient hypoxia in cardiac muscle. Expression of proton-sensing G-Proton-coupled receptor(GPCR) G2A,was increased after isoproterenol injection. In ASIC3-/- mice, transient hypoxia occurred early and G2A expression was inhibited. Therefore, G2A may participate in ASIC3-mediated pathway to protect cardiac cells from severe ischemic insults.

碩士
國立中央大學
生命科學系
107
Rheumatoid arthritis (RA), a common autoimmune disease, is characterized by chronic inflammation of the synovial joints, leading to joint damage and long-term pain. Joint inflammation is often accompanied for tissue acidosis. The previous study has found that proton-sensing receptors, ASIC3, TRPV1 and TDAG8, reduce RA-induced mechanical hyperalgesia and arthritis scores. However, it remains unclear whether these genes also affect RA-induced thermal hyperalgesia and how they regulate RA-induced pain. Previous studies have shown that satellite glial cells (SGCs) are critical to the development and maintenance of chronic pain. It is likely that proton-sensing receptors may regulate SGCs to modulate pain. To address this question, I injected 5 μg of Freund's Complete Adjuvant (CFA) into the joints every week for four weeks and performed behavioral tests and immunohistochemical staining. Repeated CFA resulted in long-term joint inflammation, bilateral thermal hyperalgesia, DRG neuron loss, and an increase in the number of SGCs. Interestingly, ASIC 3, TRPV 1 and TDAG 8 knockout mice reversed arthritis-induced thermal hyperalgesia from 7-9 weeks, and inhibited the increase in the number of SGCs.

碩士
國立臺灣大學
環境衛生研究所
102
Dengue fever is one of the most serious insect-borne infectious diseases in Taiwan. It has no vaccine or medicine for dengue now, so it should be relying on vector control. This study is aimed to develop physical strategies for trapping Aedes aegypti and Ae. albopictus by potential attractable factors, including light, color and temperature, as well as to analyze the performance of heat-activated channel gene expression. The results obtained from our laboratory experiment showed that ultraviolet light (360 nm) was more effective to attract diurnal Ae. mosquitoes than blue (450 nm), green (510 nm) and red (630 nm) light be. The attraction rate of female Ae. aegypti (n=50) and male (n=50) were 42.00±7.94% and 10.00±6.24%, and female Ae. albopictus (n=50) and male (n=50) were 46.33±3.78% and 15.67±4.04%. The preferences of female Ae. aegypti (n=25) to colors was shown in the following: black (24.53±2.02%) > deep blue (21.87±1.52%) > red (14.13±1.19%) > green (6.67±2.11%) > light blue (4.53±1.79%) > yellow (1.33±0.00%). Similarly, female Ae. albopictus to colors was: red (25.6±2.89%) > black (23.73±1.16%) > deep blue (20.53±0.73%) > light blue (6.4±1.16%) > green (5.6±1.11%) > yellow (2.4±2.89%). The combination of black color and ultraviolet for light trap led to higher attraction rate (62.42±8.03%) toward Ae.aegypti, and rate (76.05±10.34%) toward Ae. albopictus. Temperatures ranging from 26 ° C to 38 ° C could attract both Ae. aegypti and Ae. albopictus. The attraction rates of female Ae. aegypti (n=25) and Ae. albopictus (n=25) were 45.14±3.02% and 40.57±6.70%, respectively. The condition of color paper covered on heater at 32 °C brought higher attraction rate: black (70.67±4.62%) > deep blue (65.33±8.33%) > red (50.67±9.24%) > uncovered (45.33±10.07%) > white (38.67±8.33%). Similarly, female Ae. albopictus was：black (70.67±8.32%) > deep blue (72.00±6.92%) > red (65.33±8.32%) > uncovered (48.00±6.92%) > white (41.22±6.11%). In the studies on Drosophila, TRPA was affected by ambient temperature. In other studies, it was found that TRPA1 on Anopheles gambiae antennae acted as a kind of channels which would be excited by heat. Therefore, not only do we want to figure out attraction effect of heat on mosquitoes, but to understand levels of heat-induced gene expression in Ae. aegypti and Ae. albopictus. So this study separated the heads from mosquitoes, which were subsequently sorted into heat-attractable and non-attractable group. Then the total RNA was extracted from the heads followed by TRPA1 gene amplification using RT-PCR. The results showed that the level of TRPA1 expression in Ae. aegypti heads of attractable group were 1.48 times higher than non-attractable group, and those in heads of Ae. albopictus was 1.59 times as much as non-attractable group. Based on the these results, we got a specific wavelength, color and temperature that are able to efficiently attract Ae. aegypti and Ae. albopictus. And TRPA1 gene was found to be activated with heat excitement in Ae. aegypti. In the future work, based on our findings, we hope to develop a novel mosquito trap with more effective capacity for diurnal mosquitoes control, which could be expected to be applied in practical control of mosquitoes and prevention of dengue fever.