Submitted by Andreza Leão (andrezaflh@gmail.com) on 2018-08-20T19:16:26Z No. of bitstreams: 1 Tese_PreferenciaClaroEscuro.pdf: 2322742 bytes, checksum: 3409e14bcee0fac27fbb7c9afba7401e (MD5) / Approved for entry into archive by Celia Santana (celiasantana@ufpa.br) on 2018-12-12T18:01:20Z (GMT) No. of bitstreams: 1 Tese_PreferenciaClaroEscuro.pdf: 2322742 bytes, checksum: 3409e14bcee0fac27fbb7c9afba7401e (MD5) / Made available in DSpace on 2018-12-12T18:01:20Z (GMT). No. of bitstreams: 1 Tese_PreferenciaClaroEscuro.pdf: 2322742 bytes, checksum: 3409e14bcee0fac27fbb7c9afba7401e (MD5) Previous issue date: 2014-12-12 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / O Danio rerio é um modelo animal amplamente utilizado e com protocolos bem estabelecidos para experimentos comportamentais em aquários branco/preto. O lado branco do aquário parece causar aversão e a preferência parece ser por permanecer maior tempo no lado escuro. A melatonina é um indolamina com influência no funcionamento fisiológico de animais. Os objetivos deste trabalho são: fazer um levantamento na literatura sobre melatonina e sua correlação com peixes e com o Danio rerio; comparar a eficiência de dois aparatos de isolamento em testes comportamentais; testar a influência de quatro períodos do dia e de cinco fotoperíodos na variação da resposta em Zebrafish submetidos ao teste claro-escuro; comparar o tempo de permanência em cada lado do aquário teste no período noturno, com sujeitos isolados em fotoperíodo de 12/12 horas; comparar o tempo de permanência em cada lado do aquário teste no período, com sujeitos isolados em fotoperíodo de 12/12 horas, com exposição a sete diferentes concentrações de melatonina; comparar estatisticamente a amostra em quartis dentro de cada concentração; e comparar estatisticamente a amostra em quartis entre os primeiros, segundos e terceiros quartis de cada concentração. O Zebrafish e os peixes parecem ter sua fisiologia e comportamento influenciados pela melatonina (tanto endógena quanto exógena). Ambos os aparatos de isolamento demonstraram ser igualmente eficazes para testes comportamentais. A luminosidade parece arrastar o ciclo circadiano do Zebrafish, diminuindo sua aversão ao lado branco do aquário, no fotoperíodo 12/12 horas e período de coleta noturno. Ela parece influir na alteração causada pelo fotoperíodo, diminuindo ou anulando o arrastamento. Na amostra analisada parece haver subpopulações que respondem de maneira diferente a exposição à mesma concentração de melatonina. Estudos com cepas específicas de Zebrafish e com um leque maior de concentrações de melatonina parecem ser necessários para identificar a possível dose específica para contrabalancear a atuação luminosa no arrastamento do ciclo circadiano em Danio rerio. / Danio rerio is a widely used animal model for behavioral experiments in white / black tanks. The white side of the aquarium seems to cause aversion and the preference seems to be to stay longer on the dark side. Melatonin is a indoleamine which influence the physiological functioning of fish. The aims of this work are: to survey the literature on melatonin and its correlation with fishes and Danio rerio; compare the efficiency of two apparatuses insulation in behavioral tests; testing the influence of four periods of the day and five photoperiods response variation in Zebrafish to light-dark test; compare the time spent on each side of the aquarium test period, subjects isolated on a photoperiod of 12/12 hours with exposure to seven different concentrations of melatonin; statistically comparing the sample into quartiles within each concentration; and statistically compare the sample into quartiles among the first, second and third quartiles of each concentration. The Zebrafish and the fishes seem to have their physiology and behavior influenced by melatonin (both endogenous as exogenous). Both apparatuses isolation proved to be equally effective for behavioral tests. Luminosity seems to drag the Zebrafish circadian cycle, reducing their aversion to the white side of the aquarium, photoperiod 12/12 hour period and night collection. Melatonin seems to influence the change caused by photoperiod, decreasing or canceling the dragging. In our sample seems to be subpopulations that respond differently to exposure to the same concentration of melatonin. Studies with specific strains of Zebrafish and with a wider range of concentrations of melatonin appear to be necessary to identify the specific dosage possible to counteract the performance in light entrainment of the circadian cycle in Danio rerio.

Loss of hair cells due to acoustic trauma results in the loss of hearing. In humans, unlike other vertebrates, the mechanism of hair cell regeneration is not possible. The molecular mechanisms that underlie this regeneration in nonmammalian vertebrates remain elusive. To understand the gene regulation during hair cell regeneration, our previous microarray study on zebrafish inner ears found that growth hormone (GH) was significantly upregulated after noise exposure. In this current study, we utilized Next Generation Sequencing (NGS) to examine the genes and pathways that are significantly regulated in the zebrafish inner ear following sound exposure and GH injection. Four groups of 20 zebrafish each were exposed to a 150 Hz tone at 179 dB re 1μPa RMS for 40 h. Zebrafish were injected with either salmon GH, phosphate buffer or zebrafish GH antagonist following acoustic exposure, and one baseline group received no acoustic stimulus or injection. RNA was extracted from ear tissues at 1 and 2 days post-trauma, and cDNA was synthesized for NGS. The reads from Illumina Pipeline version SCS 2.8.0 were aligned using TopHat and annotated using Cufflinks. The statistically significant differentially expressed transcripts were identified using Cuffdiff for six different pairwise comparisons and were analyzed using Ingenuity Pathway Analysis. I found significant regulation of growth factors such as GH, prolactin and fibroblast growth factor receptor 2, different families of solute carrier molecules, cell adhesion molecules such as CDH17 and CDH23, and other transcription factors such as Fos, FosB, Jun that regulate apoptosis. Analysis of the cell proliferation network in the GH-injected condition compared to buffer-injected day 1 showed significant up-regulation of GH while downregulation of apoptotic transcription factors was found. In contrast, the antagonist-injected condition compared to the GH-injected condition showed an opposite pattern in which up-regulation of apoptotic transcription factors were found while GH was down-regulated. A number of other transcripts (e.g., POMC, SLC6A12, TMEM27, HNF4A, CDH17 and FGFR2) that showed up-regulation in GH-injected condition showed down-regulation in antagonist-injected condition. These results strongly suggest that injection of exogenous GH potentially has a protective role in the zebrafish inner ear following acoustic trauma.

Les PCB (polychlorobiphényles) sont des molécules connues pour leur longue demi-vie et leur forte liposolubilité qui conduisent à une bioaccumulation et une bioamplification dans les réseaux trophiques, menant à un potentiel risque pour les prédateurs de haut niveau tel que l'Homme. De plus, il a été démontré que leur affinité avec les composés lipidiques conduisaient à une transmission de la femelle à l'œuf chez les poissons. Dans ce contexte, et comme d'autres travaux ont déjà montré des effets des PCB sur la physiologie et le comportement d'animaux contaminés de différentes façons, nous avons observé les effets de ces molécules chez deux espèces, la sole commune et le poisson zèbre. La contamination a été réalisée via l'alimentation avec deux mélanges de PCB et deux concentrations qui correspondent à des situations environnementales, en termes de dose ou de choix et de proportion des congénères retenus. La dose la plus haute est équivalente à celle mesurée dans de la chair de molusques en baie de Seine et la dose intermédiaire à celle mesurée en estuaire de Loire. Les soles contaminées ont montré une diminution du niveau d'activité locomoteur après 30 jours (j) de contamination et une altération des capacités cryptiques après 60 j de contamination. Les poissons zèbre contaminés ont montré une augmentation de l'activité locomotrice après 250 j de contamination. La génération issue de cette génération de poisson zèbre contaminée a elle aussi montré une augmentation de l'activité locomotrice au stade larvaire et adulte. Chez les adultes, cela s'est traduit par une diminution de l'utilisation de la zone de fond des bacs et une augmentation du nombre de transition de zones, ce qui s'explique par une perte d'inhibition comportementale. Dans les deux cas, les phénotypes comportementaux observés chez les groupes PCB sont associés à une altération de la locomotion dans le sens d'une baisse d'activité pour une espèce placide comme la sole et dans le sens de l'augmentation pour une espèce mobile comme le poisson zèbre.

Cryopreservation of germplasm of aquatic species offers many benefits to the fields of aquaculture, conservation and biomedicine. Although successful fish sperm cryopreservation has been achieved with many species, there has been no report of successful cryopreservation of fish embryos and late stage oocytes which are large, chilling sensitive and have low membrane permeability. In the present study, the sensitivity to chilling and toxicity of cryoprotectants of early stage zebrafish ovarian follicles were studied before designing protocols for their cryopreservation using controlled slow cooling. The effect of cryoprotectant, freezing medium, cooling rate, method for cryoprotectant removal, post-thaw incubation time and ovarian follicle developmental stage were investigated. In vitro culture method for early stage zebrafish ovarian follicles were also developed. The studies showed that stage I and II ovarian follicles are less sensitive to chilling than stage III follicles and methanol was the least toxic cryoprotectant. 4M methanol in potassium chloride (KCl) buffer was found to be the optimal cryoprotective solution and the optimum cooling rate was 4 °C/min for stage I and II follicles. Although the highest survivals after 2 h post-thawed incubation were 50.7 ± 4.0% for stage II ovarian follicles obtained with FDA+PI staining, ADP/ATP ratios of the cryopreserved follicles were significantly increased indicating increased cell death. Furthermore, in vitro culture experiments showed that there was no growth for stage I and II ovarian follicles after cryopreservation, indicating that successful cryopreservation of early stage zebrafish ovarian follicles at liquid nitrogen still remains elusive. From in vitro culture study, 90% L-15 medium at pH 9.0 containing 10 IU/ml hCG was effective for in vitro culture of stage I and II ovarian follicles. Systematic study on cryopreservation of early stage fish ovarian follicles at liquid nitrogen temperature is reported ii here for the first time. The results will provide useful information on the future development of protocol design for successful cryopreservation of early stage fish ovarian follicles.

High quality fish oocytes are essential for in vitro maturation (IVM), in vitro fertilization (IVF) protocols, and for use in cryopreservation. It is important to develop methods for assessing oocyte quality for applications in aquaculture, the preservation of endangered species and managing fish models used in biomedical research. The lack of reliable methods of evaluating oocyte quality limits progress in these areas. The present study was undertaken to develop new methods to assess ovarian follicle viability and quality of stage III zebrafish (Danio rerio) ovarian follicles. The methods developed were then applied to study the impact of cryoprotectant and/or cryopreservation procedures. A vital staining procedure, not previously used with zebrafish oocytes, has been investigated. FDA-PI (Fluorescein diacetate-Propidium Iodide) staining was found to be a more sensitive then currently used viability tests and it could also be applied to all ovarian follicles developmental stages. Mitochondrial activity and distribution as biological markers was investigated with the mitochondrial membrane potentialsensitive dye JC-1- (5,5’,6,6’-tetrachloro-1,1’,3,3’-tetraethylbenzimidazolylcarbocyanine iodide). Confocal microscopy, Cryo-scanning and electron microscopy studies were undertaken to determine mitochondria distributional arrangement within the ovarian follicle. This provided new information on zebrafish ovarian follicle structure, and showed that mitochondria exhibited a contiguous distribution at the margin of the granulosa cell layer surrounding stage III zebrafish oocytes. Cryoscanning results showed a polygonal structure of the vitelline envelope, which is reported here for the first time with the mitochondrial distributional arrangement in the granulosa cell layer. Mitochondrial distribution and the evaluation of mitochondrial activity proved to be sensitive markers for ovarian follicle quality, providing more detailed information on cryoprotectant impact. The measurement of ATP levels, ADP/ATP ratio and mtDNA copy number were also undertaken following cryoprotectant exposure. These findings, together with the observation of mitochondrial distribution, suggested that even cryoprotectant treatments that are considered to have little or no toxicity can have a deleterious effect on mitochondrial activity, potentially compromising oocyte growth and embryo development. Therefore, a further optimization of the currently used protocol may need to be considered. The study of organelle distribution and organisation would support in vitro maturation and oocyte development fields, as well as their use as biological markers for quality determination. These findings will contribute to a better understanding of oogenesis/folliculogenesis processes in fish.

In aquaculture, short term chilled storage has been used to transport brood stock fish embryos for genetic improvement programmes. It is therefore important to understand the effect of chilling on embryos at both developmental and molecular levels. In the present study, gene expression patterns in zebrafish embryos were studied before investigations were carried out on the effect of chilling on gene and protein expression in these embryos. The gene expression results obtained in different developmental stages using conventional PCR showed that, only sox genes were expressed throughout the tested developmental stages from 30% epiboly to 6 somites. Quantitative RT-PCR was then used to investigate sox gene expression patterns during chilling of 50% epiboly stage embryos at 0°C for up to 180 min and also after warming. Significant decreases in sox2 and sox3 expressions were observed when compared to those of controls following chilling whilst significant increases of expressions of the two genes were observed after warming in the embryos chilled for 30 and 60 min. Studies on the impact of cryoprotectant MeOH on sox genes and protein expression showed that 50% epiboly stage zebrafish embryos could tolerate chilling for up to 6 h with or without MeOH. It was observed that expression of all three sox genes were significantly decreased following chilling for 3 h at 0°C. However the degree of decrease was less pronounced in embryos chilled with different concentrations of MeOH. Significant increases in sox genes were observed in hatching stage embryos chilled with 1 M MeOH for 3h but subsequent sox2 and sox19a protein expression was not affected. The effect of long term chilling (18h) on sox gene and protein expression in 50% epiboly stage embryos was also investigated. Improved hatching rates (56% ± 5) were achieved when embryos were chilled with 1 M MeOH + 0.1 M sucrose. Results from gene expression studies showed a stable sox2 gene expression in 18 h chilled embryos in cryoprotectant mixture when compared to that of embryos chilled without cryoprotectant mixture. Similar patterns were observed when the expression of sox2 and sox3 protein was investigated. This is the first study carried out on the effect of chilling in early stage zebrafish embryos at the molecular level. The results obtained from the present study provided useful information on the molecular mechanisms of the effect of chilling on zebrafish embryos and will have important implications in designing chilled storage protocols for fish embryos.

Heat shock proteins (hsp) and heat shock transcription factors (HSF) have important roles in the development of the eye lens. Our lab previously demonstrated that knockdown of hsp70 gene expression using morpholino antisense technology (MO) resulted in a small lens phenotype in zebrafish (Danio rerio) embryos. A less severe phenotype was seen with knockdown of hsf1, suggesting other factors that regulate hsp70 are involved during lens formation. Both HSF1 and HSF4 are known to play a role in mammalian lens development. An expressed sequence tag encoding zebrafish HSF4, named hsf4a, has been identified and a second splice variant, hsf4b, has been predicted in the Ensembl database. The objectives of this thesis were to characterize the zebrafish HSF4 and compare its expression to other HSFs as well as investigate its role in lens development. Analysis of zebrafish HSF4 sequence was performed using standard in silico analytical software. The deduced amino acid sequence of HSF4a shares structural similarities with mammalian HSF4 including the lack of an HR-C domain. This domain is absent due to a C-terminal truncation within zebrafish HSF4a relative to the mammalian protein. HSF4b is identical to the HSF4a sequence with the exception of an additional 155 amino acids at the carboxyl end of the protein which contains an HR-C domain, unlike mammalian HSF4. Surprisingly, electrophoretic mobility shift assays (EMSA) demonstrated that the binding affinity of zebrafish HSF4 to discontinuous HSEs is more similar to HSF1 than to other HSF4 proteins. The amino acid sequence of zebrafish HSF4 DNA binding domain was also more similar to HSF1 than other HSF4 proteins. These results, along with a phylogenetic analysis of HSF proteins from eleven species, suggest that HSF1 was an evolutionary precursor of HSF4 and that functions of this protein may differ between zebrafish and mammals. The expression level for each of the three zebrafish HSFs was determined in adult tissues and in developing embryos by quantitative reverse transcription polymerase chain reaction (qPCR) analysis. Expression of both hsf4 transcripts was observed predominantly in the eye but only observed in developing embryonic tissue at 60 hours post fertilization or later. This, together with the lack of an observable phenotype following MO knockdown of hsf4, suggests that HSF4 likely has a role in later stages of lens development. Additionally, hsf1 and hsf2 expression were detected in all tissues and in all stages of development as well as being present as maternal transcripts in zebrafish eggs. The results presented in this thesis demonstrate that while zebrafish HSFs share some similarity with HSF proteins from other species, they also have structural characteristics and expression patterns unique to the zebrafish.

Alzheimer’s disease (AD) is the most prevalent form of dementia. There is considerable evidence that AD is caused by accumulating amyloid beta peptides in the brain, as a result of amyloid precursor protein (APP) cleavage by secretase enzymes. The presenilin proteins are central to the gamma-secretase cleavage of the intramembrane domain of APP. Aberrant splicing and point mutations in the human presenilin genes, PSEN1 and PSEN2, have been linked to familial forms of AD, through aberrant APP cleavage resulting in irregular amyloid beta formation. Paper 1 gives a review of the literature on AD research and how animal models are used to elucidate mechanisms of AD pathogenesis. The zebrafish model is used in this thesis to investigate genes with potential relevance to AD initiation and pathogenesis. Paper 2 demonstrates that lowlevel aberrant splicing of exon 8 in psen1 transcripts in zebrafish embryos produces potent dominant negative effects that increased psen1 transcription, cause a dramatic hydrocephalus phenotype, decreased pigmentation and other developmental defects. Similar effects are also observed after low-level interference with splicing of exon 8 in psen2 transcripts. In paper 3, a microarray analysis was performed to analyse global gene expression changes to illuminate the molecular aetiology of the phenotypic effects described in paper 2. Of the 100 genes that showed greatest dysregulation after psen1 or psen2 manipulation, 12 genes were common to both treatments. Five of these have known function and showed increased expression. Cyclin G1 (ccng1) was of particular interest as the human CCNG1 protein shows increased immunoreactivity in the cytoplasm of neurons in human AD brains. Phylogenetic and conserved synteny analysis confirmed the orthology of zebrafish ccng1 with human CCNG1. Expression of zebrafish ccng1 in developing embryos at 24 hours post fertilization (hpf) was observed in the eye, tectum and somites. Decreased Ccng1 expression does not lead to any developmental defects and also cannot rescue the hydrocephalus or pigmentation phenotypes of embryos with aberrant splicing of psen1 exon 8. An analysis of zebrafish ccng1 function in paper 4 (thesis chapter in the form of a manuscript) indicates that truncation of Ccng1 appears to cause developmental defects in the brain, notochord and somites, however, it does not decrease the level of normal ccng1 transcript. The CCNG1 paralogue, Cyclin G2, (CCNG2), is also expressed in zebrafiish (ccng2). Decreasing the expression of Ccng2 results in similar effects on embryo development as truncating Ccng1. Therefore, the truncated forms of Ccng1 potentially interfere with Ccng2 function in a dominant negative manner. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1342482 / Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 2008

Alzheimer’s disease (AD) is the most prevalent form of dementia. There is considerable evidence that AD is caused by accumulating amyloid beta peptides in the brain, as a result of amyloid precursor protein (APP) cleavage by secretase enzymes. The presenilin proteins are central to the gamma-secretase cleavage of the intramembrane domain of APP. Aberrant splicing and point mutations in the human presenilin genes, PSEN1 and PSEN2, have been linked to familial forms of AD, through aberrant APP cleavage resulting in irregular amyloid beta formation. Paper 1 gives a review of the literature on AD research and how animal models are used to elucidate mechanisms of AD pathogenesis. The zebrafish model is used in this thesis to investigate genes with potential relevance to AD initiation and pathogenesis. Paper 2 demonstrates that lowlevel aberrant splicing of exon 8 in psen1 transcripts in zebrafish embryos produces potent dominant negative effects that increased psen1 transcription, cause a dramatic hydrocephalus phenotype, decreased pigmentation and other developmental defects. Similar effects are also observed after low-level interference with splicing of exon 8 in psen2 transcripts. In paper 3, a microarray analysis was performed to analyse global gene expression changes to illuminate the molecular aetiology of the phenotypic effects described in paper 2. Of the 100 genes that showed greatest dysregulation after psen1 or psen2 manipulation, 12 genes were common to both treatments. Five of these have known function and showed increased expression. Cyclin G1 (ccng1) was of particular interest as the human CCNG1 protein shows increased immunoreactivity in the cytoplasm of neurons in human AD brains. Phylogenetic and conserved synteny analysis confirmed the orthology of zebrafish ccng1 with human CCNG1. Expression of zebrafish ccng1 in developing embryos at 24 hours post fertilization (hpf) was observed in the eye, tectum and somites. Decreased Ccng1 expression does not lead to any developmental defects and also cannot rescue the hydrocephalus or pigmentation phenotypes of embryos with aberrant splicing of psen1 exon 8. An analysis of zebrafish ccng1 function in paper 4 (thesis chapter in the form of a manuscript) indicates that truncation of Ccng1 appears to cause developmental defects in the brain, notochord and somites, however, it does not decrease the level of normal ccng1 transcript. The CCNG1 paralogue, Cyclin G2, (CCNG2), is also expressed in zebrafiish (ccng2). Decreasing the expression of Ccng2 results in similar effects on embryo development as truncating Ccng1. Therefore, the truncated forms of Ccng1 potentially interfere with Ccng2 function in a dominant negative manner. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1342482 / Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 2008

Caveolae are small plasma membrane invaginations present in many different cell types, which have been linked to diverse cellular functions, including cell signalling, membrane rearrangements and lipid regulation. The caveolae markers, members of the caveolin family of proteins, are essential for caveola formation and function. Recently, however, a protein named PTRF (Polymerase I and Transcript Release Factor) or cavin, originally identified as a nuclear factor that regulates transcription in vitro, was shown to be associated with caveolae in adipocytes. In the first chapter of this thesis, I have used the zebrafish Danio rerio to investigate the relation of PTRF/cavin to caveolae as well as caveola function in vivo. During zebrafish development, PTRF/cavin was highly expressed in the notochord in 18 h, 24 h and 35 h post-fertilization embryos, as detected by in situ hybrydization. Analysis of later development stages showed that PTRF/cavin is also present in the otic vesicle, brachial arches, and periderm. Disruption of PTRF/cavin expression, via morpholino-mediated inhibition, caused severely defective development of the notochord as well as heart edema, in a dose-dependent manner. PTRF/cavin knockdown embryos had curved notochords and were shorter than the controls. Examination of the notochord by electron microscopy showed that the number of caveolae was greatly reduced in PTRF/cavin-morpholino-injected embryos. Similar effects were observed when caveolin-1, the major protein of caveolae in non-muscle cells, was down-regulated. Altogether, these results indicated a role for PTRF/cavin during formation and/or stabilization of caveolae as well as an essential role for caveolae during zebrafish embryo development. Combined with results obtained in mammalian cells, these findings identify PTRF/cavin as the first component of a caveolar coat, required for caveola formation and function (Hill et al., 2008). We subsequently identified a family of PTRF/cavin-related proteins, the cavins, that all associate with caveolae. Using biochemistry, light microscopy, and FRET-based approaches we characterised PTRF/cavin and the new members of this family of proteins SDR/cavin-2, SRBC/cavin-3 and MURC/cavin-4. We have shown that the four members of the cavin family form a multi-protein complex that associates with caveolae. This complex can constitutively assemble in the cytosol and then associate with caveolin at the plasma membrane caveolae; interestingly, caveolin is essential for the plasma membrane translocation of the cavin complex, and in caveolin-1 knockout cells the four cavin proteins are restricted to the cytosol. PTRF/cavin-1, but not other cavins, can induce caveola formation in a heterologous system and is required for the recruitment of the cavin complex to caveolae. The four cavin proteins present distinct patterns of tissue expression, which suggests that caveolae may perform tissue-specific functions regulated by the composition of the cavin complex. MURC/cavin-4 is expressed predominantly in muscle and its distribution is perturbed in human muscle disease associated with caveolin-3 dysfunction, identifying MURC/cavin-4 as a novel muscle disease candidate caveolar protein. To functionally investigate the relation of cavins and caveolae, we explored a caveolar function in mechanosensation. Through the use of hypo-osmotic media, we induced membrane-stretch and showed that the increased membrane tension leads to dissociation of the caveolin-cavin module and caveola disassembly as observed by immunofluorescence and FLIM/FRET techniques. Once released from caveolae, caveolin was seen internalized in late endosomes and lysosomes. Cavin-1, on the other hand, was found to be diffused in the cytosol and from there it was translocated to the nuclear compartment. The nuclear translocation was observed in several different cell types, which suggests a universal role for nuclear cavin-1, and was independent of caveolin expression. Analysis of live cells using real-time FLIM/FRET showed that cells quickly respond to variations in membrane tension by dissociation/re-association of caveolin and cavin-1. Altogether, in the course of this project, I was able to show that cavin-1 is an essential regulator of caveola biogenesis in cultured cells and in vivo. Cavin-1 and the other members of the PTRF/Cavin family form a multiprotein complex that is recruited to caveolae by caveolin and coats plasma membrane caveolae. The association between cavin-1 and caveolin is crucial for caveolae assembly and this interaction has a role in the cellular sensation of plasma membrane tension. Under high membrane tensions, caveolin and cavin-1 dissociate with the consequent flattening of caveolae. Under these circumstances, caveolin is internalized into enlarged endosomes and lysosomes while cavin-1 is translocated to the nucleus, identifying for the first time a caveola- to nucleus signalling pathway. The exact role of nuclear cavin-1 under plasma membrane stretch is now amenable to analysis.