Sonic hedgehog signaling regulates mode of cell division of early cerebral cortex progenitors and increases

The morphogen Sonic Hedgehog (SHH) plays a critical role in the development of different tissues. In the central nervous system, SHH is well known to contribute to the patterning of the spinal cord and separation of the brain hemispheres. In addition, it has recently been shown that SHH signaling also contributes to the patterning of the telencephalon and establishment of adult neurogenic niches. In this work, we investigated whether SHH signaling influences the behavior of neural progenitors isolated from the dorsal telencephalon, which generate excitatory neurons and macroglial cells in vitro. We observed that SHH increases proliferation of cortical progenitors and generation of astrocytes, whereas blocking SHH signaling with cyclopamine has opposite effects. In both cases, generation of neurons did not seem to be affected. However, cell survival was broadly affected by blockade of SHH signaling. SHH effects were related to three different cell phenomena: mode of cell division, cell cycle length and cell growth. Together, our data in vitro demonstrate that SHH signaling controls cell behaviors that are important for proliferation of cerebral cortex progenitors, as well as differentiation and survival of neurons and astroglial cells.

Development of A Flow Cytometry Assay for Measurement of CD4+ T Cell Proliferation

Thesis (Master's)--University of Washington, 2016-06

Regenerative Cues of Myeloid Cells on Pancreatic Epithelium

Thesis (Ph.D.)--University of Washington, 2016-07

Avaliação do potencial cancerigénico de microcistinas (cianotoxinas)

Tese de doutoramento em Farmácia (Toxicologia), apresentada à Faculdade de Farmácia da Universidade de Lisboa, 2009.

Soft tissue sarcoma: biology and therapeutic correlates

Soft tissue sarcomas (STS) comprise a heterogenenous group of greater than 50 malignancies of putative mesenchymal cell origin and as such they may arise in diverse tissue types in various anatomical locations throughout the whole body. Collectively they account for approximately 1% of all human malignancies yet have a spectrum of aggressive behaviours amongst their subtypes. They thus pose a particular challenge to manage and remain an under investigated group of cancers with no generally applicable new therapies in the past 40 years and an overall 5-year survival rate that remains stagnant at around 50%. From September 2000 to July 2006 I undertook a full time post-doctoral level research fellowship at the MD Anderson Cancer Center, Houston, Texas, USA in the department of Surgical Oncology to investigate the biology of soft tissue sarcoma and test novel anti- sarcoma adenovirus-based therapy in the preclinical nude rat model of isolated limb perfusion against human sarcoma xenografts. This work, in collaboration with colleagues as indicated herein, led to a number of publications in the scientific literature furthering our understanding of the malignant phenotype of sarcoma and reported preclinical studies with wild-type p53, in a replication deficient adenovirus vector, and oncolytic adenoviruses administered by isolated limb perfusion. Additional collaborative and pioneering preclinical studies reported the molecular imaging of sarcoma response to systemically delivered therapeutic phage RGD-4c AAVP. Doxorubicin chemotherapy is the single most active broadly applicable anti-sarcoma chemotherapeutic yet only has an approximate 30% overall response rate with additional breakthrough tumour progression and recurrence after initial chemo-responsiveness further problematic features in STS management. Doxorubicin is a substrate for the multi- drug resistance (mdr) gene product p-glycoprotein drug efflux pump and exerts its main mode of action by induction of DNA double-strand breaks during the S-phase of the cell cycle. Two papers in my thesis characterise different aspects of chemoresistance in sarcoma. The first shows that wild-type p53 suppresses Protein Kinase Calpha (PKCα) phosphorylation (and activation) of p-glycoprotein by transcriptional repression of PKCα through a Sp-1 transcription factor binding site in its -244/-234 promoter region. The second paper demonstrates that Rad51 (a central mediator of homologous recombination repair of double strand breaks) has elevated levels in sarcoma and particularly in the S- G2 phase of the cell cycle. Suppression of Rad51 with small interfering RNA in sarcoma cell culture led to doxorubicin chemosensitisation. Reintroduction of wild-type p53 into STS cell lines resulted in decreased Rad51 protein and mRNA expression via transcriptional repression of the Rad51 promoter through increased AP-2 binding. In light of poor response rates to chemotherapy, escape from local control portends a poor prognosis for patients with sarcoma. Two papers in my thesis characterise aspects of sarcoma angiogenesis, invasion and metastasis. Human sarcoma samples were found to have high levels of matrix metalloproteinase-9 (MMP-9) with expression levels that correlated with p53 mutational status. MMP-9 is known to degrade extracellular collagen, contribute to the control of the angiogenic switch necessary in primary tumour progression and facilitate invasion and metastasis. Reconstitution of wild-type p53 function led to decreased levels of MMP-9 protein and mRNA as well as zymography-assessed MMP-9 proteolytic activity and decreased tumour cell invasiveness. Reintroduction of wild-type p53 into human sarcoma xenografts in-vivo decreased tumour growth and MMP-9 protein expression. Wild-type p53 was found to suppress mmp-9 transcription via decreased binding of NF-κB to its -607/-595 mmp-9 promoter element. Studies on the role of the VEGF165 in sarcoma found that sarcoma cells stably transfected with VEGF165 formed more aggressive xenografted tumours with increased vascularity, growth rate, metastasis, and resistance to chemotherapy. Use of the anti-VEGFR2 antibody DC101 enhanced doxorubicin sensitivity at sub-conventional dosing, inhibited tumour growth, decreased development of metastases, and reduced tumour micro-vessel density while increasing the vessel maturation index. These effects were explained primarily through effects on endothelial cells (e.c.s), rather than the tumour cells per se, where DC101 induced e.c. sensitivity to doxorubicin and suppressed e.c. production of MMPs. The p53 tumour suppressor pathway is the most frequently mutated pathway in sarcoma. Recapitulation of wild-type p53 function in sarcoma exerts a number of anti-cancer outcomes such as growth arrest, resensitisation to chemotherapy, suppression of invasion, and attenuation of angiogenesis. Using a modified nude rat-human sarcoma xenograft model for isolated limb perfusion (ILP) delivery of wild-type p53 in a replication deficient adenovirus vector I showed that functionally competent wild-type p53 could be delivered to and detected in human leiomyosarcoma xenografts confirming preclinical feasibility - although not efficacious due to low transgene expression. Viral fibre modification to express the RGD tripeptide motif led to greater viral uptake by sarcoma cells in vitro (transductional targeting) and changing the transgene promoter to a response element active in cells with active telomerase expression restricted the transgene expression to the tumour intracellular environment (transcriptional targeting). Delivery of the fibre-modified, selectively replication proficient oncolytic adenovirus Ad.hTC.GFP/ E1a.RGD by ILP demonstrated a more robust, and tumour-restricted, transgene expression with evidence of anti-sarcoma effect confirmed microscopically. Collaborative studies using the fibre modified phage RGD-4C AAVP confirmed that systemic delivery specifically, efficiently, and repeatedly targets human sarcoma xenografts, binds to αv integrins in tumours, and demonstrates a durable, though heterogeneous, transgene expression of 1-4 weeks. Incorporation of the Herpes Simplex Virus thymidine kinase (HSVtk) transgene into RGD-4C AAVP permitted CT-PET spatial and temporal molecular imaging in vivo of transgene expression and allowed quantification of tumour metabolic activity both before and after interval administration of a systemic cytotoxic with predictable and measurable response to treatment before becoming apparent clinically. These papers further the medical and scientific community’s understanding of the biology of soft tissue sarcoma and report preclinical studies with novel and promising anti- sarcoma therapeutics.

Cell-cell communication in three dimensional microenvironments

Cellular behavior is dependent on a variety of extracellular cues required for normal tissue function, wound healing, and activation of the immune system. Removed from their in vivo microenvironment and cultured in vitro, cells lose many environmental cues and that may result in abberant behavior, making it difficult to study cellular processes. In order to mimic native tissue environments, optical tweezer and microfluidic technologies were used to place cells within defined areas of the culture environment. To provide three dimensional supports found in natural tissues, hydrogel scaffolds of poly (ethylene glycol) diacrylate and the basement membrane matrix Matrigel were used. Optical tweezer technology allowed precision placement and formation of homotypic and heterotypic arrays of human U937, HEK 293, and porcine mesenchymal stem cells. Alternatively, two microfluidic devices were designed to pattern Matrigel scaffolds. The first microfluidic device utilized laminar flow to spatially pattern multiple cell types within the device. Gradients of soluble molecules were then be formed and manipulated across the Matrigel scaffolds. Patterning Matrigel using laminar flow techniques require microfluidic expertise and do not produce consistent patterning conditions, limiting their use difficult in most cell culture laboratories. Thus, a buried Matrigel polydimethylsiloxane (PDMS) device was developed for spatial patterning of biological scaffolds. Matrigel is injected into micron sized channels of PDMS fabricated by soft lithography and allowed to thermally cure. Following curing, a second PDMS device was placed on top of the buried Matrigel channels to support media flow. In order to validate these systems, a cell-cell communication model system was developed utilizing LPS and TNFα signaling with fluorescent reporter systems to monitor communication in real time. We demonstrated the utility of microfluidic devices to support the cell-cell communication model system by co culturing three cell types within Matrigel scaffolds and monitoring signaling activity via fluorescent reporters.

Sonic hedgehog signaling regulates mode of cell division of early cerebral cortex progenitors and increases

The morphogen Sonic Hedgehog (SHH) plays a critical role in the development of different tissues. In the central nervous system, SHH is well known to contribute to the patterning of the spinal cord and separation of the brain hemispheres. In addition, it has recently been shown that SHH signaling also contributes to the patterning of the telencephalon and establishment of adult neurogenic niches. In this work, we investigated whether SHH signaling influences the behavior of neural progenitors isolated from the dorsal telencephalon, which generate excitatory neurons and macroglial cells in vitro. We observed that SHH increases proliferation of cortical progenitors and generation of astrocytes, whereas blocking SHH signaling with cyclopamine has opposite effects. In both cases, generation of neurons did not seem to be affected. However, cell survival was broadly affected by blockade of SHH signaling. SHH effects were related to three different cell phenomena: mode of cell division, cell cycle length and cell growth. Together, our data in vitro demonstrate that SHH signaling controls cell behaviors that are important for proliferation of cerebral cortex progenitors, as well as differentiation and survival of neurons and astroglial cells.

Soybean lunasin mediates colon carcinogenesis by inducing apoptosis and preventing outgrowth of metastasis

Colorectal cancer (CRC) is the third most common cancer worldwide. Various factors such as age, lifestyle and dietary patterns affect the risk of having CRC. Epidemiological studies showed a chemopreventive effect of soy consumption against CRC. However, which component(s) of soybean is associated with this reduced risk is not yet fully delineated. The objective of this research was to evaluate the anti-colon cancer potential of lunasin isolated from defatted soybean flour using in vitro and in vivo models of CRC. Lunasin was isolated from defatted soybean flour by a combination of different chromatographic and ultrafiltration techniques. The anti-colon cancer potential of lunasin was determined using different human colon cancer cell lines in vitro and a CRC liver metastasis model in vivo. Lunasin caused cytotoxicity to different human colon cancer cells with an IC50 value of 13.0, 21.6, 26.3 and 61.7 µM for KM12L4, RKO, HCT-116 and HT-29 human colon cancer cells, respectively. This cytotoxicity correlated with the expression of the α5 integrin on human colon cancer cells with a correlation coefficient of 0.78. The mechanism involved in the cytotoxic effect of lunasin was through cell cycle arrest and induction of the mitochondrial pathway of apoptosis. In KM12L4 human colon cancer cells, lunasin caused a G2/M phase arrest increasing the percentage of cells at G2/M phase from 12% (PBS-treated) to 24% (treated with 10 µM lunasin). This arrest was attributed to the capability of lunasin to increase the expression of cyclin dependent kinase inhibitors p21 and p27. At 10 µM, lunasin increased the expression of p21 and p27 in KM12L4 colon cancer cells by 2.2- and 2.3-fold, respectively. Flow cytometric analysis showed that lunasin at 10 µM increased the percentage of cells undergoing apoptosis from 13.6% to 24.7%. This is further supported by fluorescence microscopic analysis of KM12L4 cells treated with 10 µM lunasin showing chromatin condensation and DNA fragmentation. The mechanism involved is through modification of proteins involved in the mitochondrial pathway of apoptosis in KM12L4 cells as 10 µM lunasin reduced the expression of the anti-apoptotic Bcl-2 protein by 2-fold and increased the expression of the pro-apoptotic proteins Bax, cytochrome c and nuclear clusterin by 2.2-, 2.1- and 2.3- fold, respectively. This led to increased expression and activity of the executioner of apoptosis, caspase-3 by 1.8- and 2.3-fold, respectively. This pro-apoptotic property of lunasin can be attributed to its capability to internalize into the cytoplasm and nucleus of colon cancer cells 24 h and 72 h after treatment, respectively. In addition, lunasin mediated metastasis of colon cancer cells in vitro by inhibiting the focal adhesion kinase activation thereby reducing expression of extracellular regulated kinase and nuclear factor kappa B and finally inhibiting migration of colon cancer cells. In KM12L4 colon cancer cells, 10 µM lunasin resulted in the reduction of phosphorylation of focal adhesion kinase and extracellular regulated kinase by 2.5-fold, resulting in the reduced nuclear translocation of p50 and p65 NF-κB subunits by 3.8- and 1.4-fold, respectively. In an in vivo model of CRC liver metastasis, daily intraperitoneal administration of lunasin at 4 mg/kg body weight resulted in the inhibition of KM12L4 liver metastasis as shown by the reduction of the number of liver metastases from 28 (PBS-treated) to 14 (lunasin-treated, P = 0.047) and reduction in tumor burden as measured by liver weight/body weight from 0.13 (PBS-treated) to 0.10 (lunasin-treated, P = 0.039). Moreover, lunasin potentiated the anti-metastatic effect of the chemotherapeutic drug oxaliplatin given at 5 mg/kg body weight twice per week. Lunasin and oxaliplatin combination resulted in a more potent inhibition of outgrowth of KM12L4 cell metastases to the liver reducing the number of liver metastases by 6-fold and reducing the tumor burden in the liver by 3-fold when compared to PBS-treated group. This can be attributed by the capability of lunasin and oxaliplatin to reduce expression of proliferating cell nuclear antigen in liver-tumor tissue as measured by immunohistochemical staining. The results of this research for the first time demonstrated the anti-colon cancer potential of lunasin isolated from defatted soybean flour which might contribute to the chemopreventive effect of soybean in CRC as seen in different epidemiological studies. In conclusion, lunasin isolated from defatted soybean flour mediated colon carcinogenesis by inducing apoptosis and preventing outgrowth of metastasis. We suggest that the results of this research serve as a basis for further study on the chemopreventive effect of lunasin against CRC and a possible adjuvant role for lunasin in therapy of patients with metastatic CRC.

From genes to radioresistance in head and neck squamous cell carcinoma

Head and Neck Cancers (HNC) are a group of tumours located in the upper aero-digestive tract. Head and Neck Squamous Cell Carcinoma (HNSCC) represent about 90% of all HNC cases. It has been considered the sixth most malignant tumour worldwide and, despite clinical and technological advances, the five-year survival rate has not improved much in the last years. Nowadays, HNSCC is well established as a heterogeneous disease and that its development is due to accumulation of genetic events. Apart from the majority of the patients being diagnosed in an advanced stage, HNSCC is also a disease with poor therapeutic outcome. One of the therapeutic approaches is radiotherapy. However, this approach has different drawbacks like the radioresistance acquired by some tumour cells, leading to a worse prognosis. A major knowledge in radiation biology is imperative to improve this type of treatment and avoid late toxicities, maintaining patient quality of life in the subsequent years after treatment. Then, identification of genetic markers associated to radiotherapy response in patients and possible alterations in cells after radiotherapy are essential steps towards an improved diagnosis, higher survival rate and a better life quality. Not much is known about the radiation effects on cells, so, the principal aim of this study was to contribute to a more extensive knowledge about radiation treatment in HNSCC. For this, two commercial cell lines, HSC-3 and BICR-10, were used and characterized resorting to karyotyping, aCGH and MS-MLPA. These cell lines were submitted to different doses of irradiation and the resulting genetic and methylation alterations were evaluated. Our results showed a great difference in radiation response between the two cell lines, allowing the conclusion that HSC-3 was much more radiosensitive than BICR-10. Bearing this in mind, analysis of cell death, cell cycle and DNA damages was performed to try to elucidate the motifs behind this difference. The characterization of both cell lines allowed the confirmation that HSC-3 was derived from a metastatic tumour and the hypothesis that BICR-10 was derived from a dysplasia. Furthermore, this pilot study enabled the suggestion of some genetic and epigenetic alterations that cells suffer after radiation treatment. Additionally, it also allowed the association of some genetic characteristics that could be related to the differences in radiation response observable in this two cell lines. Taken together all of our results contribute to a better understanding of radiation effects on HNSCC allowing one further step towards the prediction of patients’ outcome, better choice of treatment approaches and ultimately a better quality of life.

Molecular and biochemical mechanisms involved in the toxicity of a marine cyanobacteria compound on cancer cell lines

In recent years marine biotechnology has revealed a crucial role in the future of bioindustry. Among the many marine resources, cyanobacteria have shown great potential in the production of bioactive compounds with diverse applicability. The pharmacological potential of these organisms has been one of the most explored areas in particular its antibacterial, antifungal and anticancer potential. This work was based on the assessment of potential anticancer compound E13010 F 5.4 isolated from marine cyanobacteria strain Synechocystis salina LEGE 06099. Thus the aim of this work was to explore molecular and biochemical mechanisms underlying the bioactivity detected in human cancer cells, specifically in lines RKO colon carcinoma and HT-29. The isolation of the compound was performed from biomass obtained by large-scale culture. To obtain the compound fractionation was carried and confirmation and isolation performed by Nuclear Magnetic Resonance (NMR), Thin Layer Chromatography (TLC) and High-Performance Liquid Chromatography (HPLC). Cell viability assays were performed based on reduction of 3- (4,5-dimetiltiaziol-2-yl) -2,5-diphenyltetrazolium bromide (MTT) to assess the cytotoxic potential of the compound. From the battery of cell lines RKO (colon carcinoma), HT-29 (colorectal adenocarcinoma), MG-63 (osteosarcoma) and T47D (breast carcinoma) the cell lines RKO and HT-29 were selected for elucidation of mechanisms of cytotoxicity. For the elucidation of the mechanisms involved in cytotoxicity the cell lines RKO and HT29 were exposed to the compound. A genomic approach based in the mRNA expression of genes involved in apoptosis and cell cycle by Real-Time PCR and a proteomic approach based on the separation of proteins by two-dimensional electrophoresis (2DGE) was performed. For mRNA expression were selected the genes RPL8, HPRT1, VDAC, SHMT2, CCNE, CCNB1, P21CIP, BCL-2 and BAD and for proteomics isoelectric focussing between 3 – 10 and molecular weight of 19 – 117 kDa separated by polyacrylamide gels (2DGE). The MTT results confirmed the reduction of the cell viability. The RT-PCR results for the expression of genes studied were not yet fully elucidative. For the cell line RKO there was a significant reduction in the expression of the gene P21CIP, and a tendency for reduction in the BAD gene expression and for increased expression of gene CCNB1, pointing to an effort for cell proliferation. In HT-29 cell line, there was a tendency for increase in the expression of P21CIP and BAD, which may explain the reduction in cell viability. The 2DGE results indicate proteomic patterns with differentially altered spots in the treated and control cells with both qualitative and quantitative differences, and differences in response between the RKO and HT-29 cell lines.

Characterization of TRIB2-mediated resistance to anti-cancer drugs

Dissertação de Mestrado, Oncobiologia: Mecanismos Moleculares do Cancro, Departamento de Ciências Biomédicas e Medicina, Universidade do Algarve, 2016

Life-cycle portfolio choice with liquid and illiquid financial assets

Traditionally, quantitative models that have studied households׳ portfolio choices have focused exclusively on the different risk properties of alternative financial assets. We introduce differences in liquidity across assets in the standard life-cycle model of portfolio choice. More precisely, in our model, stocks are subject to transaction costs, as considered in recent macroliterature. We show that when these costs are calibrated to match the observed infrequency of households׳ trading, the model is able to generate patterns of portfolio stock allocation over age and wealth that are constant or moderately increasing, thus more in line with the existing empirical evidence.

Inhibition of proliferation, migration and invasion of human non-small cell lung cancer cell line A549 by phlomisoside F from Phlomis younghusbandii Mukerjee

Purpose: To determine the effect of phlomisoside F (PMF) on the proliferation, migration and invasion of human non-small cell lung cancer cell line A549 and explore the possible mechanisms. Methods: The anti-proliferative effect of PMF on A549 cells was determined by CCK-8. Subsequently, migration and invasion were evaluated by Transwell and Transwell with matrigel assays, respectively. Furthermore, cell cycle and apoptosis were assessed by flow cytometry, while the mechanisms of action were determined by Western blotting. Results: PMF exhibited significant anti-proliferative effect on A549 cells in concentration-dependent and time-dependent manners, with half maximal inhibitory concentration (IC50) of 54.51 μM. Treatment with PMF (10, 20 and 40 μM) for 48 h resulted in significantly decreased migration and invasion in A549 cells. In addition, PMF at concentrations of 25, 50 and 75 μM induced cell cycle arrest in G0/G1phase and enhanced cell apoptosis in A549 cells. Furthermore, caspase-3, caspase-9 and Bax protein expressions were up-regulated while Bacl-2 and COX-2 protein expressions were significantly downregulated at 10, 20 and 40 μM concentrations of PMF. Conclusion: PMF suppresses A549 cell growth, migration and invasion. The mechanism may be related to the induction of mitochondria-mediated apoptosis pathway via regulation of caspase-3, caspase-9, Bcl-2 and Bax expressions, and inhibition of PGE2 synthesis by reducing COX-2 expression.

Understanding the role of CAP proteins in the polarization process of C. elegans

La division cellulaire asymétrique est un processus crucial dans le développement des organismes multicellulaires puisqu’elle permet la génération de la diversité cellulaire. Les cellules qui se divisent de façon asymétrique doivent tout d’abord se polariser et correctement orienter leur fuseau mitotique pour ségréger des déterminants cellulaires en deux entités distinctes. L’embryon du nématode C. elegans est un modèle robuste et largement utilisé pour étudier la division cellulaire asymétrique. Dans cet embryon, le point d'entrée du spermatozoïde détermine l'axe de polarité antéro-postérieur. Suite à la fécondation, le cortex embryonnaire est uniformément contractile et un complexe conservé formé des protéines PAR-3, PAR-6 et PKC-3 (nommé complexe PAR-3 ci-dessous) est localisé sur l'ensemble du cortex. La complétion de la méiose maternelle induit une relaxation corticale au postétieur et un flux cortical vers l’antérieur de l’embryon. Ces contractions corticales asymétriques mènent à la formation d'un domaine antérieur contenant le complexe PAR-3, tandis que le cortex postérieur, dont le complexe PAR-3 s’est délocalisé, est enrichi avec les protéines PAR-2 et PAR-1. Par conséquent, les domaines formés par les protéines PAR définissent un pôle antérieur et un pôle postérieur dans l'embryon suite au remodelage du cytosquelette. Les protéines PAR-4 et PAR-5 restent localisées de façon uniforme dans l'embryon. Curieusement, les protéines PAR exercent une régulation par rétroaction sur la contractilité corticale. Il a été montré qu’une des protéines PAR récemment identifiée, PAR-5, est orthologue à la protéine adaptatrice 14-3-3 et joue un rôle important dans la contractilité corticale. En dépit de son rôle central dans la contractilité corticale et le processus de polarisation cellulaire, le mécanisme par lequel PAR-5 régule la contractilité corticale n’est pas bien compris. Le but de ce projet est de mieux comprendre comment PAR-5 et ses interacteurs contrôlent la régulation des contractions corticales et, de ce fait, la polarité cellulaire. Dans un essai de capture de la protéine GST (GST pull-down), nous avons identifié plusieurs nouveaux interacteurs de PAR-5. Parmi ceux-ci, nous avons trouvé CAP-2 (protéine de coiffage de l'actine), qui a été identifiée dans des éxpériences de capture de 14-3-3 dans trois systèmes modèles différents. CAP-2 est un hétérodimère des protéines CAP, qui sont impliquées dans la régulation de l'actine. Nous avons trouvé que la déplétion des protéines CAP par interférence à l’ARN dans des vers de type sauvage mène à une augmentation létalité embryonnaire, ce qui suggère que ces protéines jouent un rôle important dans le développement embryonnaire. L'imagerie en temps réel d'embryons déplétés pour les protéines CAP montre qu’ils ont une diminution des contractions corticales avec un sillon de pseudoclivage mois stable, suggérant un défaut dans la régulation du cytosquelette d'actine-myosine. Ceci a également été confirmé par la diminution de la vitesse et du nombre de foci de NMY-2::GFP. En outre, ces embryons montrent une légère diminution de la taille du croissant cortical de PAR-2 lors de la phase d’établissement de la polarité. Les embryons déplétés en CAP-2 montrent également un retard dans la progression du cycle cellulaire, mais le lien entre ce phénotype et la régulation des contractions corticales reste à être précisé. La caractérisation des protéines CAP, des régulateurs du remodelage du cytosquelette, permettra d'améliorer notre compréhension des mécanismes qui sous-tendent l'établissement et le maintien de la polarité cellulaire, et donc la division cellulaire asymétrique.

L'acide valproïque inhibe la progression dans le cycle cellulaire chez Saccharomyces cerevisiae

L’acétylation est une modification post-traductionnelle des protéines essentielles. Elle est impliquée dans bon nombre de processus cellulaires importants comme la régulation de la structure de la chromatine et le recrutement de protéines. Deux groupes d’enzymes, soient les lysines acétyltransférases et les lysines désacétylases, régulent cette modification, autant sur les histones que sur les autres protéines. Au cours des dernières années, de petites molécules inhibitrices des désacétylases ont été découvertes. Certaines d’entre elles semblent prometteuses contre diverses maladies telles le cancer. L’acide valproïque, un inhibiteur de deux des trois classes des désacétylases, a un effet antiprolifératif chez plusieurs organismes modèles. Toutefois, les mécanismes cellulaires sous-jacents à cet effet restent encore méconnus. Ce mémoire met en lumière l’effet pH dépendant de l’acide valproïque sur différentes voies cellulaires importantes chez la levure Saccharomyces cerevisiae. Il démontre que ce composé a la capacité d’inhiber la transition entre les phases G1 et S par son action sur l’expression des cyclines de la phase G1. De plus, il inhibe l’activation de la kinase principale de la voie activée suite à un stress à la paroi cellulaire. L’acide valproïque occasionne également un arrêt dans la réplication de l’ADN sans y causer de dommage. Il s’agit là d’un effet unique qui, à notre connaissance, n’est pas observable avec d’autres agents qui inhibent la progression en phase S.