Colin D Clyne

Oita University, Ōita, Oita, Japan

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Publications (78)348.99 Total impact

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    ABSTRACT: Ethnopharmacological relevance: Ficus umbellata Vahl. (Moraceae) is a medicinal plant used in Cameroon to treat amenorrhea as well as other physiological disorders related to menopause. Aim of study: In order to justify scientifically its traditional use, the estrogen-like properties of the aqueous (AE) and methanol (MeOH) extracts of F. umbellata were investigated. Material and methods: In vitro, the ability of different extracts of F. umbellata to activate estrogen receptors α (ERα) and β (ERβ) in cell-based reporter gene assays using human embryonic kidney (HEK293T) cells transfected with ERs was tested. In vivo, a 3-day uterotrophic assay and the capacity of the extracts to alleviate hot flushes in ovariectomized adult rats were tested. Using a bioassay-guided fractionation the major compound of F. umbellata was isolated and tested in vitro on HEK293T-ERα and ERβ cells. Results: AE and MeOH extracts significantly altered ERα as well as ERβ activities. In vivo, both extracts significantly increase the uterine and vaginal epithelium thickness, and uterine total protein levels in a dose dependent manner. Interestingly, both extracts of F. umbellata at the dose of 100 mg/kg BW significantly decreased the total number, average duration as well as frequency of hot flushes in experimental rats compared to age-matched OVX controls. Finally, 7-methylumbelliferone, a coumarin was characterized as the major compound of F. umbellata; however this compound did not transactivate ERα as well ERβ in vitro. Conclusion: These aforementioned results suggest that F. umbellata extracts as used by the traditional practitioner have estrogen-like effects and may alleviate some menopausal problems such as vaginal dryness and hot flushes.
    Full-text · Article · Jan 2016 · Journal of ethnopharmacology
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    Reidun Aesoy · Colin D Clyne · Ashwini L Chand
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    ABSTRACT: There is emerging evidence asserting the importance of orphan nuclear receptors (ONRs) in cancer initiation and progression. In breast cancer, there is a lot unknown about ONRs in terms of their expression profile and their transcriptional targets in the various stages of tumor progression. With the classification of breast tumors into distinct molecular subtypes, we assess ONR expression in the different breast cancer subtypes and with patient outcomes. Complementing this, we review evidence implicating ONR-dependent molecular pathways in breast cancer progression to identify candidate ONRs as potential prognostic markers and/or as therapeutic targets.
    Full-text · Article · Aug 2015 · Frontiers in Endocrinology

  • No preview · Article · Aug 2015 · Cancer Research
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    ABSTRACT: Flavonoids are secondary metabolites abundantly present in commonly consumed fruits and vegetables. They possess diverse properties such as anti-inflammatory, anti-oxidant and anti-cancer. Epidemiologic studies suggest that an enrich flavonoids diet is linked to a decreased risk of breast cancer. These protective properties are due to the alteration of numerous signaling pathways involved in cancer-related phenomena such as inflammation and proliferation. Human clinical trials examining the effect of supplementation of some flavonoids on disease prevention have been conducted. There is no natural flavonoid that has been approved for the treatment of breast cancer. However, natural flavonoids served as lead compounds in the synthesis of cancer chemo preventive and/or therapeutic agents.
    Full-text · Article · Jul 2015 · Current Medicinal Chemistry
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    ABSTRACT: Aromatase is the critical enzyme that converts androgens to estrogens. It is frequently highly expressed in the tumour bearing breast of women diagnosed with estrogen receptor positive tumours, resulting in dramatically increased local estrogen production to drive tumour progression. Expression of aromatase is regulated primarily at the transcriptional level of its encoding gene CYP19A1, located on chromosome 15 of the human genome. A characteristic feature of CYP19A1 expression is its use of alternative promoters to regulate transcription in a tissue-specific manner. In breast cancer, the increase in aromatase expression is mediated via higher expression of the distal adipose-specific promoter I.4 and a switch to the preferential use of proximal promoters I.3 and II. This results in a net increase of CYP19A1 transcripts in tumour-bearing breast up to 3–4-fold higher than normal breast. Current aromatase inhibitors – whilst efficacious – exhibit significant side effects that reduce patient compliance. Understanding the transcription factors and signalling pathways that control aromatase expression will lead to opportunities to develop breast-specific inhibitors with an improved side-effects profile.
    No preview · Article · Jan 2015 · The Journal of steroid biochemistry and molecular biology
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    ABSTRACT: The mineralocorticoid receptor (MR) is a member of the nuclear receptor superfamily. Pathological activation of the MR causes cardiac fibrosis and heart failure, but clinical use of MR antagonists is limited by the renal side effect of hyperkalemia. Coregulator proteins are known to be critical for nuclear receptor-mediated gene expression. Identification of coregulators which mediate MR activity in a tissue-specific manner may allow the development of novel tissue-selective MR modulators that confer cardiac protection without adverse renal effects. Our earlier studies identified a consensus motif amongst MR-interacting peptides, MPxLxxLL. Gem-associated protein 4 (Gemin4) is one of the proteins which contains this motif. Transient transfection experiments in HEK293 and H9c2 cells demonstrated that Gemin4 repressed agonist-induced MR transactivation in a cell-specific manner. Furthermore, overexpression of Gemin4 significantly decreased, while knockdown of Gemin4 increased, the mRNA expression of specific endogenous MR target genes. A physical interaction between Gemin4 and MR is suggested by their nuclear co-localisation upon agonist treatment. These findings indicate that Gemin4 functions as a novel coregulator of the MR.
    No preview · Article · Jan 2015 · Journal of Molecular Endocrinology

  • No preview · Article · Oct 2014 · Cancer Research
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    ABSTRACT: The mineralocorticoid receptor (MR) plays a central role in salt and water homeostasis via the kidney; however, inappropriate activation of the MR in the heart can lead to heart failure. A selective MR modulator that antagonizes MR signaling in the heart but not the kidney would provide the cardiovascular protection of current MR antagonists but allow for normal electrolyte balance. The development of such a pharmaceutical requires an understanding of coregulators and their tissue-selective interactions with the MR, which is currently limited by the small repertoire of MR coregulators described in the literature. To identify potential novel MR coregulators, we used T7 phage display to screen tissue-selective cDNA libraries for MR-interacting proteins. Thirty MR binding peptides were identified, from which three were chosen for further characterization based on their nuclear localization and their interaction with other MR-interacting proteins, or in the case of XRCC6, its known status as an androgen receptor coregulator. Eukaryotic elongation factor 1A1 (EEF1A1), structure-specific recognition protein 1 (SSRP1), and x-ray repair cross-complementing protein 6 (XRCC6) modulated MR-mediated transcription in a ligand-, cell- and/or promoter-specific manner, and co-localized with the MR upon agonist treatment when imaged using immunofluorescence microscopy. These results highlight the utility of phage display for rapid and sensitive screening of MR binding proteins, and suggest that EEF1A1, SSRP1 and XRCC6 may be potential MR coactivators whose activity is dependent on ligand, cellular context and target gene promoter.
    Full-text · Article · Jul 2014 · Molecular Endocrinology
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    ABSTRACT: The cytokine Tumor Necrosis Factor-α is critical to Estrogen Receptor positive breast cancer pathology, stimulating estrogen-biosynthesis pathways and preventing the differentiation of estrogen-producing fibroblasts. High concentrations of TNFα are detected in the tumor microenvironment, and infiltrating immune cells are thought to be a major source. This study identifies that TNFα is also a tumor-derived factor, expressed in ER+ tumour epithelial cells and regulated by 17-β-estradiol (E2). Treatment of MCF-7, T47D and ZR-75 breast cancer cells with E2 increased TNFα mRNA and protein expression and secretion. This effect was mitigated with the use of ERα inhibitors 4-hydroy-tamoxifen and ICI-182780, indicating that E2-mediated TNFα induction was via the actions of ERα. Chromatin immunoprecipitation reveals ERα binding to the TNFα promoter upon stimulation with E2. This study demonstrates for the first time a positive feedback loop between estradiol and TNFα, critical in maintaining high levels of the hormone within the ER+ breast tumour microenvironment.
    No preview · Article · Jul 2014 · Molecular and Cellular Endocrinology
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    ABSTRACT: Nelumal A, the active principle of Ligularia nelumbifolia was preliminarily tested as an aromatase inhibitors in HEK293 cells transfected with aromatase cDNA and using anastrazole as the reference drug. This screening revealed that it showed an appreciable level of inhibition. Subsequent experiments aimed to evaluate the aromatase activity and expression in KGN cells confirmed that the title natural product, after an incubation of 48 h, compared favourably with anastrazole (1 microM) in the concentration range 10-30 microM. Moreover, nelumal A (30 microM) abolished the aromatase mRNA expression in the same cell line.
    Full-text · Article · Jun 2014 · Natural product communications
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    ABSTRACT: Although molecular signatures based on transcript expression in breast cancer samples have provided new insights into breast cancer classification and prognosis, there are acknowledged limitations in current signatures. To provide rational, pathway-based signatures of disrupted physiology in cancer tissues that may be relevant to prognosis, this study has directly quantitated changed gene expression, between normal breast and cancer tissue, as a basis for signature development. The nuclear receptor (NR) family of transcription factors, and their coregulators, are fundamental regulators of every aspect of metazoan life, and were rigorously quantified in normal breast tissues and ERα positive and ERα negative breast cancers. Coregulator expression was highly correlated with that of selected NR in normal breast, particularly from postmenopausal women. These associations were markedly decreased in breast cancer, and the expression of the majority of coregulators was down-regulated in cancer tissues compared with normal. While in cancer the loss of NR-coregulator associations observed in normal breast was common, a small number of NR (Rev-ERBβ, GR, NOR1, LRH-1 and PGR) acquired new associations with coregulators in cancer tissues. Elevated expression of these NR in cancers was associated with poorer outcome in large clinical cohorts, as well as suggesting the activation of ERα -related, but ERα-independent, pathways in ERα negative cancers. In addition, the combined expression of small numbers of NR and coregulators in breast cancer was identified as a signature predicting outcome in ERα negative breast cancer patients, not linked to proliferation and with predictive power superior to existing signatures containing many more genes. These findings highlight the power of predictive signatures derived from the quantitative determination of altered gene expression between normal breast and breast cancers. Taken together, the findings of this study identify networks of NR-coregulator associations active in normal breast but disrupted in breast cancer, and moreover provide evidence that signatures based on NR networks disrupted in cancer can provide important prognostic information in breast cancer patients.
    No preview · Article · Apr 2014 · Molecular oncology
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    ABSTRACT: Liver Receptor Homolog-1 (LRH-1) is an orphan nuclear receptor that belongs to the NR5A subgroup of nuclear receptors. LRH-1 induces key genes to regulate metabolic process, ovarian function, cancer cell proliferation and steroidogenesis. In the breast, LRH-1 modulates and synergizes with endogenous estrogen signalling to promote breast cancer cell proliferation. We used siRNA knockdown strategies in order to deplete LRH-1 in breast cancer cells and followed with microarray analysis to identify LRH-1 dependent mechanisms. We identified key genes involved in TGF-β signalling to be highly responsive to LRH-1 knockdown. This relationship was validated in two breast cancer cell lines over-expressing LRH-1 in vitro and in a novel transgenic mouse with targeted LRH-1 over-expression in mammary epithelial cells. Notably, TGF-β signalling was activated in LRH-1 over expressing breast cancer cells and mouse mammary glands. Further analyses of mammary gross morphology revealed a significant reduction in mammary lateral budding after LRH-1 over expression. These findings suggest that the altered mammary morphogenesis in LRH-1 transgenic animals is mediated via enhanced TGF-β expression. The regulation of TGF-β isoforms and SMAD2/3-mediated downstream signalling by LRH-1 also implicates a potential contribution of LRH-1 in breast cancer. Collectively this data demonstrates that LRH-1 regulates TGF-β expression and downstream signalling in mouse mammary glands.
    No preview · Article · Feb 2014 · Endocrinology
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    ABSTRACT: The heritable component of breast cancer accounts for only a small proportion of total incidences. Environmental and lifestyle factors are therefore considered one of the major influencing components increasing breast cancer risk. Endocrine disrupting chemicals (EDCs) are ubiquitous in the environment. The estrogenic property of EDCs has thus seen many associations between ongoing exposures and the development of endocrine-related diseases, including breast cancer. The environment consists of a heterogenous population of EDCs and despite many identified modes of action, including that of altering the epigenome, drawing definitive correlations to breast cancer has been a point of much discussion. In this review we describe in detail well characterised EDCs and their actions in the environment; their ability to disrupt mammary gland formation in animal and human experimental models; and their associations with exposure and breast cancer risk. We also highlight the susceptibility of early-life exposure of each EDC to mediate epigenetic alterations, and where possible describe how these epigenome changes influence breast cancer risk.
    No preview · Article · Feb 2014 · Endocrine Related Cancer
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    ABSTRACT: Estrogens are known to play a role in modulating metabolic processes within the body. The Aromatase knockout (ArKO) mice have been shown to harbor factors of Metabolic syndrome with central adiposity, hyperinsulinemia and male-specific hepatic steatosis. To determine the effects of estrogen ablation and subsequent replacement in males on whole body glucose metabolism, three- and six-month-old male ArKO mice were subjected to whole body glucose, insulin and pyruvate tolerance tests and analyzed for ensuing metabolic changes in liver, adipose tissue, and skeletal muscle. Estrogen-deficient male ArKO mice showed increased gonadal adiposity which was significantly reduced upon 17β-estradiol (E2) treatment. Concurrently, elevated ArKO serum leptin levels were significantly reduced upon E2 treatment and lowered serum adiponectin levels were restored to wild type levels. Three-month-old male ArKO mice were hyperglycemic, and both glucose and pyruvate intolerant. These phenotypes continued through to 6 months of age, highlighting a loss of glycemic control. ArKO livers displayed changes in gluconeogenic enzyme expression, and in insulin signaling pathways upon E2 treatment. Liver triglycerides were increased in the ArKO males only after 6 months of age, which could be reversed by E2 treatment. No differences were observed in insulin-stimulated ex vivo muscle glucose uptake nor changes in ArKO adipose tissue and muscle insulin signaling pathways. Therefore, we conclude that male ArKO mice develop hepatic glucose intolerance by the age of 3 months which precedes the sex-specific development of hepatic steatosis. This can be reversed upon the administration of exogenous E2.
    Full-text · Article · Feb 2014 · PLoS ONE
  • Kevin C Knower · Ashwini L Chand · Colin D Clyne

    No preview · Article · Jan 2014 · Breast Cancer Research and Treatment
  • [Show abstract] [Hide abstract]
    ABSTRACT: Although molecular signatures based on transcript expression in breast cancer samples have provided new insights into breast cancer classification and prognosis, there are acknowledged limitations in current signatures. To provide rational, pathway-based signatures of disrupted physiology in cancer tissues that may be relevant to prognosis, this study has directly quantitated changed gene expression, between normal breast and cancer tissue, as a basis for signature development. The nuclear receptor (NR) family of transcription factors, and their coregulators, are fundamental regulators of every aspect of metazoan life, and were rigorously quantified in normal breast tissues and ERα positive and ERα negative breast cancers. Coregulator expression was highly correlated with that of selected NR in normal breast, particularly from postmenopausal women. These associations were markedly decreased in breast cancer, and the expression of the majority of coregulators was down-regulated in cancer tissues compared with normal. While in cancer the loss of NR-coregulator associations observed in normal breast was common, a small number of NR (Rev-ERBβ, GR, NOR1, LRH-1 and PGR) acquired new associations with coregulators in cancer tissues. Elevated expression of these NR in cancers was associated with poorer outcome in large clinical cohorts, as well as suggesting the activation of ERα -related, but ERα -independent, pathways in ERα negative cancers. In addition, the combined expression of small numbers of NR and coregulators in breast cancer was identified as a signature predicting outcome in ERα negative breast cancer patients, not linked to proliferation and with predictive power superior to existing signatures containing many more genes. These findings highlight the power of predictive signatures derived from the quantitative determination of altered gene expression between normal breast and breast cancers. Taken together, the findings of this study identify networks of NR-coregulator associations active in normal breast but disrupted in breast cancer, and moreover provide evidence that signatures based on NR networks disrupted in cancer can provide important prognostic information in breast cancer patients.
    No preview · Article · Jan 2014 · Molecular Oncology
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    ABSTRACT: The interaction between breast tumor epithelial and stromal cells is vital for initial and recurrent tumor growth. While breast cancer-associated stromal cells provide a favorable environment for proliferation and metastasis, the molecular mechanisms contributing to this process are not fully understood. Nuclear receptors (NRs) are intracellular transcription factors that directly regulate gene expression. Little is known about the status of NRs in cancer-associated stroma. Nuclear Receptor Low-Density Taqman Arrays were used to compare the gene expression profiles of all 48 NR family members in a collection of primary cultured cancer-associated fibroblasts (CAFs) obtained from estrogen receptor (ER)α positive breast cancers (n = 9) and normal breast adipose fibroblasts (NAFs) (n = 7). Thirty-three of 48 NRs were expressed in both the groups, while 11 NRs were not detected in either. Three NRs (dosage-sensitive sex reversal, adrenal hypoplasia critical region, on chromosome X, gene 1 (DAX-1); estrogen-related receptor beta (ERR-β); and RAR-related orphan receptor beta (ROR-β)) were only detected in NAFs, while one NR (liver receptor homolog-1 (LRH-1)) was unique to CAFs. Of the NRs co-expressed, four were significantly down-regulated in CAFs compared with NAFs (RAR-related orphan receptor-α (ROR-α); Thyroid hormone receptor-β (TR-β); vitamin D receptor (VDR); and peroxisome proliferator-activated receptor-γ (PPAR-γ)). Quantitative immunohistochemistry for LRH-1, TR-β, and PPAR-γ proteins in stromal fibroblasts from an independent panel of breast cancers (ER-positive (n = 15), ER-negative (n = 15), normal (n = 14)) positively correlated with mRNA expression profiles. The differentially expressed NRs identified in tumor stroma are key mediators in aromatase regulation and subsequent estrogen production. Our findings reveal a distinct pattern of NR expression that therefore fits with a sustained and increased local estrogen microenvironment in ER-positive tumors. NRs in CAFs may provide a new avenue for the development of intratumoral-targeted therapies in breast cancer.
    No preview · Article · Oct 2013 · Breast Cancer Research and Treatment
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    ABSTRACT: The present study aims to determine the estrogenicity of Millettia macrophylla, a Cameroonian medicinal plant, in ovariectomized rats and to investigate the underlying mechanisms, in order to justify scientifically its traditional use. To accomplish this objective, we used dichloromethane (DCM) and methanol (MeOH) extracts of the stem bark of M. macrophylla. In the cell culture based assay, the MeOH extract significantly transactivated estrogen receptor α (ERα) and estrogen receptor β (ERβ); in addition, the estrogen-like effects of both, DCM and MeOH extracts, could be inhibited in vitro by the pure ER antagonist ICI 182,780, indicating that these effects were primarily mediated through ERs. In animal experiments, both DCM and MeOH extracts significantly increased the uterine and vaginal epithelial heights in the 3-day treatment assay, while only the MeOH extract exhibited such effects in the sub-chronic treatment regimen. Furthermore, the MeOH extract significantly decreased fasting serum triglycerides, total cholesterol levels and artherogenic risk in the sub-chronic treatment. These results indicate that M. macrophylla extracts have estrogen-like effects supporting their traditional use in Cameroon to alleviate some menopausal problems (See graphical abstract in Supplementary Fig. 1, available in the online version only).
    Full-text · Article · Sep 2013 · Journal of Pharmacological Sciences
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    ABSTRACT: The present study aims to determine the estrogenicity of Millettia macrophylla , a Cameroonian medicinal plant, in ovariectomized rats and to investigate the underlying mecha -nisms, in order to justify scientifically its traditional use. To accomplish this objective, we used dichloromethane (DCM) and methanol (MeOH) extracts of the stem bark of M. macrophylla. In the cell culture based assay, the MeOH extract significantly transactivated estrogen receptor a (ER a) and estrogen receptor b (ER b); in addition, the estrogen-like effects of both, DCM and MeOH extracts, could be inhibited in vitro by the pure ER antagonist ICI 182,780, indicating that these effects were primarily mediated through ERs. In animal experiments, both DCM and MeOH extracts significantly increased the uterine and vaginal epithelial heights in the 3-day treatment assay, while only the MeOH extract exhibited such effects in the sub-chronic treatment regimen. Furthermore, the MeOH extract significantly decreased fasting serum triglycerides, total cholesterol levels and artherogenic risk in the sub-chronic treatment. These results indicate that M. macrophylla extracts have estrogen-like effects supporting their traditional use in Cameroon to alleviate some menopausal problems (See graphical abstract in Supplementary Fig. 1, available in the online version only).
    Full-text · Article · Sep 2013 · Journal of Pharmacological Sciences
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    ABSTRACT: The expression of orphan nuclear receptor Liver Receptor Homolog-1 (LRH-1) is elevated in breast cancer and promotes proliferation, migration and invasion in vitro. LRH-1 expression is regulated by oestrogen (E2), with LRH-1 mRNA transcript levels higher in oestrogen receptor α (ERα) positive (ER+) breast cancer cells compared to ER- cells. However, the presence of LRH-1 protein in ER- cells suggests discordance between mRNA transcript levels and protein expression. To understand this, we investigated the impact of mRNA and protein stability in determining LRH-1 protein levels in breast cancer cells. LRH-1 transcript levels were significantly higher in ER+ versus ER- breast cancer cells lines; however LRH-1 protein was expressed at similar levels. We found LRH-1 mRNA and protein was more stable in ER- compared to ER+ cell lines. The tumour-specific LRH-1 variant isoform, LRH-1v4, which is highly responsive to E2, showed increased mRNA stability in ER- versus ER+ cells. In addition, in MCF-7 and T47-D cell lines, LRH-1 total mRNA stability was reduced with E2 treatment, this effect mediated by ERα. Our data demonstrates that in ER- cells, increased mRNA and protein stability contribute to the abundant protein expression levels. Expression and immunolocalisation of LRH-1 in ER- cells as well as ER- tumours suggests a possible role in the development of ER- tumors. The modulation of LRH-1 bioactivity may therefore be beneficial as a treatment option in both ER- and ER+ breast cancer.
    Full-text · Article · Aug 2013 · Biochemical and Biophysical Research Communications

Publication Stats

3k Citations
348.99 Total Impact Points

Institutions

  • 2015
    • Oita University
      Ōita, Oita, Japan
  • 2002-2015
    • University of Vic
      Vic, Catalonia, Spain
  • 2002-2014
    • Prince Henry's Institute
      • Laboratory of Cancer Drug Discovery
      Melbourne, Victoria, Australia
  • 1996-2009
    • University of Texas Southwestern Medical Center
      • • Department of Obstetrics and Gynecology
      • • Division of Reproductive Endocrinology and Infertility
      Dallas, Texas, United States
  • 2006
    • Duke University Medical Center
      • Department of Pharmacology and Cancer Biology
      Durham, NC, United States
  • 2002-2005
    • Monash University (Australia)
      • Department of Biochemistry and Molecular Biology
      Melbourne, Victoria, Australia
  • 2004
    • Università della Calabria
      • Department of Pharmaco-Biology
      Rende, Calabria, Italy