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Raghida Abed Almawla Abou Merhi

Full professor
Life & Earth Sciences department - Section I - Hadath
Speciality: Biology
Specific Speciality: Molecular and Cellular physiopathology / Cytogenetics
Interests: History, exotic culture, music, landscape

Teaching 9 Taught Courses
(2014-2015) BioA 402 - Cancerology

M1 Applied Animal Science

(2014-2015) GNSA 501 - Functional Genomics

M2 Genomics and Health

(2014-2015) GNSA 503 - Applied Genetics

M2 Genomics and Health

(2014-2015) BioA 414 - Genetics of organisms and populations

M1 Applied Animal Science

(2014-2015) BioA 415 - Genetics of organisms and populations Lab

M1 Applied Animal Science

(2014-2015) GNSA 580 - Master thesis

M2 Genomics and Health

(2014-2015) GNSA 580 - Master thesis

M2 Genomics and Health

(2014-2015) Biol 380 - Molecular Genetics

BS Biochemistry

(2014-2015) Biol 328 - General Biotechnology

BS Earth and life sciences

Education
1993 - 1997: Phd (doctoral thesis)

Pierre and Marie Curie University (Paris VI)
Molecular and Cellular Physiopathology

Very Honorable

1992 - 1993: DEA (Saint antoine/ Kremlin Bicetre Hospital)

Pierre et Marie Curie University (Paris VI)
Molecular and Cellular Physiopathology

1985 - 1988:

Lebanese University- Faculty of Sciences
Natural Sciences

Good (high Honors)

Conferences 23 participations
4ème Rencontre Internationale en « Cancer Biology and Stem Cells Research Meeting » EDST-LU

Seminar
2014-10-16 to 2014-10-17

20th LAAS

Poster session
2014-04-27 to 2014-04-29

4th Annual Basic Biomedical Research Day-AUB

Poster session
2014-02-15 to 2014-02-15

3rd International Meeting in Cellular and Molecular Biology, BioBeirut. LU- EDST

Congress
2013-10-30 to 2013-10-31

19th LAAS International Science Conference- LAU

Conference
2013-04-05 to 2013-04-06

2nd International Meeting in Cellular and Molecular Biology LU- EDST

Congress
2012-11-16 to 2012-11-17

Symposium about Biomedical and Translational Research- Qatar

Symposium
2012-05-16 to 2012-05-17

18th LAAS International Science Meeting: New Discoveries in Science, NDU

Conference
2012-03-22 to 2012-03-24

1st International Meeting in Cellular and Molecular Immunology LU- EDST

Congress
2011-11-17 to 2011-11-18

GDR meeting (Genome Dynamics Retrait in Brissac, Mas de Luziere, St. Andre de Buege

Symposium
2011-06-27 to 2011-06-28

Cancer meeting- at AUB/DTS

Seminar
2011-06-07 to 2011-06-07

1st Annual AUB Basic Biomedical Research Day

Poster session
2011-02-19 to 2011-02-19

First Kamal A. Shair Central Research Science Laboratory Research Conference

Poster session
2010-06-17 to 2010-06-17

Scientific Research Day-AUB

Poster session
2010-05-22 to 2010-05-22

Scientific Research Day 2010

Poster session
2010-05-08 to 2010-05-08

Faculty of Health Science at AUST on Thursday, June 4th, 2009.

Seminar
2009-06-04 to 2009-06-04

Joint US-NCI/Jordan oncology society/King Hussein cancer center cancer conference. Amman, Jordan, 2004.

Congress
2004-01-01 to 2004-01-01

8th congress of the European heamatology association. Lyon, France, 2003

Poster session
2003-01-01 to 2003-01-01

7th congress of the European haematology association. Florence Italy, 2002. The haematology journal, 2002.

Conference
2002-01-01 to 2002-01-01

10th international conference on human retrovirology: HTLV and related viruses, Dublin, Ireland, 2001.

Poster session
2001-01-01 to 2001-01-01

ESHRE 15th Annual, 27 June-30 June 1999, Tours, France.

Poster session
1999-06-27 to 1999-06-30

Colloque Club Français de la différenciation et du déterminisme du sex.12 décembre 1996- Nancy France.

Colloquium
1996-12-12 to 1996-12-12

Symosium Sex differenciation: Clinical and Biological aspects.1-3 July 1995 Cambridge-UK.

Congress
1995-07-01 to 1995-07-03

Publications 18 publications
Ghewa A. El-Achkar, Mariam Jouni, May F. Mrad, Taghreed Hirz, Nehme El Hachem, Ali Khalaf, Soukaina Hammoud, Hussein Fayyad-Kazan, Assaad A. Eid, Bassam Badran, Raghida Abou Merhi, Ali Hachem, Eva Hamade, Aïda Habib Thiazole derivatives as inhibitors of cyclooxygenases in vitro and in vivo Eur J Pharmacol. 2015 Jan 21. pii: S0014-2999(15)00030-8. doi: 10.1016/j.ejphar.2015.01.008. [Epub ahead of print 2015

Cyclooxygenases (COXs) are important membrane-bound heme containing enzymes important in platelet activation and inflammation. COX-1 is constitutively expressed in most cells whereas COX-2 is an inducible isoform highly expressed in inflammatory conditions. Studies have been carried out to evaluate thiazole derivatives as anti-inflammatory molecules. In this study, we investigated the in vitro and in vivo effects of two novel thiazole derivatives compound 1 (N-[4-(4-hydroxy-3-methoxyphenyl)-1,3-thiazol-2-yl] acetamide) and compound 2 (4-(2-amino-1,3-thiazol-4-yl)-2-methoxyphenol) on PGE2 production and COX activity in inflammatory settings. Our results reveal a potent inhibition of both compound 1 (IC50 9.01±0.01µM) and 2 (IC50 11.65±6.20µM) (Mean±S.E.M.) on COX-2-dependent PGE2 production. We also determined whether COX-1 activity was inhibited. Using cells stably over-expressing COX-1 and human blood platelets, we showed that compound 1 is a specific inhibitor of COX-1 with IC50 (5.56×10-8±2.26×10-8µM), whereas compound 2 did not affect COX-1. Both compounds exhibit anti-inflammatory effect in the dorsal air pouch model of inflammation as shows by inhibition of PGE2 secretion. Modeling analysis of docking in the catalytic site of COX-1 or COX-2 further confirmed the difference in the effect of these two compounds. In conclusion, this study contributes to the design of new anti-inflammatory agents and to the understanding of cyclooxygenase inhibition by thiazole.

Racha Al Halabi, Raghida Abou Merhi, Sahar Al-ayyoubi, Chirine El Baba, Eva Hamade, Saritha Shakilam, Matthias Ocker, Regine Schneider-Stock, Hala Gali-Muhtasib. Gallotannin is a DNA damaging compound that induces senescence independently of p53 and p21 in human colon cancer cells. Molecular Carcinogenesis; DOI 10.1002/mc.22172, 2014, Published online in Wiley Online Library 2014

he plant secondary metabolite gallotannin (GT) is the simplest hydrolyzable tannin shown to have anti-carcinogenic properties in several cell lines and to inhibit tumor development in different animal models. Here, we determined if GT induces senescence and DNA damage and investigated the involvement of p53 and p21 in this response. Using HCT116 human colon cancer cells wildtype for p53+/+/p21+/+ and null for p53+/+/p21−/− or p53−/−/p21+/+, we found that GT induces senescence independently of p21 and p53. GT was found to increase the production of reactive oxygen species (ROS) by altering the redox balance in the cell, mainly by reducing the levels of glutathione and superoxide dismutase (SOD). Using the key antioxidants N-acetyl cysteine, dithiothreitol, SOD, and catalase, we showed that ROS were partially involved in the senescence response. Furthermore, GT-induced cell cycle arrest in S-phase in all HCT116 cell lines. At later time points, we noticed that p53 and p21 null cells escaped complete arrest and re-entered cell cycle provoking higher rates of multinucleation. The senescence induction by GT was irreversible and was accompanied by significant DNA damage as evidenced by p-H2AX staining. Our findings indicate that GT is an interesting anti colon cancer agent which warrants further study

Hiba El Hajj, Raghida Abou Merhi, Ali Bazarbachi. Human Herpes Virus type 8/Kaposi Sarcoma Herpes Virus: Overview on viral pathogenesis and treatment of associated diseases. Boca Raton, FL : Taylor & Francis/CRC Press, [2014] Cancer Causing Viruses and Their Inhibitors, 1st edition - Introduction - Chapter 10 2014

1 online resource : text file, PDF "Cancer-causing viruses, also called oncoviruses, play a key role in the development of certain cancers. They contribute to genetic changes that disrupt the cell cycle machinery, interfering with functions such as cell growth. Cancer-Causing Viruses and Their Inhibitors presents a plethora of research from internationally reputed contributors who discuss different types of oncoviruses, their mechanisms of invasion and growth, and their life cycles. The book begins with an overview of the oncoviruses discovered to date and includes a brief description of their structures, genotypes, replication, and mechanisms of infection leading to cancers. It then explores several of these viruses in detail, including: Human T-cell leukemia virus type 1 (HTLV-1), Hepatitis C virus (HCV), Epstein-Barr virus (EBV), Human papilloma virus (HPV), Human herpes virus 8 (HHV-8)/Kaposi's sarcoma-associated herpes virus (KSHV), Human immunodeficiency virus (HIV/AIDS), Oncolytic viruses. This book is an essential reference for those working in virology, oncology, and biotechnology. The discoveries presented will enable researchers and clinicians to optimize both historical and current approaches to anti-viral therapies"--Provided by publisher.

Dina Abdallah, Eva Hamade, Raghida Abou Merhi, Bassam Badran, Rene Buchet, Saida Mbarek. Fatty acid composition in matrix vesicles and in microvilli from femurs of chicken embryos revealed selective recruitment of fatty acids. Biochemical and Biophysical Research Communications BBRC; Apr 18;446(4):1161-4. 2014

Hypertrophic chondrocytes participate in matrix mineralization by releasing matrix vesicles (MVs). These MVs, by accumulating Ca(2+) and phosphate initiate the formation of hydroxyapatite. To determine the types of lipids essential for mineralization, we analyzed fatty acids (FAs) in MVs, microvilli and in membrane fractions of chondrocytes isolated from femurs of chicken embryos. The FA composition in the MVs was almost identical to that in microvilli, indicating that the MVs originated from microvilli. These fractions contained more monounsaturated FAs especially oleic acid than in membrane homogenates of chondrocytes. They were enriched in 5,8,11-eicosatrienoic acid (20:3n-9), in eicosadienoic acid (20:2n-6), and in arachidonic acid (20:4n-6). In contrast, membrane homogenates from chondrocytes were enriched in 20:1n-9, 18:3n-3, 22:5n-3 and 22:5n-6. Due to their relatively high content in MVs and to their selective recruitment within microvilli from where MV originate, we concluded that 20:2n-6 and 20:3n-9 (pooled values), 18:1n-9 and 20:4n-6 are essential for the biogenesis of MVs and for bone mineralization.

Jiovanni A. Di Battista, Wassim Shebaby, Ozge Kizilay, Eva Hamade, Raghida Abou Merhi, Saida Mebarek, Dina Abdallah, Bassam Badran, Fady Saad, Eddie K. Abdalla, Wissam H. Faour. Proliferation and differentiation of human adipose-derived mesenchymal stem cells (ASCs) into osteoblastic lineage are passage dependent. Inflamm Res. 2014 Nov;63(11):907-17. doi: 10.1007/s00011-014-0764-y. Epub 2014 Aug 7 2014

OBJECTIVE: The effect of in vitro expansion of human adipose-derived stem cells (ASCs) on stem cell properties is controversial. We examined serial subcultivation with expansion on the ability of ASCs to grow and differentiate into osteoblastic lineages. DESIGN: Flow cytometric analysis, growth kinetics, cell population doubling time, light microscopy and confocal analysis, and osteogenesis induction were performed to assess growth and osteogenic potential of subcultivated ASCs at passages 2 (P2), P4 and P6. RESULTS: Flow cytometric analysis revealed that ASCs at P2 express classical mesenchymal stem cell markers including CD44, CD73, and CD105, but not CD14, CD19, CD34, CD45, or HLA-DR. Calcium deposition and alkaline phosphatase activity were the highest at P2 but completely abrogated at P4. Increased passage number impaired cell growth; P2 cultures exhibited exponential growth, while cells at P4 and P6 showed near linear growth with cell population doubling times increased from 3.2 at P2 to 4.8 d at P6. Morphologically, cells in various subcultivation stages showed flattened shape at low density but spindle-like structures at confluency as judged by phalloidin staining. CONCLUSIONS: Osteogenic potential of ASCs is impaired by successive passaging and may not serve as a useful clinical source of osteogenic ASCs past P2.

Maamoun Fatfat, Raghida Abou Merhi, Omar Rahal, Detcho A Stoyanovsky, Angela Zaki,1 Hazar Haidar, Valerian E Kagan, Hala Gali-Muhtasib, and Khaled Machaca Copper chelation selectively kills colon cancer cells through redox cycling and generation of reactive oxygen species. BMC Cancer. 2014 Jul 21;14:527. doi: 10.1186/1471-2407-14-527. 2014

Abstract BACKGROUND: Metals including iron, copper and zinc are essential for physiological processes yet can be toxic at high concentrations. However the role of these metals in the progression of cancer is not well defined. Here we study the anti-tumor activity of the metal chelator, TPEN, and define its mechanism of action. METHODS: Multiple approaches were employed, including cell viability, cell cycle analysis, multiple measurements of apoptosis, and mitochondrial function. In addition we measured cellular metal contents and employed EPR to record redox cycling of TPEN-metal complexes. Mouse xenografts were also performed to test the efficacy of TPEN in vivo. RESULTS: We show that metal chelation using TPEN (5μM) selectively induces cell death in HCT116 colon cancer cells without affecting the viability of non-cancerous colon or intestinal cells. Cell death was associated with increased levels of reactive oxygen species (ROS) and was inhibited by antioxidants and by prior chelation of copper. Interestingly, HCT116 cells accumulate copper to 7-folds higher levels than normal colon cells, and the TPEN-copper complex engages in redox cycling to generate hydroxyl radicals. Consistently, TPEN exhibits robust anti-tumor activity in vivo in colon cancer mouse xenografts. CONCLUSION: Our data show that TPEN induces cell death by chelating copper to produce TPEN-copper complexes that engage in redox cycling to selectively eliminate colon cancer cells.

Hiba El Hajj, Jihane Ali, Akram Ghantous, Dana Hodroj, Ahmad Daher, Kazem Zibara, Chloé Journo5, Zaher Otrock, Ghazi Zaatari, Renaud Mahieux, Marwan El Sabban, Ali Bazarbachi*, and Raghida Abou Merhi*. Combination of Arsenic and Interferon-α Inhibits expression of KSHV Latent Transcripts and Synergistically Improves Survival of Mice with Primary Effusion Lymphomas. PLOS1, V.8;11. 2013

BACKGROUND: Kaposi sarcoma-associated herpesvirus (KSHV) is the etiologic agent of primary effusion lymphomas (PEL). PEL cell lines infected with KSHV, but negative for Epstein-Barr virus have a tumorigenic potential in non-obese diabetic/severe combined immunodeficient mice and result in efficient engraftment and formation of malignant ascites with notable abdominal distension, consistent with the clinical manifestations of PEL in humans. METHODOLOGY/PRINCIPAL FINDINGS: Using this preclinical mouse model, we demonstrate that the combination of arsenic trioxide and interferon-alpha (IFN) inhibits proliferation, induces apoptosis and downregulates the latent viral transcripts LANA-1, v-FLIP and v-Cyc in PEL cells derived from malignant ascites. Furthermore, this combination decreases the peritoneal volume and synergistically increases survival of PEL mice. CONCLUSION/SIGNIFICANCE: These results provide a promising rationale for the therapeutic use of arsenic/IFN in PEL patients.

Taghreed Hirz, Ali Khalaf, Nehme El-Hachem, May F Mrad, Hassan Abdallah, Christophe Créminon, Raghida Abou Merhi, Aïda Habib, Ali Hachem, Eva Hamade. New analogues of 13-hydroxy-octadecadienoic acid and 12-hydroxyeicosatetraenoic acid block human blood platelet aggregation and cyclooxygenase-1 activity. Chemistry Central Journal 6:152. 2012

BACKGROUND: Thromboxane A2 is derived from arachidonic acid through the action of cyclooxygenases and thromboxane synthase. It is mainly formed in blood platelets upon activation and plays an important role in aggregation. Aspirin is effective in reducing the incidence of complications following acute coronary syndrome and stroke. The anti-thrombotic effect of aspirin is obtained through the irreversible inhibition of cyclooxygenases. Analogues of 12-hydroxyeicosatetraenoic acid and 13-hydroxyocatdecadienoic acid were shown previously to modulate platelet activation and to block thromboxane receptors. RESULTS AND DISCUSSION: We synthesized 10 compounds based on the structures of analogues of 12-hydroxyeicosatetraenoic acid and 13-hydroxyocatdecadienoic acid and evaluated their effect on platelet aggregation triggered by arachidonic acid. The structure activity relationship was evaluated. Five compounds showed a significant inhibition of platelet aggregation and highlighted the importance of the lipidic hydrophobic hydrocarbon chain and the phenol group. Their IC50 ranged from 7.5 ± 0.8 to 14.2 ± 5.7 μM (Mean ± S.E.M.). All five compounds decreased platelet aggregation and thromboxane synthesis in response to collagen whereas no modification of platelet aggregation in response to thromboxane receptor agonist, U46619, was observed. Using COS-7 cells overexpressing human cyclooxygenase-1, we showed that these compounds are specific inhibitors of cyclooxygenase-1 with IC50 ranging from 1.3 to 12 μM. Docking observation of human recombinant cyclooxygenase-1 supported a role of the phenol group in the fitting of cyclooxygenase-1, most likely related to hydrogen bonding with the Tyr 355 of cyclooxygenase-1. CONCLUSIONS: In conclusion, the compounds we synthesized at first based on the structures of analogues of 12 lipoxygenase metabolites showed a role of the phenol group in the anti-platelet and anti-cyclooxygenase-1 activities. These compounds mediate their effects via blockade of cyclooxygenase-1.

Racha Al-Halabi (1), MirellaBouChedid (1), Raghida Abou Merhi, Hiba El-Hajj, Hind Zahr, Regine Schneider-Stock, Ali Bazarbachi and HalaGali-Muhtasib. Gallotannin inhibits NF-kappa B signaling and Growth of human colon cancer xenografts. Cancer biology and therapy Volume 12, Issue 1, July 1 2011

Gallotannin (GT), the polyphenolic hydrolyzable tannin, exhibits anti-inflammatory and anticancer activities through mechanisms that are not fully understood. Several effects modulated by GT have been shown to be linked to interference with inflammatory mediators. Considering the central role of nuclear factor kappa B (NF-ĸB) in inflammation and cancer, we investigated the effect of GT on NF-ĸB signaling in HT-29 and HCT-116 human colon cancer cells. DNA binding assays revealed significant suppression of tumor necrosis factor (TNF-α)-induced NFĸB activation which correlated with the inhibition of IĸBα phosphorylation and degradation. Sequentially, p65 nuclear translocation and DNA binding were inhibited. GT also down-regulated the expression of NFĸB-regulated inflammatory cytokines (IL-8, TNF-α, IL-1α) and caused cell cycle arrest and accumulation of cells in pre-G 1 phase. In vivo, GT (25 mg/kg body weight) injected intraperitoneally (i.p.) prior to or after tumor inoculation significantly decreased the volume of human colon cancer xenografts in NOD/SCID mice. GT-treated xenografts showed significantly lower microvessel density (CD31) as well as lower mRNA expression levels of IL-6, TNF-α and IL-1α and of the proliferation (Ki-67) and angiogenesis (VEGFA) proteins, which may explain GTs in vivo anti-tumorigenic effects. Overall, our results indicate that the anti-inflammatory and antitumor activities of GT may be mediated in part through the suppression of NF-ĸB activation.

L. Hadad, H. Hajj, R. Abou Merhi, Y. Kfoury, R. Mahieux, M Sabban and A. Bazarbachi. KSHV Transformed Primary Effusion Lymphoma Cells Induce a VEGF dependent Angiogenesis and Establish Functional Gap Junctions with Endothelial Cells. Leukemia Apr;22(4):826-34. Epub 2007 Dec 20. 2008

Kaposi's sarcoma-associated herpesvirus (KSHV) is the causative agent of primary effusion lymphoma (PEL) and of Kaposi's sarcoma. PEL is an aggressive proliferation of B cells with poor prognosis. We evaluated both in vitro and in vivo the potential role of angiogenic factors secreted by PEL cells, that is, their interaction with endothelial cells and their implication in the invasive behavior of tumoral cells. In vitro, PEL-induced angiogenesis is dependent on vascular endothelial growth factor (VEGF) and VEGF receptors. However, although PEL cells produce VEGF and basic fibroblast growth factor (b-FGF) transcripts, they only secrete VEGF in vitro. In vivo, very high levels of both VEGF and b-FGF were found in the ascitic fluid of NOD/SCID mice injected with PEL cells. We then show evidence of cell adhesion and gap junction-mediated heterocellular communication between PEL cells and endothelial cells. Finally, we show that PEL cells extravasate through the endothelial barrier and that the specific tyrosine kinase inhibitor of VEGF receptors, PTK-787/ZK-222584, the anti-VEGF antibody, bevacizumab or the gap junction inhibitor 18-alpha-glycyrrhetinic acid, partially attenuate PEL cell extravasation. Angiogenesis, cell adhesion and communication likely contribute to the development of PEL and represent potential therapeutic targets.

R Abou-Merhi, R Khoriaty, D Arnoult, H El Hajj, H Dbouk, S Munier, ME El-Sabban, O Hermine, A Gessain, H de The, R Mahieux and A Bazarbachi. PS-341 or combination of arsenic trioxide and interferon-a inhibit growth and induce caspase-dependent apoptosis in KSHV/HHV-8-infected primary effusion lymphoma cells. Leukemia 21, 1792–1801 2007

Kaposi's sarcoma (KS)-associated herpes virus (KSHV) is the causative agent of primary effusion lymphoma and of KS. Primary effusion lymphoma (PEL) is an aggressive proliferation of B cells. Conventional chemotherapy has limited benefits in PEL patients, and the prognosis is very poor. We previously reported that treatment of human T-cell leukemia virus type 1 (HTLV-1)-associated adult T-cell leukemia/lymphoma cells either with arsenic trioxide (As) combined to interferon-alpha (IFN-alpha) or with the bortezomib (PS-341) proteasome inhibitor induces cell cycle arrest and apoptosis, partly due to the reversal of the constitutive nuclear factor-kappaB (NF-kappaB) activation. PEL cells also display an activated NF-kappaB pathway that is necessary for their survival. This prompted us to investigate the effects of PS-341, or of the As/IFN-alpha combination on PEL cells. A dramatic inhibition of cell proliferation and induction of apoptosis was observed in PS-341 and in As/IFN-alpha treated cells. This was associated with the dissipation of the mitochondrial membrane potential, cytosolic release of cytochrome c, caspase activation and was reversed by the z-VAD caspase inhibitor. PS-341 and As/IFN-alpha treatment abrogated NF-kappaB translocation to the nucleus and decreased the levels of the anti-apoptotic protein Bcl-X(L). Altogether, these results provide a rational basis for a future therapeutic use of PS-341 or combined As and IFN-alpha in PEL patients.

H. Dbouk, A. Tawil, F. Nasr, L. Kandakarjian and R. Abou Merhi. Significance of CEA and VEGF as diagnostic markers of colorectal cancer in Lebanese patients. The open clinical cancer (2007), I, 1-5. The open clinical cancer I, 1-5. 2007

Carcinoembryonic antigen and vascular endothelial growth factors are among the most important prognostic markers of colorectal cancer. Testing for these markers independently has been of limited value in screening for this tumor. The aim of this study is to determine the importance of simultaneous blood CEA and VEGF level determinations in diagnosis of colorectal cancer. Thirty-six patients diagnosed with colorectal cancer along with eight healthy controls were tested by ELISA for CEA and VEGF levels in serum and plasma, respectively. The positive predictive value of these markers was 95.4% for CEA and 89.5% for VEGF, and for combined CEA and VEGF was also high at 88%. Combined CEA and VEGF blood level assay constitutes a useful panel in detecting patients with colorectal cancer. Positive results allow selection of a subgroup of patients with a high tumor risk; therefore, such tests comprise valuable tumor diagnostic tests to add to current detection methods.

Bazarbachi A, Abou Merhi R., Gessain A., Talhouk R., El-Khoury H., Nasr R., Gout O., Sulahian R., Homaidan F., de The H., hermine O., El Sabban M. HTLV-1 infected cells extravasate through the endothelial barrier using a local angiogenesis-like mechanism. Cancer research; 64: 2039-46. 2004

Extravasation of tumor cells through the endothelial barrier is a critical step in cancer metastasis. Human T-cell lymphotropic virus type I (HTLV-I)-associated adult T-cell leukemia/lymphoma (ATL) is an aggressive disease characterized by visceral invasion. We show that ATL and HTLV-I-associated myelopathy patients exhibit high plasma levels of functional vascular endothelial growth factor and basic fibroblast growth factor. The viral oncoprotein Tax transactivates the promoter of the gap-junction protein connexin-43 and enhances gap-junction-mediated heterocellular communication with endothelial cells. The interaction of HTLV-I-transformed cells with endothelial cells induces the gelatinase activity of matrix metalloproteinase (MMP)-2 and MMP-9 in endothelial cells and down-regulates the tissue inhibitor of MMP. This leads to subendothelial basement membrane degradation followed by endothelial cell retraction, allowing neoplastic lymphocyte extravasation. We propose a model that offers a mechanistic explanation for extravasation of HTLV-I-infected cells: after specific adhesion to endothelia of target organs, tumor cells induce a local and transient angiogenesis-like mechanism through paracrine stimulation and direct cell-cell communication with endothelial cells. This culminates in a breach of the endothelial barrier function, allowing cancer cell invasion. This local and transient angiogenesis-like sequence that may facilitate visceral invasion in ATL represents a potential target for ATL therapy.

Taher A., Khalil I., Abou Merhi R., Shamseddine A., BazarbachiA. High prevalence of prothrombin G20210A mutation among patients with deep venous thrombosis in Lebanon. Thrombosis and haemostasis ; 89 (5):945-6. 2003

M. El Sabban, R. Abou Merhi, H. AbiHaidar, B. Arnulf, H. Khoury, J. Basbous, J. Nejmeh, H. de the, O. Hermine and A. Bazarbachi. Human T-cell lymphotropic virus type-1-transformed cells induce angiogenesis and establish functional gap junctions with endothelial cells. Blood -V.99, N 9, 3383-338. 2002

The role of angiogenesis in the growth and metastasis of solid tumors is well established. However, the role of angiogenesis in hematologic malignancies was only recently appreciated. We show that HTLV-I-transformed T cells, but not HTLV-I-negative CD4(+) T cells, secrete biologically active forms of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) and, accordingly, induce angiogenesis in vitro. Furthermore, fresh ATL leukemic cells derived from patients with acute ATL produce VEGF and bFGF transcripts and proteins. The viral transactivator Tax activates the VEGF promoter, linking the induction of angiogenesis to viral gene expression. Angiogenesis is associated with the adhesion of HTLV-I-transformed cells to endothelial cells and gap junction-mediated heterocellular communication between the 2 cell types. Angiogenesis, cell adhesion, and communication likely contribute to the development of adult T-cell leukemia-lymphoma and represent potential therapeutic targets.

R. Abou Merhi, L.Guillaud, C. Delouis and C. Cotinot. Establishement and characterisation of immortalized ovine sertoli cell lines In Vitro Cell. Dev. Biol- Animal 37: 581-588 2001

The objective of this study was to generate immortalized Sertoli cell lines from prepubertal lamb testes to facilitate investigations during the course of testicular differentiation. The Sertoli cells were enzymatically isolated and immortalized by transfection, with the sequences coding for the SV40 large T-antigen fused downstream of regulatory elements from the human vimentin gene. The different cell lines were positively stained with antibodies to vimentin and transferrin, in agreement with their Sertoli origin. Reverse transcriptase polymerase chain reaction was used to analyze the specific expression of molecular markers (clusterin/sulfated glycoprotein ISGP-2], follicle-stimulating hormone [rFSH], alpha-inhibin, anti-Müllerian hormone, Wilms' tumor gene [WT-1], steroidogenic factor 1 [SF-1], SRY-related HMG box gene g [SOX9], and sex-determining region of Y chromosome) normally expressed in this cellular type. All were shown to express messenger ribonucleic acids for SGP-2, alpha-inhibin, WT-1, SOX9, and SF-1 (except SF-1 for clone no. 1). Moreover, we performed alkaline phosphatase and receptor tyrosine kinase p145 (c-kit) detection to ensure the absence of contamination by peritubular, germ cells, and Leydig cells. Both tests were negative for all the seven cell lines. These ovine Sertoli cell lines are the first ones obtained from livestock that exhibit specific Sertoli cell characteristics resembling different stages of phenotypic development. They provide useful in vitro model systems for toxicological investigations, coculture, and transfection experiments, making it possible to study signal transduction pathways, cell-cell interactions, and gene expression in species other than rodents.

V. Rouiller-fabre, S. Carmona, R. Abou Merhi, R. Cate, R. Habert and B. Vigier. Effect of Anti-Mullerian hormon on Sertoli and Leydig cell functions in fetal and immature rats. Endocrinology-vol.139, No3: 1213-1220 1998

Anti-Mullerian hormone (AMH) is mainly involved in the regression of Mullerian ducts in male fetuses, but it may have other functions linked to gonadal development. The present study examines the effect of AMH on steroidogenesis by Sertoli and Leydig cells in fetal and immature rats during the period where AMH is physiologically produced in the testis. The basal aromatase activity of Sertoli cells in primary culture was strongly stimulated (77-91%) by cAMP. AMH (35 nM) reduced cAMP-stimulated aromatase activity by 49-69% as early as fetal day 16 and until postnatal day 20. This effect was dose dependent and was seen after 48 h in culture. AMH also blocked the Sertoli cell aromatase activity stimulated by FSH, but LH did not stimulate this activity, confirming that the aromatase activity effectively resulted from Sertoli cells and not from contaminating Leydig cells. RT-PCR analysis showed that AMH reduced aromatase activity by decreasing the amount of aromatase messenger RNA. AMH also inhibited the LH-stimulated testosterone production by dispersed fetal Leydig cells in culture in a dose-dependent manner. The inhibitory effect of AMH did not depend on the fetal stage studied (16 or 20 days postconception) and resulted from a drop in the steroidogenic activity of each Leydig cell without affecting the number of 3beta-hydroxysteroid dehydrogenase-positive cells. These data provide the first evidence that AMH, like other members of the transforming growth factor-beta family, has an autocrine/paracrine effect on testicular steroidogenic function during the fetal and prepubertal periods.

E. Payen, E. Pailhoux, R. Abou Merhi, L. Gianquinto, M. Kirszenbaumm, A. Locatelli and C. Cotinot. Characterisation of ovine SRY transcript and developmental expression of gene involved sexual differenciation. Int.J.Dev.Biol.40: 567-575 1995

In mammals, the presence of SRY, the sex-determining gene located on the Y chromosome is required to induce the gonadal anlage to differentiate as a testis, whereas its absence leads to the development of an ovary. We report here the characterization by 5' and 3' RACE analysis of several SRY transcripts which are expressed in the ovine male developing gonads. These transcripts were not detected in any other fetal tissues and were expressed only in the genital portion of the urogenital ridge. The temporal profile of SRY expression analyzed by RT-PCR suggests that in the sheep fetus the role of SRY is not limited to initiating Sertoli cell differentiation as in mice. Indeed, SRY transcripts persist after the full differentiation of the testis. In addition to SRY, other genes are known to be involved in mammalian sex determination: Wilms' tumor gene WT-1, steroidogenic factor gene Ftz-F1 (SF-1) and anti-Müllerian hormone (AMH). We investigated the expression patterns of these genes by RT-PCR during fetal development in sheep gonads. Concerning WT-1 and SF-1, our results are consistent with those described in mice where the earliest expression was detected before the sexual differentiation in both sexes. In male, the ontogenesis of AMH transcription corresponds to the seminiferous cords formation (30 dpc). In female, we have observed the presence of SF-1 transcripts from the undifferentiated stage until birth. In addition, P450 aromatase expression is detected from 30 dpc and is correlated with the presence of 17-beta estradiol in sheep ovary. These data reveal significant differences between rodent and ruminant models concerning the sex-determining pathway.

Supervision 11 Supervised Students
Role of DdX19 helicase in genome stability maintenance and sensitivity of cancer cell to DNA damaging agents

Mariam Ali Jaafar
Master M2 Thesis: Genomics and Health in 2015

Identification of the role of FANCM / DHP complex in cell proliferation: DNA Damage and Fanconi Anemia

Elie Azab Nassour
Master M2 Thesis: Genomics and Health in 2016

Regulation of signaling pathways by the tumor suppressor PTPN13

William Fadi Bakhache
Master M2 Thesis: Genomics and Health in 2016

Zebrafish: a model to study bacterial virulence

Mohamad Hekmat Saleh
Master M2 Thesis: Genomics and Health in 2016

MicroRNA expression profile in Imitanib-resistant Chronic Myeloid Leukemia patients

Lara Farouk Hammoud
Master M2 Thesis: Genomics and Health in 2016

Epigenetic modulations and Anti-tumor Molecular Events of Pathenolide, a NFkappaB inhibitor, on PEL

Wafaa Maarouf Assaad
Master M2 Thesis: Genomics and Health in 2016

Gallotannin targets NF-ĸB signaling and induces DNA damage and senescence in human colon cancer

Racha Al Halabi, PhD (graduated July 2014)
Co-supervision thesis For the degree of Doctor Philipps-Marburg University and Lebanese University (DSST/ LU) Speciality : Molecular Biology Thesis defense: 22 July 2014 In collaboration with AUB

Colorectal cancer is one of the leading cancer killers worldwide and is strongly associated with environmental causes, such as smoking, heavy alcohol use, high intake of red meat and no physical activities. The plant secondary metabolite Gallotannin (GT) is the simplest hydrolyzable tannin shown to have anti-carcinogenic properties in several cell lines and to inhibit tumor development in different animal models. Gallotannin are widely distributed in nature and in fruits such as in mangoes, pomegranates and acorns. GT exhibits anti-inflammatory and anti-cancer activities through mechanisms that are not fully understood. Thus we aimed to unravel some of its mechanisms of action against human colon cancer. We first investigated the effect of GT on NF-ĸB signaling in HT29 and HCT116 human colon cancer cells, considering the central role of nuclear factor kappa B (NF-ĸB) in inflammation and cancer. Indeed several effects modulated by GT were shown to be linked to interference with inflammatory mediators. DNA binding assays revealed significant suppression of tumor necrosis factor (TNFα)-induced NF-ĸB activation which correlated with the inhibition of IĸBα phosphorylation and degradation. Sequentially, p65 nuclear translocation and DNA binding were inhibited. GT also down-regulated the expression of NF-ĸB regulated inflammatory cytokines (IL-8, TNFα, IL-1α) and caused cell cycle arrest and accumulation of cells in Pre-G1 phase. In vivo, GT (25mg/kg body weight) injected intraperitoneally (i.p.) prior to or after tumor inoculation significantly decreased the volume of human colon cancer xenografts in NOD/SCID mice. GT-treated xenografts showed significantly lower microvessel density (CD31) as well as lower mRNA expression levels of IL-6, TNFα and IL-1α and of the proliferation (Ki-67) and angiogenesis (VEGFA) proteins, which may explain GTs in vivo anti-tumorigenic effects. In the second part of our study, we determined if GT induces senescence and DNA damage and investigated the involvement of p53 and p21 in this response. Using HCT116 human colon cancer cells wildtype for p53+/+/p21+/+ and null for p53+/+ /p21-/- or p53-/-/p21+/+, we found that GT induces senescence independently of p21 and p53. GT was found to increase the production of reactive oxygen species (ROS) by altering the redox balance in the cell, mainly by reducing the levels of glutathione and superoxide dismutase (SOD). Using the key antioxidants N-acetyl cysteine, dithiothreitol, SOD and catalase, we showed that ROS were partially involved in the senescence response. Furthermore, GT induced cell cycle arrest in S-phase in all HCT116 cell lines. At later time points, we noticed that p53 and p21 null cells escaped complete arrest and re-entered cell cycle provoking higher rates of multinucleation. The senescence induction by GT was irreversible and was accompanied by significant DNA damage as evidenced by p-H2AX staining. Overall, our findings indicate that GT is capable of modulating the interconnected matrix of pathways (suppression of NF-ĸB activation, senescence, DNA damage and ROS) that together are capable of driving and restraining tumor development. Thus this readily available and inexpensive plant derived compound is an interesting anti colon cancer agent which warrants further investigation for translation to the clinic. Key words: colon cancer, herbal medicine, Gallotannin, inflammation, NF-ĸB, senescence, reactive oxygen species, DNA damage, xenograft.

From the nuclear pore to DNA damage: the ATR-mediated shuttling of Ddx19 to resolve transcription-replication conflicts

Dana Hodroj, PhD (graduated December 2014)
Thèse en cotutelle pour obtenir le grade deDocteur Délivré par Université Montpellier II (École Doctorale Sciences Chimiques et Biologiques pour la Santé) et Université Libanaise (École Doctorale Sciences et Technologie) Spécialité : Biochimie et Biologie Moléculaire Soutenue publiquement le 9 Décembre 2014

Cells are continuously challenged by DNA damage resulting from external cues as UV light, ã- irradiation and exposure to genotoxic chemicals, as well as from endogenous stress caused by cellular metabolism. Growing evidence points to transcription as a biological process that could adversely affect genome integrity. One currently highly investigated mechanism by which transcription can induce genome instability is through the formation of R-loops, RNA:DNA hybrid structures exposing a displaced single-stranded DNA tract. These aberrant structures occur as byproducts of transcription and/or upon interference between replication and transcription, and more recently were also shown to accumulate upon disruption of mRNA biogenesis and processing. Persistent unresolved R-loops are a potent source of genomic instability as they ultimately generate double strand breaks and promote recombination events. To deal with the deleterious consequences of DNA damage, cells activate elaborate DNA damage response (DDR) pathways to delay cell division and stimulate repair of lesions, thus preserving genome stability. Recently in yeast transient DDR activation has also been proposed to be important in the coordination of transcription and replication, in order to avoid topological constraints and the formation of aberrant structures generated upon collision of their machineries. By means of an in vitro screen aimed at identifying new DDR genes, we isolated Ddx19, a DEAD-Box helicase known to be involved in mRNA export, as a novel DNA damage responsive gene. Ddx19 interacts with the nucleopore complex via nucleoporin CAN/Nup214, and is involved in mRNA remodelling and export through its ATPase and helicase activities, stimulated by IP6 and the Gle1 factor. My present thesis work unravels a novel function of Ddx19 in preserving genome stability in mammalian cells, distinct from its known role in mRNA export. I show that upon UV-induced damage, Ddx19 transiently relocalizes from the cytoplasmic face of the nucleopore to the nucleus in an ATRdependent manner. Downregulation of Ddx19 gives rise to spontaneous, proliferation-dependent DNA damage, as determined by the specific activation of the ATM-Chk2 pathway and formation of ãH2AX and 53BP1 nuclear foci. This is concomitant with the slowing down of replication forks that are unable to restart after being stalled with camptothecin. In addition, cells depleted of Ddx19 display strong accumulation of nuclear R-loops, enriched in the nucleolar compartment, and around the nuclear periphery. Moreover, these cells show low viability and exhibited synthetic lethality when combined with inhibition of topoisomerase I expression. I propose Ddx19 as a second helicase required for R-loops resolution, functioning alongside but independently of Senataxin, the first known RNA helicase to resolve these structures in vivo in mammalian cells. I provide evidence that this new function of Ddx19 does not depend on its interaction with the nuclear pore, but rather on its helicase activity and on a serine residue phosphorylated by Chk1 which promotes its relocalization into the nucleus upon damage. These data put forward Ddx19 as a novel RNA helicase that facilitates ATR-dependent coordination of DNA replication and transcription through R-loops resolution, thus preserving genome integrity. Keywords: genome instability, replication stress, ATR, RNA helicase, nucleopore, R-loops.

Identification and characterization of mutations of ACVRL1 gene in a cohort of patients affected by Hereditary Hemorrhagic Telangiectasia

Ferdos Alaadine, PhD student
Co-supervison thesis: expected defense in Septembre 2015 - Laboratoire de Génomique et Santé / PRASE Plateforme, Université Libanaise, Hadath, Liban. - Equipe des maladies génétiques rares, Universitéde Poitiers/ CHU de Poitiers, France

Hereditary Hemorrhagic Telangiectasia syndrome (HHT) or Rendu-Osler-Weber (ROW) syndrome is an autosomal dominant vascular disorder. Two most common forms of HHT, HHT1 and HHT2, have been linked to mutations in the endoglin (ENG) and activin receptor-like kinase 1 (ACVRL1or ALK1) gene respectively. This work was designed to examine the patogenicity of 23 nucleotide variations in ACVRL1 gene detected in more than 400 patients. Among them, 14 missense mutations and one intronic variant were novels, and 8 missense mutations were previously identified with questionable implication in HHT2. The functionality of missense mutations was analyzed in response to BMP9 (specific ligand of ALK1), the expression of their proteins and their localization were analyzed by Western Blot and fluorescence microscopy. The splicing impairment of the intronic and two missense mutations was examined by minigene assay using pSplice Express. 18 out of 22 missense mutations were defective by functional analysis. Splicing studies revealed that one missense mutation (c.733A>G, p.Ile245Val) affects the splicing of the harboring exon 6 as well as an intronic mutation outside the consensus splicing sites (c.1048+5G>A in intron 7). Both mutations induce a frame shift creating a premature stop codon likely to result in mRNA degradation by NMD surveillance mechanism. In conclusion, functional and splicing analyses together, represent two robust diagnostic tools to be used by geneticists confronted with novel or conflicted ACVRL1 mutations. Furthermore, our results confirm that mutations of ACVRL1 adapt to the happloinsufficiency model inducing the loss of one allele product via mRNA degradation or synthesis of a non functional protein. Keywords: ACVRL1; missense mutation; functional analysis; splicing study; NMD

Mode of Action of ST1926 and Parthenolide in Treatment of HHV8-associated PEL

Louna Karam, PhD student
Co-supervision thesis: expected thesis defense 2017 - Faculty of Sciences/EDST, Lebanese University, R. Hariri Campus, Lebanon. - Biology department, Faculty of natural sciences, University of Erlangen, Germany. - Department of Molecular Genetics and Biochemistry, AUB, Lebanon.

Introduction: Primary effusion lymphoma (PEL) is a rare AIDS-associated B-cell neoplasm, caused by Human Herpes Virus 8 (HHV8) infection and manifested as malignant effusions in body cavities. PEL cells don't present conventional genetic cancer mutation, however its oncogenesis is attributed to HHV-8 latent genes, LANA-1/2, v-cyclin and v-FLIP. PEL is life threatening to immunocompromised and elderly people since it relapses after standard chemotherapy treatments, thus the need for new effective, targeted drugs. Among studied drugs, parthenolide (PTL), a natural sesqueterpene lactone and a known NF-kB inhibitor, was reported to have important anti-cancer activities against a variety of hematopoietic malignancies. Also, ST1926, a novel orally available, synthetic retinoid, exhibited a targeted apoptotic and genotoxic effect on a large panel of human cancer cells. The aim of this study is to elucidate the anti-tumor activities and underlying molecular mechanisms, of PTL and ST1926 on PEL in vivo/in vitro study levels. Methods: Human PEL (BC1, BC3), non-PEL cell lines (RAJI) and ascites derived from PEL-like mice model were used. The anti-proliferative effect of ST1926 and PTL was detected using MTT cell proliferation assay. Flow cytometry was used to detect cell cycle distribution and apoptosis using Propidium iodide and Annexin V-FITC kit. The expression of apoptosis-regulated genes was examined at the protein level using Western blot. The drugs effect on latent viral transcripts expression was studied via qRT-PCR. Results: Preliminary results, show that ST1926 and PTL hold a potent anti-proliferative effect on PEL cell lines and ascites. Both drugs individually show to increase preG1-population in cell cylce, in P53 and PARP cleavage on PEL cells and ascites. As well ST1926 downregulated all viral latent transcripts expression in ex-vivo PEL ascites. conclusion: The promising apoptosis promoting and anti-viral effects of these drugs could provide a new basis for its clinical application in PEL. Keywords: Primary Effusion Lymphoma, HHV8, Parthenolide, ST1926, PEL ascites, apoptosis

Role of Epidermal Growth Factor and their Receptors in Bladder Cancer Progression and Invasion of Lebanese Patients Smokers

Ihssan sayed Joumaa, PhD student
Cosupervision thesis: Directors Ahmad Daher and Francis Vacherot Co director: Raghida abou Merhi Expected thesis defense 2016 a. Genomic and Health laboratory/PRASE-EDST, Research team ER031, R. Hariri campus, Lebanese University. b. Affiliation institutionnelle : Hopital Henri Mondor, Université Paris Est, Paris, France Adresse du laboratoire : Equipe n°7 "Recherche translationnelle en oncogenèse génito-urinaire", IMRB -Inserm U955, 8 rue du général Sarrail- 94010 Créteil Research area 2. Molecular bases of cancer and genome surveillance Subject 2.2 Role of Epidermal Growth Factor and their Receptors in Bladder Cancer Progression and Invasion of Lebanese Patients Smokers

Bladder cancer is one of the most common urological malignancies worldwide generally and in Lebanon specifically and a frequent cause of cancer-related death, thus it appears a demanding necessity to investigate and identify the molecular targets that are implicated in the progression of bladder cancer. There is great evidence based on preclinical and clinical data that suggests the role of Epidermal Growth Factor Receptor family in a variety of cancers since the dysregulation of EGFR enhances aberrant signaling which most exclusively lead to solid cancers. Investigating the role of EGFR in progression of bladder cancer and correlation with tumor stage and grade, age, gender and smoking exposure in Lebanese patients is the aim of this project. The expression of EGFR was examined at the level of both RNA, by reverse-transcriptase polymerase chain reaction, and protein, by western blot, of tumor samples obtained from Lebanese patients who have been submitted to bladder cystectomy. We have demonstrated that EGFR is expressed at mRNA and protein levels in all our tumor samples. Real-time PCR and more western blot experimental procedures were performed to better quantify EGFR expression at RNA and protein levels. Real time PCR preliminary results will be presented later. This work helps to develop new therapeutic strategies especially those targeted against EGFR and other downstream molecules being critical in bladder cancer progression and invasion. Key words: EGFR; bladder cancer progression; smoking.

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