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Clinical relevance of free peritoneal tumor cells detection in gastric and colorectal cancer: a multiple molecular approach
No full textBackground
Free disseminated peritoneal tumor cells derive from the detachment from primary cancer and may result in peritoneal carcinomatosis. Peritoneal lavage cytology has low sensitivity in detecting free peritoneal tumor cells; reverse-transcriptase polymerase reaction showed higher sensitivity but low specificity. Our study introduces the combination of the RT-PCR, the immunomagnetic enrichment and the immunofluorescence for the detection of peritoneal free tumor cells.
Materials and Methods
Samples of peritoneal lavage were collected from 22 gastric and 45 colorectal cancer patients; samples were also obtained from 6 patients who underwent abdominal surgery for non-malignant diseases. CEA and CK20 mRNA levels were quantified using a real-time qRT-PCR system. Immunomagnetic enrichment followed by immunofluorescence analysis was performed using monoclonal antibody against the pan-epithelial marker EpCAM/CD326 and polyclonal antibodies against the carcinoembryonic antigen.
Results
For gastric carcinoma the positivity rate for cytology, immunofluorescence and qRT-PCR was 14%, 18% and 77% respectively; for colorectal carcinoma the positivity rate for cytology, immunofluorescence and qRT-PCR was 0%, 18% and 44% respectively. All patients except one when positive at immunofluorescence were also positive at qRT-PCR. All samples of peritoneal lavage from the control group resulted negative for cytology, IF and real time qRT-PCR.
Conclusion
The combination of conventional real time qRT-PCR with immunoenrichment and immunofluorescence, which permit morphological assessment and unequivocal identification of the DTCs as well as validation of the molecular analysis, could be an useful and more powerful procedure for the detection of free peritoneal tumor cells.Non
identification of molecular targets in the human genome
No full textThe purpose of the present study is to contribute to the field of functional genomics by developing, testing and applying computational methods to the problem of the evaluation of the effects of environmental and pharmacological molecules on genome expression.
Original results are described in four independent sections
The first two sections are devoted to the investigation of the coding potential of alternative splicing products in the human genome.
Sections three and four are devoted to the application of computational techniques to investigate the molecular targets and the effects on their expression of molecules known to interfere with the physiological functions
of a cell. In particular these techniques were applied on a class of compounds (tocotrienols) constituents of the Vitamin E
Vasoactive Intestinal Peptide Receptor 1 in immune response and autoimmunity: genetic and functional aspects
No full textThe human Vasoactive Intestinal Peptide (VIP) is a potent immunosuppressive agent, affecting both innate and adaptive immunity. VPAC1, one of its receptors, has been found to be down-modulated in cells of the immune system in response to activating stimuli. We have studied the role of VPAC1 SNPs in the genetic predisposition to Ankylosing Spondylitis (AS), Idiophatic Achalasia (IA) and Type 2 diabetes (TD2), three multifactorial diseases for which the inflammatory component contribute to their pathogenesis. The Ankylosing Spondylitis association studied performed showed that independently from diseases HLA-B*2705, the AS-associated allele, co-occurs more frequently with allele T at SNP rs896 in the 3’-UTR of VPAC1 gene (p=0.001). For Idiophatic Achalasia we have found an association between SNPs rs437876 and rs896 and the patients with late onset (p=0.0005). We have, also, reported an association between type 2 diabetes and rs9677 in females (ptrend=6x10-4) which correlates with worse clinical parameters. These genetic studies have indicated an association especially with SNPs mapping in the 3'-UTR of VPAC1, prompting us to perform functional studies in monocytes. We have found an association between the presence of a T at rs896 in VPAC1 3’-UTR, and a sharp reduction of VPAC1 expression, both at mRNA and protein level after LPS treatment. Moreover, we have selected a microRNAs, miR-525-5p, as having its putative target sequence next to SNP rs896. We have found that this miRNA is upregulated during inflammation in monocytes and that it has, among its targets, VPAC1, therefore inducing its down-regulation. Taken together, these studies shed light in some of the aspects of the complex regulatory network controlling VPAC1 expression and inflammation
An upper bound for the genus of a curve without points of small degree
No full textIn this paper we prove that for any prime p and for any p-power q
there is a constant C_p > 0 such that for any n > 0 there is a smooth,
projective, absolutely irreducible curve over F_q of genus g ≤ C q^n
without points of degree smaller than n.Phd thesisUniversità di Roma 'Sapienza
Use of Mouse Models for the Study of Hedgehog Pathway's Role in Tumorigenesis
No full textErasmus Mundus BASILEUS - Balkans Academic Scheme for the Internationalisation of Learning in cooperation with EU universitie
ROLE OF NOT4 AND JHD2 IN REGULATION OF HISTONE H3 METHYLATION AND TRANSCRIPTION OF RIBOSOMAL PROTEIN GENES IN YEAST
No full textInnovative materials and methods for HPLC and UHPLC: design of new bidentate urea-type stationary phases. Stereodynamic studies of artemisinin derivatives.
No full textThis Ph.D. thesis focuses on three main topics:
1) Design and evaluation of new stable bidentate urea-type stationary phases for HPLC and UHPLC. New totally synthetic bidentate urea-type phases were envisioned for HPLC and UHPLC, characterized by a high stability, a wide application range and an excellent suitability for LC-MS. Four conceptually new stationary phases have been developed for the separation of a wide variety of polar compounds by hydrophilic interaction chromatography (HILIC) through a facile one-pot two-step procedure with the aim of obtaining high hydrolytic stability in a variety of elution conditions. A full chemical characterization of the new materials has been obtained through solid-state NMR (both 29Si and 13C CPMAS) spectroscopy. A major application field of the bidentate urea-type stationary phase with free amino groups USP-HILIC-NH2-sil was sugars analysis, usually hampered by anomer peak splitting and instability of the stationary phases under conditions normally employed to suppress it. Complex mixtures of mono-, di- and oligosaccharides were successfully resolved under mild chromatographic conditions, which also allowed an
easy interface with mass spectrometry. The potential of such materials was shown in the separation of other highly polar compounds, including polyols, hydroxybenzoic acids, nucleobases, and vitamins. A parallel project was developed using a very similar synthetic scheme where enantiopure diamines were treated with (3-isocyanatopropyl) triethoxysilane to yield reactive ureido selectors that were eventually attached to unmodified silica particles through a stable, bidentate tether. The two steps were part of an easy and time-saving one-pot procedure, as was the case for the HILIC phases. These new monomeric brush-type chiral stationary phases, hereafter called “Crab-like” CSPs, are characterized by an excellent stability and enantioselectivity, a high permeability and efficiency, as well as an extended compatibility with all solvents and no memory effect, which allowed us to operate under normal phase, reversed phase and polar organic mode conditions. Comparative studies were performed with commmercial polymeric brush-type CSPs bearing the same chiral selector (1,2-diaminocyclohexane and 1,2-diphenyl-1,2-ethylenediamine) in order to evaluate the resolution and performance skills of the new “Crab-like” CSPs in different elution modes.
2) Extending the use of “Inverted Chirality Columns Approach (ICCA)” for enantiomeric excess determination in absence of reference samples: application to a water-soluble camptothecin derivative. The aim of the present study was to extend the use of the “Inverted Chirality Columns Approach (ICCA)” previously developed for the identification and quantitation of the trace enantiomer in highly enriched samples of the camptothecin (CPT) family of drugs to a novel water-soluble CPT derivative, namely namitecan (ST1968), currently undergoing phase I clinical trials as anticancer agent. Namitecan, identified from a series of hydrophilic 7-oxyiminomethyl derivatives, contains a free terminal amino group, which traditionally hampers the analysis under normal-phase HPLC conditions. Nevertheless, commercially available Pirkle-type chiral stationary phases (CSPs) available in both the enantiomeric forms (i.e., having the same bound selector with opposite configuration) mainly operate under normal-phase HPLC conditions. For this reason, namitecan was pre-columnN-derivatized with isocyanates A–D and their sulfur analogues E–H to reduce its polarity by converting the amino group into a fragment compatible with the chiral recognition mechanism (i.e., ureido and thioureido groups). Once the optimal columns system
and derivatizing agents were selected, an original enantioselective HPLC–MS/MS technique was developed on the Whelk-O1 CSPs.
3) Kinetic and thermodynamic studies of the epimerization processes in Artemisinin-derived antimalarial drugs by Dynamic High Performance Liquid Chromatography (D-HPLC). Stereodynamic investigations have been performed on two active artemisinin derivatives having labile stereogenic centres, namely dihydroartemisinin (DHA), and an in vivo degradation product of DHA containing an endoperoxide, an aldehyde and a ketonic moiety, namely MKA (monoketoaldehyde). We were able to obtain MKA through pyrolysis or basic degradation of DHA and have fully characterized its structure through a variety o analytical techinques. The stereodynamic studies performed have focused on a stereolabile hemiacetalic function present in both molecules. Linear Solvation Energy Relationships (LSER) analysis was used to study the thermodynamics of the epimerization process in DHA and MKA. Computational studies have provided further data on the differential stability and acidity of the two epimeric forms of MKA, while a kinetic investigation is introduced.Financial support from FIRB, Research program: Ricerca e Sviluppo del Farmaco (CHEM-PROFARMA-NET), grant no.RBPR05NWWC 003 is acknowledged. We are grateful for financial supports from Sapienza Università
di Roma, Italy (Funds for selected research topics 2009–2010)