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MMBio - Marie Curie Training Network

Konstantinos Chionis

 
 

Background:

I received my Bachelor of Science Degree in Chemistry from the University of Ioannina, Greece. In my Bachelor thesis I studied the synthesis and Biological role of amphipathic cationic peptides model for the development of new antimicrobial agents.

My penchant for research motivated me to fulfill my Master studies in Organic and Biochemistry. My Master Thesis was entitled “Synthesis and Biological study of natural cationic peptide Anoplin and various other analogues for the development of new antimicrobial agents”, under the directions of Professor Maria Sakarellos-Daitsioti and Assoc. Professor Eugenia Panou-Pomonis. More specifically, I synthesized new analogues of the natural peptide Anoplin. The synthesis of the peptides was performed by the stepwise solid-phase synthesis (SPPS). The peptides were purified by semi-preparative HPLC. The purity of the peptides was checked by analytical HPLC and the correct molecular masses were confirmed by ESI-MS. Moreover, all conformational characteristics of the peptides were studied by Circular Dichroism (CD). All these new analogues were studied for their antimicrobial against Gram positive/negative bacteria, their hemolytic activity and proteolytic stability.

During my master studies, I took up an Erasmus placement traineeship in the Department of Chemistry “Ugo-Schiff” at the University of Florence under the supervision of Professor Anna-Maria Papini. The research topic of this internship was focused on synthesis, purification and characterization of peptides used for in vitro diagnostic in cancer disease patients. During this internship I modified the original sequence of Anoplin and other Anoplin derivatives with sialic acid. After the synthesis, ELISA experiments were performed to evaluate the Ab recognition of the synthetic peptides. The experience gave me the opportunity to acquire knowledge on enzyme-linked immunosorbent assay (ELISA).

In terms of my military service, I worked as Chemist analyst at the Oil Analyst Department of the Greek Army. The laboratory deals with the oil and hydraulic fluids analysis of several helicopters and aircraft gears of Aviation Army using atomic emission spectroscopy method, in order to detect wear gear metal in supplied samples. Such identification predicts and prevents gear faults and contributes to the proper gear functions.

Training and Transferable Skills:

-          Solid phase peptide synthesis (SPPS)
-          HPLC (Preparative, semi-preparative and analytical HPLC)
-          HPLC-MS
-          Mass spectroscopy (ESI-MS)
-          NMR
-          IR
-          CD (Circular Dichroism)
-          ELISA (Direct, Sandwich and competitive ELISA)
-          Atomic emission spectroscopy (Oil analysis)

Research Projects:

Previous research in our group has led to the development of artificial ribonucleases based on oligonucleotide conjugates with 2-aminobenzimidazoles. The conjugates are able to selectively cleave any type of single stranded RNA. Such compounds are able to destroy mRNAs in a living cell. As a result, downregulation of a specific gene will occur. This approach is a routine operation in fuctional genomics. However, siRNAs and LNA-gapmers are used for this purpose exclusively. Today, artificial ribonucleases from this laboratory cannot compete in terms of reaction rates. Thus, the major aim of this PhD project is rate optimization of the catalyst. This will be done by:

  1. Adding electron donors and acceptors in order to modulate the pKa of the catalyst.
  2. Replacing 2-aminobenzimidazoles by other classes of guanidine analogs
  3. Using different polyamines linkers to combine two, three or even more guanidine analogs.

A second strategy for the modulation of mRNA translation relies on the natural catalysts of the cell. The half-lives and transcription efficiencies of mRNAs are strictly regulated in most cases by proteins or protein–RNA complexes interacting with their untranslated regions. miRNAs for example are bound by argonaut proteins forming RISC complexes. By base pairing of the RNA part, RISC complexes can associate with mRNAs. As a result, translation of the m RNA is stopped and degradation is initiated by decapping and removing the poly-A tail. Other regulatory sites within mRNAs are AU-rich elements. When bound by tristetraprolin their lifetime are reduced while binding of HuR increased their stability. Although regulation via AU-rich elements is complex, tristetraprolin is most often the dominant player and levels of HuR are low. In many cancer cells this ratio is inverted. High levels of HuR stabilize the mRNAs of proto-oncogenes and growth factors thereby contributing to the malignant transformation.

This project will test the possibility to capture HuR by RNA analogs which are stable against enzymatic degradation. In case of success a bifunctional oligonucleotide will be used to redirect HuR to a new m RNA target which normally has no AU-rich element in its 3’-UTR. The first part of the oligo consists of the HuR recognition site while the second part is complementary to the new target RNA. This redirection strategy may allow us to upregulate tumor inhibitor genes specifically. Furthermore, pronounced effects are expected in cell types only which express higl levels of HuR.

 Publications:

“Synthesis and biological activity of lipophilic analogs of the cationic antimicrobial active peptide Anoplin”. K.Chionis, D.Krikorian, A-I Koukkou, M Sakarellos-Daitsioti and E.Panou-Pomonis. J Pept. Sci, 22(11-12), 731-736 (2016). 

 

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