CH ARLES UNIV ERSITY
1
stFaculty of Medicine
I N S T I T U T E O F P H Y S I O L O G Y
Jaromír Mysliveček, M.D., Ph.D.
Professor of Physiology
128 00 PRAGUE, ALBERTOV 5 CZECH REPUBLIC
Evaluation of the Doctoral Thesis by Mr. Milorad Ivetić
Title: The molecular mechanisms and role of purinergic signaling in hypothalamus
The doctoral thesis of Milorad Ivetić concerns the role of extracellular ATP and purinergic P2 receptors (P2X and P2Y) in the hypothalamus. The author tested two hypotheses: first - P2X and P2Y receptors play a role in the enhanced release of hormones from magnocellular supraoptic nucleus neurons stimulated through fasting/refeeding experimental protocol.
Second - activation of P2X7 and P2Y receptors contributes to the rhythmical release of ATP from SCN astrocytes.
As a tool for testing the hypotheses, the author used a diversity of techniques: quantitative real-time PCR, ATP bioluminescent assays, immunohistochemistry, patch-clamping, and calcium imaging. His thesis also presents and discusses the results from complementary approaches performed by his colleagues.
The Introduction of the Thesis is concisely written, and it is well and easily readable. This part consists of 30 pages. The author first describes purinergic signaling including the types of purinergic receptors, their physiological function, and specific properties of P2X2, P2X4, P2X7, and P2Y purinergic receptors. The next sub-chapter of the Introduction is devoted to the hypothalamo-neurohypophyseal system with special attention to neurohypophyseal hormones vasopressin and oxytocin. This part is followed by a specific description of purinergic signaling in the hypothalamus with respect to circadian ATP release. The last part of the Introduction describes experimental protocols used to stimulate the synthesis and release of neurohypophyseal hormones.
The Material and methods section precisely describes the experimental attitude to the topic.
This part has 7 pages and reproducibly describes all methods used in the thesis (animals, slice preparation, organotypic cell culture preparation, primary cultures of SCM astrocytes, patch- clamping, ATP detection, calcium imaging, real-time PCR, immunohistochemistry and statistical evaluation of data from the experiments).
The Result section has 16 pages and is organized into small sub-chapters describing the specific objectives of each experiment and describing the results obtained. The data are well illustrated.
In the Discussion section (8 pages), the results are discussed in the context of the international literature in the field. The Discussion is well-written and relatively easy to follow.
The References (16 pages) represent the relevant list of up-to-date papers in the field.
The Ph.D. work has directly led to two articles, one in which the author signed as the first author and the second one which is co-authored by Milorad Ivetić.
I have only several minor comments (see below) that do not in any sense undermine the quality of the work and may well remain for discussion during this thesis defense.
Minor comments:
1) p. 10: there are much more functions of the hypothalamus, not only endocrine functions, and the role in circadian rhythm.
2) p. 12: typo: hzpothlamus.
3) p. 26: the role of PVN parvocellular neurons in autonomic function should be mentioned.
4) p. 38: it is not appropriate to mention “There are multiple mechanisms…” and then to give only one example (cholecystokinin).
5) p. 50: the part of the sentence: ”…were determined by two-way analysis of variance (ANOVA) and Tukey's post hoc test using SigmaPlot v10.01 with p < 0.01 (**) and p <
0.05 (*).” This sentence should be rewritten.
6) p. 59: if there are data from a previous paper (Svobodova et al., 2003) then it should not be included in the section Results. It should be moved to the Discussion.
7) p. 68: the mRNA expression of purinergic receptors need not necessarily represent the number of receptors. This should be discussed in the Thesis. Regarding this: how relevant are gene expression data for purinergic receptors with respect to the receptors present/receptor function?
In conclusion, Milorad Ivetić performed quality research work using a variety of experimental techniques. The results obtained are interesting and contribute to a better understanding of the role of the purinergic system in hypothalamus – neurohypophysis physiology.
I think this work is worth being defended in front of the thesis committee.
Jaromír Mysliveček, M.D., Ph.D.
Professor of Physiology Institute of Physiology 1st Faculty of Medicine Charles University Prague
Czech Republic
