Darbo metu atlikta A grupės farmakologiškai aktyvių medžiagų patekimo į organizmą jų sukeliamą žalą bei kiekius maisto produktuose liečiančią mokslinę literatūrą. Aptartas farmakologiškai aktyvių medžiagų teisinis reglamentavimas Lietuvoje bei ES. Išanalizuoti 2005-2011 metų literatūroje rasti farmakologiškai aktyvių medžiagų monitoringo duomenys ir aptariamaisiais metais A grupės medžiagų žaliaviniame piene, stebėsenos rezultatai. Informacija ir tyrimų medžiaga gauti iš Nacionalinės veterinarijos laboratorijos ir atlikta jų statistinė analizė SPSS statistiniu paketu (SPSS Inc, 1995-2007). Analizuojant 2005-2011 metų A grupės farmakologiškai aktyvių medžiagų aprašomosios statistikos rezultatus, nustatyta, kad iš viso buvo ištirti 114 889 mėginiai (žaliaviniame piene). Daugumos A grupės medžiagų likučių, tokių kaip stilbeno ir stilbeno produktų, antitiroidinių agentų, steroidų, rezorcilo rūgšties laktonų, beta-antagonistų aptariamuoju laikotarpiu žaliaviniame piene neaptikti. Aptikti chloramfenikolio ir nitrofurantoino likučiai. Chloramfenikolio nustatyta 2006 metais (2 mėginiai), 2008 metais (1 mėginys) ir 2011 metais (1 mėginys), o nitrofurantoinas buvo nustatytas 2008 metais (1 mėginys). Metai turi statistiškai reikšmingą (p=0,0001) chloramfenikolio likučiams žaliaviniame piene įtaką, tai rodo šių likučių tendencingą mažėjimą. Šių medžiagų aptikimas rodo, kad vyksta nelegalus jų naudojimas, kuris yra žalingas visuomenės sveikatai bei turi įtakos eksportui. / The legal regulation of pharmacologically active substances in Lithuania and the EU was discussed, the monitoring data on the pharmacologically active substances was analyzed with the monitoring results of pharmacologically active substances of group A accumulated in raw milk. The information and research’s material was received from the National Laboratory of Veterinary and its statistical analysis was done with the help of SPSS statistical package (SPSS Inc, 1995-2007). While analyzing the results of the descriptive statistics of the monitoring of pharmacologically active substances of group A in the years 2005-2011, it was determined that 114 889 samples were analyzed in search for group A pharmacological residues in raw milk. The remains of the majority of A group’s substances – stilbene and stilbene products, antithyroidal agents, steroids, resorcinol acid lactines, beta-antagonists were not found in raw milk analyzed in 2005-2011. During the monitored period the residues of chloramphenicol and nitrofurantoin were found in raw food material. The chloramphenicol residues were found in 2006 – 2 samples, in 2008 – 1 sample and in 2011 – 1 sample. And the nitrofurantoin residues were found in 2008 – 1 sample. That shows illegal use of these substances, which can cause problems on the public health and export as well. The single factor of year has a statistically significant influence (p=0,0001) on chloramphenicol residues in raw milk. It shows, that amounts of... [to full text]

Darbo tikslai: Analizuoti B grupės farmakologiškai aktyvių medžiagų liekanas maisto produktuose 2004-2011 metais. Tyrimo uždaviniai: a) surinkti ir išanalizuoti farmakologiškai aktyvių B grupės medžiagų kiekius maisto produktuose reglamentuojančius teises aktus; b) surinkti ir išanalizuoti mokslinę literatūrą apie farmakologiškai aktyvių B grupės medžiagų patekimą į maisto produktus, jų sukeliamą žalą bei kiekius maisto produktuose; c) išanalizuoti 2004–2011 metų farmakologiškai aktyvių B grupės medžiagų stebėsenos rezultatų ypatumus; d) išanalizuoti 2004–2011 metais sukauptų B grupės farmakologiškai aktyvių medžiagų stebėsenos rezultatus pagal tyrimo metus ir maisto žaliavų/produktų grupes. Magistrinį darbą sudaro šios dalys: pirma dalis – B grupės farmakologiškai aktyvios medžiagos, jų apibūdinimas ir leistini jų kiekiai maisto produktuose, antra dalis – savi tyrimai, kuriuose atlikta B grupės farmakologiškai aktyvių medžiagų 2004–2011 metų liekanų analizė . Darbe panaudotos 14 lentelių ir 4 paveikslai. Darbo apimtis 58 puslapiai. Atlikus darbą gautos šios svarbiausios išvados: 1. Kasmet Lietuvoje vykdant farmakologiškai aktyvių medžiagų stebėseną ištiriama 3441–4319 mėginių. Nustatomi 16–41 mėginiai (0,462–0,997 %), kuriuose kontroliuojamas liekanų kiekis viršija DLK. 2. Tiriant gyvūninius maisto produktus, medžiagų liekanos, viršijančios DLK, dažniau nustatytos žuvų, kiaušinių ir pieno mėginiuose. Dažniausiai DLK viršijo aplinkos teršalų (B3 grupės) ir antibakterinių... [toliau žr. visą tekstą] / Research purpose: to analyse the residue from pharmacologically active substances of group B in food products in 2004-2011. Information and the material of the research were received from the National Food and Veterinary Institute for evaluation. Research objectives: a) to collect and analyse legislations regulating the amount of pharmacologically active substances of group B in food products; b) to collect and analyse scientific literature about pharmacologically active substances of group B getting into food products, the amount and the harm caused by them; c) to analyse the features of the results made by monitoring pharmacologically active substances of group B in 2004-2011; d) to analyse the observed results of pharmacologically active substances of group B, according to the groups of food products and the year the research was carried out. The Master degree dissertation contains these parts: part 1-pharmacologically active substances of group B, their description and the safe amount of them in food products, part 2- the research into the analysis of residue from pharmacologically active substances of group B made in 2004-2011. 14 tables and 4 pictures are used in the research. The thesis contains 58 pages. The following most important conclusions were made: 1. 3441-4319 samples are examined annually by monitoring pharmacologically active substances in Lithuania. 16-41 samples, (0,462–0,997 %) which have exceeded MAC of controlled residue, are ascertained. 2. Examining... [to full text]

Modern techniques like automated microscopy and spectroscopy now make it possible to study quantitatively, across multiple phenotypic and molecular parameters, how cell populations are affected by different treatments and/or environmental disturbances. As the technology development at the instrument level often is ahead of the data analytical tools and the scientific questions, there is a large and growing need for computational algorithms enabling desired data analysis. These algorithms must have capacity to extract and process quantitative dynamic information about how the cell population is affected by different stimuli with the final goal to transform this information into development of new powerful therapeutic strategies. In particular, there is a great need for automated systems that can facilitate the analysis of massive data streams for label-free methods such as phase contrast microscopy (PCM) imaging and spectroscopy (NMR). Therefore, in this thesis, algorithms for quantitative high-throughput phenotypic and systemic profiling of in vitro growing cell populations via label-free microscopy and spectroscopy are developed and evaluated. First a two-dimensional filter approach for high-throughput screening for drugs inducing autophagy and apoptosis from phase contrast time-lapse microscopy images is studied. Then new methods and applications are presented for label-free extraction and comparison of time-evolving morphological features in phase-contrast time-lapse microscopy images recorded from in vitro growing cell populations. Finally, the use of dynamic morphology and NMR/MS spectra for implementation of a reference database of drug induced changes, analogous to the outstanding mRNA gene expression based Connectivity Map database, is explored. In conclusion, relatively simple computational methods are useful for extraction of very valuable biological and pharmacological information from time-lapse microscopy images and NMR spectroscopy data offering great potential for biomedical applications in general and cancer pharmacology in particular.

La combinaison de biomatériaux et cellules souches, a pour but de protéger des cellules endommagées et de ralentir la progression des maladies neurodégénératives, comme la maladie de Huntington (MH). Les cellules souches mésenchymateuses et particulièrement une sous-population, les cellules MIAMI, ont déjà démontré leur efficacité dans la maladie de Parkinson. Il est cependant essentiel d’améliorer leur différenciation neuronale, leur survie et évaluer leur sécrétome. L’objectif principal de ce travail fut de proposer une stratégie innovante de médecine régénératrice pour la MH associant cellules souches, nano et micro médecines. Pour l’évaluer, un nouveau modèle animale ex vivo de la MH a été mis en place. Nous avons ensuite développé et optimisé deux nano-vecteurs, des nanocapsules lipidiques et des nanoparticules solides de SPAN, et les avons associés à un inhibiteur de REST qui est un facteur de transcription qui empêche la différenciation neuronale. La transfection de ce siREST a montré une amélioration du phénotype neuronal. Ces cellules ainsi modifiées furent ensuite induites vers un phénotype GABAergic grâce à des facteurs de croissance. Puis elles ont été associées à un support 3D, les microcarriers pharmacologiquement actif (MPA) permettant une meilleure intégration des cellules après greffe. Les MPA sont des microsphères ayant une surface biomimétique de laminine et libérant de façon contrôlée un facteur trophique le « brain derived neurotrophic factor » (inducteur d’un phénotype neuronal et neuro-protecteur). Des résultats prometteurs ont été obtenus, encourageant à continuer l’évaluation de cette stratégie in vivo dans des modèles génétiques de la MH. / The combination of biomaterials and stem cells aims to protect damaged cells and slow the progression of neurodegenerative diseases such as Huntington's disease(HD). Mesenchymal stem cells, particularly a subpopulation known as MIAMI cells, have already demonstrated their effectiveness in Parkinson's disease. However, it is essential to improve their neuronal differentiation, survival, and to assess their secretome. The main objective of this work was to propose an innovative regenerative medicine strategy for HD by combining stemcells, micro and nano medicines. To perform this assessment, a new ex vivo animal model of HD has been set up. We then developed and optimized two nanovectors,lipid nanocapsules and solid SPAN nanoparticles,carrying an inhibitor of REST a transcription factor, which prevents neuronal differentiation. The transfection of this siREST showed an improvement in the neuronal phenotype. These modified cells were then induced into a GABAergic phenotype through growth factors. They were then associated with a 3D support, the pharmacologically active microcarriers (PAM) allowing a high rate of engraftment. The PAM are microspheres which have a biomimetic surface of laminin and release a trophic factor BDNF, brain derived neurotrophic factor (inducer of a neural phenotype and neuroprotective) in a controlled manner. Promising results were obtained, further encouraging continuing the evaluation of this strategy in vivo in genetic models of HD.

<p>Ispitana je direktna i indirektna razgradnja odabranih&nbsp; &beta;-blokatora (metoprolol- tartarata, MET i propranolol-hidrohlorida, PRO) kao i diuretika (hidrohlortiazida, HCTZ) i njegovog stabilnog intermedijera hidrolize 4-amino-6-hlor-1,3-benzendisulfonamida (ABSA). Praćena je i kinetika razgradnje direktnom i indirektnom fotolizom uz primenu UVA, UVC, sunčevog i simuliranog sunčevog zračenja (SSZ). Najpre je ispitana&nbsp; stabilnost MET pod dejstvom SSZ, UVA, UVC, UVA/H₂O_{2 ,} i UVA/&nbsp; BrO₃^-. Dalje je ispitana efikasnost razgradnje MET pod dejstvom O₃i UVC/O_3. Identifikovano je deset intermedijera tokom UVC, O_3&nbsp;i UVC/O₃ razgradnje, pri&nbsp; čemu samo jedan ima značajno vi&scaron;u toksičnost prema algama i bakterijama u odnosu na ostale. Efikasnost fotokatalitičke razgradnje MET je ispitana u TiO₂ suspenzijama sa komercijalnim katalizatorima (Wackherr i Degussa P25). Mehanizam fotokatalitičke razgradnje je detaljno ispitan, pri&nbsp; čemu je identifikovano&nbsp; četrnaest intermedijera. EC₅₀ vrednost MET i njegovih sme&scaron;a nastalih pri fotokatalitičkoj razgradnji su određene na tri ćelijske linije sisara (H-4-II-E, HT-29 i MRC-5). Kako bi se povećala efikasnost&nbsp; rocesa fotokatalitičke razgradnje primenom komercijalnih katalizatora, ispitan je&nbsp; uticaj prisustva elektron-akceptora u suspenziji, pri čemu je upoređen uticaj O₂/H₂O₂, i O₂/ BrO₃^-&nbsp;na mehanizam razgradnje. Na osnovu teorije funkcionala gustine stekao se uvid u promene unutar molekula MET u prisustvu reaktivnih radikala. Takođe, ispitana je efikasnost nedopiranih TiO₂ , kao i dopiranih La(III) nanoprahova sintetisanih sol&minus;gel postupkom, u razgradnji MET. Uticaj temperature &nbsp;kalcinacije na fotokatalitičku efikasnost TiO 2 nanoprahova dopiranih pomoću La(III) &nbsp;ispitana je na supstratima MET i PRO, pri čemu je analiziran i uticaj strukture&nbsp; polaznog jedinjenja na&nbsp; informacija efikasnost fotokatalitičke razgradnje. Rezultati su upoređeni sa nedopiranim TiO₂nanoprahom i TiO₂Degussa P25 (pri pH-vrednosti 9). Pored toga, ispitana je kinetika i toksičnost PRO i njegovih intermedijera nastalih tokom razgradnje sa TiO₂Degussa P25. Ispitan je i uticaj strukture polaznog jedinjenja (MET, HCTZ i ABSA) na fotorazgradnju pod dejstvom&nbsp;UVA, sunčevog i simuliranog sunčevog zračenja, u odsustvu/prisustvu TiO₂ Degussa P25. Takođe, ispitan je i sinergistički efekat MET i ABSA na proces hidrolize, direktne fotolize i fotokatalize sa SSZ/TiO₂Degussa P25. Kako bi se stekao uvid u toksičnost proučavanih sistema, ispitan je njihov uticaj na rast odabranih ćelijskih linija sisara.</p> / <p>Direct and indirect degradation of selected&nbsp; &beta;-blockers (metoprolol tartrate, MET and&nbsp;propranolol hydrochloride, PRO) and also diuretic (hydrochlorothiazide, HCTZ) in &nbsp;addition to its stable hydrolysis intermediate 4-amino-6-chloro-&nbsp;&nbsp; 1,3-benzenedisulfonamide (ABSA) were investigated. The kinetics of their degradation obtained by direct and indirect photolysis under UVA, UVC, sunlight and simulated sunlight irradiation (SSI) have been followed. Firstly, the stability of MET under influence of SSI, UVA, UVC, UVA/H₂O₂ , and UVA/&nbsp; BrO₃^- has been investigated. Further, the efficiency of MET degradation under influence of O₃, and UVC/O₃ has been explored. Ten intermediates have been identified during the UVC, O₃, and UVC/O₃ treatments, while only one intermediate had significantly higher toxicity towards the algae and bacteria in respect to the others. Efficiency of&nbsp; photocatalytic degradation of MET was investigated in TiO₂ suspensions with&nbsp;commercial catalysts (Wackherr and Degussa P25). Mechanism of photocatalytic degradation was investigated in detail according to which fourteen intermediates were identified. EC 50 &nbsp;value of MET and its mixtures formed during the photocatalytic degradation has been determined at three mammalian cell lines (H-4-II-E, HT-29, and MRC-5). In order to improve the efficiency of photocatalytic degradation process applying commercial catalysts, the influence of presence of electron acceptors in suspension has been investigated and the influence of O₂/H₂O_2&nbsp;and O₂/BrO₃^- to the mechanism of degradation has been compared. Based on the density&nbsp; functional theory an insight to the changes within MET molecule in the presence of reactive radicals has been made. Also, the efficiency of bare TiO₂, as well as&nbsp;doped La(III) nanopowders synthesized by sol-gel procedure, in the degradation of&nbsp; MET has been investigated. The influence of calcination temperature on&nbsp; hotocatalytic efficiency of TiO_2&nbsp;nanopowders doped with La(III) has been studied on the MET and&nbsp; PRO substrates, and the influence of structure of the starting compound on the&nbsp; efficiency of &nbsp;photocatalytic degradation had been analyzed. Results have been&nbsp; compared with bare TiO_2&nbsp;nanopowder and TiO₂ Degussa P25 (at pH value of 9). Besides, kinetics and toxicity of PRO and its intermediates formed during the degradation with TiO₂ Degussa P25 have been investigated. The influences of starting compound&rsquo;s structure (MET, HCTZ, and ABSA) to photodegradation under UVA, sunlight and SSI, in the absence/presence of TiO₂ Degussa P25, have been investigated. Also, the synergistic effects of MET and ABSA to the process of&nbsp; hydrolysis, direct photolysis, and photocatalysis with SSI/TiO₂ Degussa P25 have been investigated. In order to get &nbsp;an insight into the toxicity of the studied systems, their influence on the growth of selected mammalian cell lines has been investigated as well.</p>

Une approche de la médecine régénérative du système nerveux consiste à développer des substituts biologiques avec une fonction réparatrice en utilisant des cellules souches et des biomatériaux qui peuvent être recouverts des molécules de la matrice extracellulaire. Nous avons ainsi développé des microcarriers pharmacologiquements actifs, MPA. Ce sont des microsphères (MS) polymériques à base de PLGA, biodégradables et biocompatibles, recouvertes des molécules d’adhérence qui fournissent un support en 3-dimensions aux cellules. Les microcarriers ainsi associés aux cellules souches permettent, après implantation, d’augmenter la survie et de maintenir l’état de différenciation des cellules qu’ils portent,renforçant leurs effets de réparation tissulaire. Ces MPA peuvent également libérer des facteurs de croissance encapsulés et afin d’améliorer le relargage de protéines encapsulées une nouvelle combinaison de polymère : PLGA-Poloxamer188 (P188) -PLGA a été développé dans notre laboratoire. Il a aussi été montré que les MPA de PLGA-P188-PLGA fonctionnalisées avec de la fibronectine et poly-D-lysine induisaient une meilleure prolifération de cellules souches mésenchymateuses que les MPA de PLGA.Ces cellules sont très largement utilisées en médecine régénérative car elles sont faciles à prélever, se trouvant dans la moelle osseuse, et capables de se différencier vers le lignage chondrogénique, ostéogénique et dans certaines conditions, neuronale. Nous travaillons avec une sous population de ces cellules appelées cellules MIAMI (marrow isolated adult multilineage inducible) qui s’engagent vers une différenciation en cellule neuronale après un traitement avec 2 facteurs de croissance (EGF/ bFGF) et sur un support matriciel de laminine. Dernièrement, il a été mis en évidence que les propriétés physicochimiques des supports polymériques régissent également le comportement des cellules souches(adhésion, survie et différenciation). L’objectif de cette étude est d’étudier l’effet des propriétés physicochimiques et mécaniques des surfaces i) des MS sur l’adsorption de laminine et poly-D-lysine et ii) des MPA sur l’adhérence et la différenciation neuronale des cellules MIAMI. Nous avons montré que la présence du bloc hydrophile « poloxamère 188 » dans la composition du polymère PLGA-P188-PLGAdiminue l’adsorption de molécules d’adhérence en formant une couche sur ces surfaces. Sur les MPA de PLGA, les molécules d’adhérence s’adsorbent bien quelle que soit la charge globale des molécules. Cesdeux MPA ont une charge globale positive et permettent l’attachement de cellules à leur surface. Cependant, l’adhérence à court terme de cellules est plus forte sur les MPA de PLGA comparé aux MPA de PLGA-P188-PLGA mais à la longue les cellules finissent par adhérer aux deux supports. Le PLGAP188-PLGA présente une forte énergie libre de surface et ces MPA présentent une surface moins rigide que les MPA de PLGA. Nos résultats suggèrent que ces caractéristiques de surface permettent aux cellules d’adhérer malgré la faible quantité de laminine sur ces supports. A long terme les cellules présentent le même comportement quel que soit le type du support. Elles se différencient en cellule de type neuronal exprimant des marqueurs de neurone mature comme le neurofilament et nous trouvons le même nombre de cellules adhérées à leur surface. En outre, nous avons montré que les cellules sont capables de sécréter de la même manière des molécules de la matrice extracellulaire sur les deux types de MPA expliquant probablement la similitude de comportement à long terme. / An approach to regenerative nervous system medicine is to develop biological substitutes with restorative function using stem cells and biomaterials that can be coated with extracellular matrix molecules. We have developed pharmacologically active microcarriers, PAMs. These are PLGA based, biodegradable and biocompatible polymeric microspheres (MS) coated with adhesion molecules that provide 3-dimensional support for cells. The microcarriers thus associated with the stem cells make it possible, after implantation, to increase the survival and maintain the state of differentiation of the cells they carry, reinforcing their tissue repair effects. These PAMs can also release encapsulated growth factors and to enhance the release of encapsulated proteins a new polymer combination: PLGA-Poloxamer188 (P188) -PLGA has been developed in our laboratory. It has also been shown that PLGA-P188-PLGA PAMs functionalized with fibronectin and poly-Dlysineinduce better proliferation of mesenchymal stem cells than PLGA PAMs. These cells are very widely used in regenerative medicine because they are easy to collect, found in the bone marrow, and able to differentiate towards the chondrogenic lineage, osteogenic and under certain conditions,neuronal. We are working with a subpopulation of these cells called MIAMI cells (marrow isolated adult multilineage inducible) that engage in neuronal cell differentiation after treatment with 2growth factors (EGF / bFGF) and on a laminin matrix support. Recently, it has been demonstrated that the physicochemical properties of polymeric supports also regulate the behavior of stem cells (adhesion, survival and differentiation). The objective of this study is to study the effect of physicochemical and mechanical properties of surfaces i) MS on laminin and poly-D-lysineadsorption and ii) PAMs on adhesion and neuronal differentiation of MIAMI cells. We have shown that the presence of the hydrophilic "poloxamer 188" block in the PLGA-P188-PLGA polymer composition decreases the adsorption of adhesion molecules by forming a layer on these surfaces.On PLGA PAMs, the adhesion molecules adsorb well regardless of the overall charge of the molecules. These two PAMs have a positive overall charge and allow the attachment of cells to their surface. However, in short-term cell adhesion is stronger on PLGA PAMs compared to PLGA-P188-PLGA PAMs, but in the long-term the cells eventually adhere to both supports. PLGA-P188-PLGAhas a high free surface energy and these PAMs have a less rigid surface than PLGA PAMs. Our results suggest that these surface characteristics allow cells to adhere despite the low amount of laminin on these supports. In the long-term the cells exhibit the same behavior whatever the type of PAMs. They differentiate into neuronal cells expressing mature neuron markers such as the neurofilament-M and we find the same number of cells adhered to their surface. Furthermore, we have shown that cells are able to secrete extracellular matrix molecules in the same way on both types of PAMs, probably explaining the similarity of the behavior in long-term.

The human heart has poor endogenous regeneration. If myocytes are lost due to injury, the myocardium is unable to restore its myocyte content and instead undergoes compensatory hypertrophy and remodeling. Cardiac tissue engineering aims to recreate and provide functional myocardium that replaces the injured myocardium. In this study, human engineered heart muscle (EHM) from cardiomyogenically differentiated human embryonic stem cells was generated. EHMs consisted of elongated, anisotropically organized cardiomyocyte bundles and responded “physiologically” to increasing calcium concentrations. To generate large myocardium capable of encompassing the ventricles, a novel process to systematically upscale the dimensions of engineered myocardium to a humanized Biological Ventricular Assisted Device (hBioVAD) was introduced. The hBioVADs formed a “pouch-like” myocardium at rabbit heart dimensions and were beating spontaneously. Further enhancement by biomimetic pulsatile loading generated “more mature” myocardium. Additional paracrine functionality was integrated by generating insulin-like growth factor-1 (IGF-1) secreting fibroblasts for tissue engineering applications. IGF-1 release induced higher levels of Akt phosphorylation and hypertrophy in cardiomyocytes resulting in increased force generation of EHM. Finally, feasibility of “paraBioVAD” (IGF-1 cell line and cardiomyocytes) implantation was demonstrated in a healthy rat model. Histological observations demonstrated engraftment on the heart and the presence of vascular structures. In conclusion, a humanized “paraBioVAD” technology for mechanic and paracrine heart support was developed. Future studies will assess its therapeutic utility in heart failure