14 research outputs found
Mechanisms of nanoparticle formation and gene delivery of polyplex-based delivery systems
Gene therapy provides a promising option for treatment of various diseases, but the fact remains that the large number of gene delivery systems has met with little therapeutic success. Viral gene delivery has a high degree of specificity and efficacy, but it does not provide sufficient safety for clinical applications. Therefore, the search for an efficient alternative, a synthetic gene delivery vector, has been active.
Typically, non-viral delivery vectors are based on the use of cationic polymers which bind and compact DNA via electrostatic interactions into nanoparticles (polyplexes). The ability of a cationic polymer to bind and condense DNA is important for effective delivery because good packing not only protects DNA against degradation in the extracellular space, but also allows effective release of DNA inside cells. While cationic polymers are relatively nontoxic and safe, they lack significant efficacy. This major drawback of non-viral vectors is largely due to a poor understanding of the mechanism underlying the complexation and gene delivery process. Furthermore, the lack of reliable methods to study the binding between DNA and cationic polymers has hindered development in synthetic gene delivery systems.
The aim of this study was to investigate the mechanisms of DNA complex formation and gene transfer mediated by cationic polymers with different structures (poly-L-lysine, PLL; polyethylenimines, PEIs; poly-β-amino esters, PBAEs) and transfection efficiencies.
This thesis combines time-resolved fluorescence spectroscopy with cell transfection studies in order to elucidate how polymer structure can affect DNA binding and influence gene delivery outcomes. This method allows the quantitative determination of polymer–DNA interaction and binding. We showed that the mechanism of PEI–DNA and PLL–DNA complex formation was positively cooperative with a saturation limit near 100% at a polymer/DNA molar (N/P) ratio of 2, whereas most of PBAE–DNA complexes expressed negative cooperativity and reached a saturation level close to 80%. The polymer topology, the type of amines (primary, secondary and tertiary) and their density, and the environmental pH had a clear effect on the binding constants and the degree of cooperativity. The possible correlation between fluorescence parameters and transfection efficiency was investigated with a series of PBAEs. Their transfection efficiency showed an increasing trend in association with the relative efficiency of PBAE–DNA nanoparticle formation.
The role of free polymer in polyplex formation and gene delivery was examined with PEI as a model vector. For PEI polyplexes, the formation of the polyplex core was completed at N/P 2 and the excess of polymer formed a protective shell around the core. Unlike PLL, PEI molecules were able to undergo an exchange between the core and shell of the polyplexes. Such differences in structural dynamics of these polyplexes may partly provide an explanation for the differences seen in their DNA release and transfection efficacy at the cellular level. The excess of PEI in the shell had no effect on the physical state of polyplexes, suggesting that the polyplex core retains its original structure during shell formation. However, the excess of PEI was a crucial factor in successful transfection. The role of free PEI in the gene transfection process was examined in cell cultures with modified cell-surface glycosaminoglycans content. This study showed that free PEI is essential for minimizing the undesirable binding of polyplexes to cell-surface glycosaminoglycans, which may otherwise pose a barrier in non-viral gene delivery. Lastly, we focused on the role of PEI structure in PEI–liposome–DNA delivery systems (lipopolyplexes). We found that the enhancement of lipopolyplex-mediated delivery by different types of PEI species is common and associated with PEI size rather than structure.
In conclusion, the present study demonstrated that the fluorescence spectroscopy approach for the analysis of gene delivery systems can provide valuable quantitative information about the binding behaviour of various cationic polymers to DNA. The improved understanding of mechanisms behind formation of these complexes can contribute to the design of polymeric delivery vectors with improved properties. Furthermore, this study sheds light on the mechanisms by which free polymer enhances gene transfer. It explains why high N/P ratios are needed for effective transfection and how the interactions between free polymer and cell-surface GAGs lead to alterations in gene transfer by the polyplexes.Geeniterapia on lupaava vaihtoehto useiden sairauksien hoitoon. Tähän mennessä kehitetyt geenien siirtotekniikat eivät ole kuitenkaan vastanneet niille asetettuja odotuksia. Virusvektorien avulla tehty geeninsiirto on spesifistä ja tehokasta, mutta se ei ole tarpeeksi turvallista kliinisiin sovelluksiin. Siksi tehokkaita synteettisiä eli muuhun kuin viruksiin perustuvia vektoreita etsitään jatkuvasti.
Synteettiset vektorit ovat yleensä kationisia polymeerejä, jotka sitovat ja pakkaavat DNA:ta sähköstaattisilla vuorovaikutuksilla nanopartikkeleiksi (polyplekseiksi). Hyvä vektori suojaa DNA:ta hajoamiselta solun ulkopuolella, mutta päästää DNA:n vapaaksi solun sisällä. Kationisilla polymeereillä ei ole samanlaisia turvallisuus ongelmia kuin virusvektoreilla, mutta tähän mennessä tutkitut polypleksit eivät ole virusvektoreihin verrattuna kovin tehokkaita. Jotta voitaisiin kehittää tehokkaampia DNA-polypleksejä, niiden muodostumisen ja geenien siirron mekanismit tulisi ymmärtää paremmin.
Tämän työn tarkoituksena oli tutkia miten kationisen polymeerin kemiallinen rakenne vaikuttaa polypleksien muodostumiseen ja geeninsiirron tehokkuuteen. Polymeereinä käytettiin poly-L-lysiiniä (PLL), polyetyleeni-imiinejä (PEI) ja poly-β-aminoestereitä (PBAE). Aikaerotteisilla fluoresenssimittauksilla saatiin kvantitatiivista tietoa polymeerin ja DNA:n välisistä vuorovaikutuksista. PEI–DNA- ja PLL–DNA-polypleksien muodostumisen havaittiin olevan positiivisesti kooperatiivista ja niiden sitoutumisisotermit saturoituivat noin 100%:iin N/P-suhteen ollessa noin kaksi. Sen sijaan PBAE–DNA-kompleksien muodostuminen oli negatiivisesti kooperatiivista ja niiden sitoutumisisotermit saturoituivat noin 80% kohdalle. Polymeerien topologia, amiiniryhmien tiheys ja tyyppi (primaarinen, senkundaarinen ja tertiaarinen) sekä ympäristön pH vaikuttivat sekä sitoutumisvakioon että kooperatiivisyyteen. PBAE-polymeereillä havaittiin transfektiotehokkuuden korreloivan PBAE–DNA-nanopartikkelien muodostumisen suhteellisen tehokkuuden kanssa.
Polymeerin määrän vaikutusta polypleksien muodostumiseen ja geenisiirtoon tutkittiin PEI:n ja PLL:n avulla. Näillä polymeereillä polypleksin ydin on valmis, eli muodostunut nanopartikkeli on sähköisesti neutraali, kun N/P-suhde on noin kaksi. Suuremmilla N/P-suhteilla ytimen muodostumisesta ylijäävä polymeeri muodostaa suojaavan kuoren polypleksin ytimen ympärille. PEI-polyplekseissä molekyylit pääsivät vaihtumaan ytimen ja kuoren välillä, mutta PLL-polyplekseille vaihtoa ei tapahdu. Tällaiset erot polypleksien rakenteellisessa dynamiikassa voivat osaltaan selittää solutasolla havaittuja eroja näiden vektoreiden transfektiotehdokkuudessa. Kuoren polymeeri ei vaikuta polypleksin fysikaaliseen tilaan, joten polypleksin ydin säilyttää alkuperäisen rakenteensa kuoren muodostuessa sen ympärille. Kuoren polymeerillä on kuitenkin tärkeä rooli transfektion onnistumisessa. Tämän ns. vapaan polymeerin roolia geenin siirrossa tutkittiin PEI:n avulla käyttämällä solulinjaa, jonka pinnan glykosanimoglykaanimäärää oli muunneltu. Saatujen tulosten perusteella vapaa PEI estää polypleksien haitallista sitoutumista glykosanimoglykaaneihin. Tämä havainto selittää osaltaan miksi tehokas geeninsiirto vaatii korkeita N/P-suhteita.
Lopuksi tutkittiin PEI:n kemiallisen rakenteen vaikutusta lipopolyplekseissä eli PEI-liposomi-DNA-polyplekseissä. PEI:n havaittiin edistävän lipopolypleksi-välitteistä geeninsiirtoa ja PEI:n koolla havaittiin olevan suurempi merkitys kuin sen kemiallisella rakenteella.
Yhteenvetona voidaan todeta tämän tutkimuksen osoittavan, että fluoresenssispektroskopian avulla voidaan saada arvokasta kvantitatiivista tietoa kationisten polymeerien ja DNA:n välisestä sitoutumisesta. Lisäksi tämän tutkimuksen aikana saatiin lisätietoa mekanismeista, joiden avulla vapaa polymeeri edistää geeninsiirtoprosessia. Saatuja tuloksia voidaan hyödyntää parempien geeninsiirtovektoreiden kehittämisessä
Academic self-efficacy development of psychology students at FF UK
This bachelor thesis is divided into two sections. Theoretical part of this thesis is focused on more detailed introduction of both concepts: self-efficacy and academic self-efficacy. Aim of the suggestion of research project is to propose a procedure of detection the development of academic self-efficacy at psychology students. For research purposes, I will use a test battery, consisting of a specially designed academic self-efficacy questionnaire, emotional intelligence test and student engagement test. The study is conceived as a quantitative and longitudinal. It will capture academic self-efficacy development of psychology students at Charles University philosophical faculty from the beginning of their studies to their graduation.Tato bakalářská práce je rozdělena do dvou oddílů. Literárně přehledová část práce je zaměřena na podrobnější teoretické vymezení konceptu vnímané osobní a akademické účinnosti. Návrh výzkumného projektu si klade za cíl vytvořit postup zjišťování vývoje vnímané akademické účinnosti u studentů psychologie na Filozofické fakultě Univerzity Karlovy. Pro potřeby výzkumu je použita testová baterie, sestávající ze speciálně navrženého dotazníku vnímané akademické účinnosti, testu emoční inteligence a testu studentova zapojení. Jedná se o kvantitativní longitudinální studii, která zachycuje vývoj vnímané akademické účinnosti u studenů psychologie Filozofické fakulty Univerzity Karlovy od počátku studia až po zdárné absolvování oboru.Katedra psychologieDepartment of PsychologyFilozofická fakultaFaculty of Art
A comparison between novel FPGA-based pad monitoring system using ballistocardiography and the conventional systems for synchronization and gating of CMRI at 3 tesla: A pilot study
This pilot pre-clinical study demonstrates the applicability of a new type of pneumatic cardiac triggering (PCT) for cardiac imaging. The pilot research compares the novel FPGA-based pad monitoring system for cardiac triggering using ballistocardiography (BCG) with conventional systems based on electrocardiography (ECG) and photoplethysmography (PPG). The implemented system enables cardiac triggering without the need to fix the sensors to the patient's body. This unique approach has the potential to reduce the preparation time for examination and the examination itself and to increase patient's comfort. The pilot pre-clinical study was conducted on 10 subjects at the Siemens Prisma 3T MRI Scanner within the CEITEC Multimodal and Functional Imaging Laboratory - Central European Institute of Technology, Masaryk University, upon the approval of the Ethics Committee. In total, 748 peaks (heart beats) were detected, with 7.347 correctly identified as true positive peaks, 140 incorrectly detected as false positive peaks, and 106 missed peaks (false negative). For all subjects, the total accuracy reached 96.31% and F1 score reached 98.18%. The applicability of the proposed BCG system was also analyzed in terms of objective (BRISQUE, NIQE, PIQE) and subjective evaluation of the images by 10 experts. The study compares images from two basic cardiac sequences - TRUE FISP (Free Induction Decay Steady-State Precession) and PSIR (Phase Sensitive Inversion Recovery) sequences. The BCG system achieves comparable results with the most frequently used and most accurate clinical ECG system used as gold standard. The results prove that the BCG signal captured by our new sensor can be used as a substitute for ECG signal during MRI exam with reliability of 97%.Web of Science84170414
A comparison of alternative approaches to MR cardiac triggering: A pilot study at 3 Tesla
This pilot comparative study evaluates the usability of the alternative approaches to magnetic resonance (MR) cardiac triggering based on ballistocardiography (BCG): fiber-optic sensor (O-BCG) and pneumatic sensor (P-BCG). The comparison includes both the objective and subjective assessment of the proposed sensors in comparison with a gold standard of ECG-based triggering. The objective evaluation included several image quality assessment (IQA) parameters, whereas the subjective analysis was performed by 10 experts rating the diagnostic quality (scale 1 - 3, 1 corresponding to the best image quality and 3 the worst one). Moreover, for each examination, we provided the examination time and comfort rating (scale 1 - 3). The study was performed on 10 healthy subjects. All data were acquired on a 3 T SIEMENS MAGNETOM Prisma. In image quality analysis, all approaches reached comparable results, with ECG slightly outperforming the BCG-based methods, especially according to the objective metrics. The subjective evaluation proved the best quality of ECG (average score of 1.68) and higher performance of P-BCG (1.97) than O-BCG (2.03). In terms of the comfort rating and total examination time, the ECG method achieved the worst results, i.e. the highest score and the longest examination time: 2.6 and 10:49 s, respectively. The BCG-based alternatives achieved comparable results (P-BCG 1.5 and 8:06 s; OBCG 1.9, 9:08 s). This study confirmed that the proposed BCG-based alternative approaches to MR cardiac triggering offer comparable quality of resulting images with the benefits of reduced examination time and increased patient comfort.Web of Science2662605259
A low-cost system for seismocardiography-based cardiac triggering: A practical solution for cardiovascular magnetic resonance imaging at 3 tesla
This study describes a pilot clinical validation of a new low-cost system for the continuous monitoring of the human body's cardiorespiratory activities within the magnetic resonance examination area. This study primarily focuses on monitoring cardiac activity and the related cardiac triggering. The patented system tested by the authors is based on seismocardiography (SCG). The study was conducted on 18 subjects on a Siemens Prisma 3T MR scanner. Standard anatomical and diffusion sequences were used to test cardiac activity monitoring. A wide range of commonly used diagnostic sequences were used to test imaging of the heart by means of cardiac triggering. System functionality was verified against a commercially available electrocardiography (ECG) system. Monitoring of cardiac activity (detection of the R-R interval in ECG and the AO-AO interval in SCG) was objectively evaluated by determining the overall probability of correct detection (ACC), sensitivity (SE), positive predictive value (PPV), and harmonic mean between SE and PPV, i.e. F1. Imaging quality control using Cardiac Triggering was performed by subjective evaluation of images by the physicians. The study conducted clearly confirmed the functionality of the system in terms of continuous cardiac activity monitoring. In all 18 subjects, a mean PPV > 99% was achieved; F1 > 99 %; SE > 99 %; ACC > 98 %; 1.96 sigma < 3.5 bpm. Also, Cardiac Triggering functionality was confirmed by the physicians on the basis of analyzing cardiac images using the T1/T2 balanced echo sequences and the T1 flash sequence measured natively.Web of Science711862911860
Evaluation of the Company Správa nemovitostí Olomouc, a. s.
This Master Thesis deals with the evaluation of the company Správa nemovitostí Olomouc, a. s. as of December 20, 2011.The value of the company was determined based on detail strategic and financial analyses which illuminated the core business and future potential of the company. Results of the analyses and interviews with the management of the company allowed preparing thorough five year financial plan. After the fifth year I have assumed a stable growth. The final value was determined as an average between two income methods, Free Cash Flow to the Firm and Economic Value Added. For comparison I have also prepared a property method valuation using the book value of the company
Academic self-efficacy development of psychology students at FF UK
This bachelor thesis is divided into two sections. Theoretical part of this thesis is focused on more detailed introduction of both concepts: self-efficacy and academic self-efficacy. Aim of the suggestion of research project is to propose a procedure of detection the development of academic self-efficacy at psychology students. For research purposes, I will use a test battery, consisting of a specially designed academic self-efficacy questionnaire, emotional intelligence test and student engagement test. The study is conceived as a quantitative and longitudinal. It will capture academic self-efficacy development of psychology students at Charles University philosophical faculty from the beginning of their studies to their graduation
Financial analysis Fujitsu Siemens Computers, s. r. o.
Financial analysis is the analysis of the financial health of company. This can be analised in many ways - rentability, liquidity, activity, etc. Also some bonite and bankrot models can be used. This work is about the financial analysis of Fujitsu Siemens Computers, s. r. o
Non-specific positive reaction in pre-transfusion examination
Pre-transfusion examination is a set of immunohematological examinations before administration of a transfusion preparation to a patient. The purpose of this examination is to ensure safe and effective transfusion to the recipient. The pre- administration examination of the blood product must include an examination of the recipient's blood type, screening of the recipient's anti-erythrocyte antibodies, and a transfusion product compatibility test. The test is performed by the agglutination method and in some cases the enzyme is used in the screening
Eigenfrequencies of a physical model of human vocal folds
Tato bakalářská práce se zaměřuje na numerickou simulaci vlastních frekvencí a vlastních tvarů kmitání lidských hlasivek. Jejím cílem je vytvoření numerického modelu, jehož prostřednictvím budou výpočty vlastních frekvencí a tvarů uskutečněny. Teoretická část seznamuje čtenáře s anatomií a fyziologií fonačního ústrojí a fyzikální podstatou vzniku lidského hlasu. Experimentální část se zabývá vývojem samotného modelu a jeho výsledky. Výstupem z práce je funkční numerický model hlasivky v softwaru COMSOL Multiphysics.This bachelor thesis is focused on numerical simulation of eigenfrequencies and eigenmodes of human vocal folds. The goal of the thesis is the creation of a numerical model based on which the calculations would be realized. In the theoretical part there is an introduction into the anatomy and physiology of human phonatory system and basic physical principles explaining the creation of human voice. The experimental part consists of development of the physical model and its results. The result of the thesis is a functional numerical model constructed in COMSOL Multiphysics