Vähi-testis antigeen MAGE-A4 inkorporeeritakse ekstratsellulaarsetesse vesiikulitesse ja on eksponeeritud selle pinnal

Väitekirja elektrooniline versioon ei sisalda publikatsiooneVähkkasvaja on väga kompleksne haigus, mis on endiselt üks suurimaid surma põhjusi maailmas. Seetõttu on vähi-uuringud olnud prioriteetne suund teaduses pikka aega. Vähivastane immuunteraapia on paljulubav uudne tehnoloogia, mis seisneb inimese enda immuunsüsteemi ergutamises vähiga tõhusamalt võitlemiseks. Üheks immuunteraapia eelduseks on sihtmärgi ehk antigeenide olemasolu, mis on spetsiifilised just vähkkoe suhtes. Vähi-testis antigeenid (ingl lüh CTA) on valgud, mida tavapäraselt rakud toodavad ehk ekspresseerivad peamiselt munandites ehk testistes, kuid haiguslikult võivad need olla kõrgel tasemel ekspresseeritud ka erinevates vähkkasvajates. On näidatud, et CTA-d osalevad vähi arengut soodustavates protsessides, kuid antigeenidena võivad need esile kutsuda ka vähivastast immuunvastust. MAGE-A4, kui tuntud CTA, on tsütoplasmas või rakutuumas lokaliseeruv osalise korrapäratu struktuuriga valk, millel on kirjeldatud nii vähi kasvu soodustavaid kui ka pärssivaid omadusi. MAGE-A4 täpsest rakulisest funktsioonist on endiselt vähe teada. Üks moodus, kuidas vähk oma arengut ning levikut kehas toetab on rakuväliste ehk ekstratsellulaarsete vesiikulite (EV-d) rakendamine soodsate tingimuste tekitamiseks kehas. EV-d on membraaniga ümbritsetud nanoskaalas osakesed, mida rakud vabastavad endast rakuvälisesse ruumi. EV-sid võib leida kõigist kehavedelikest nagu näiteks verest või uriinist. EV-sid peetakse rakuvahelise suhtluse vahendajateks, sest need on võimelised edastama bioaktiivseid molekule ühest rakust teise. Sellise laia rolli tõttu osalevad EV-d pea kõikides füsioloogilistes ja patoloogilistes protsessides kehas, sh ka vähis. Viiruslaadsed partiklid (VLP-d) on EV-de sarnase struktuuri ja suurusega osakesed, mis erinevad ainult viiruse kapsiidi poolest, mis paikneb vesiikuli sees. VLP-d viiruse genoomi ei sisalda ja pole seetõttu nakkusohtlikud. VLP-de moodustumist on võimalik esile kutsuda kui viia rakkudesse viiruse kapsiidi valgu geen. Tänu biosobivusele ja -stabiilsusele peetakse EV-sid ja VLP-sid potentsiaalseteks vahenditeks uudsetes vähivastastes teraapia ja diagnostika tehnoloogiates. Käesolevas lõputöös avastati, et MAGE-A4 paigutatakse Moloney hiire leukeemia viiruse VLP-desse ja natiivsetesse EV-desse rakkude poolt, mis MAGE-A4 ekspresseerivad. Nii kunstlikult indutseeritud VLP-d kui ka natiivselt moodustunud EV-d eksponeerivad MAGE-A4 nende välispinnal, mis on üllatav nähtus kui arvestada MAGE-A4 rakusisest lokalisatsiooni. MAGE-A4 oli tuvastatav erinevate suurustega EV populatsioonidest, mis viitab MAGE-A4 inkorporeerimisele EV-desse erinevatest alatüüpidest. Tüüpilistes EV-de hoiustamistingimustes olid MAGE-A4 kandvad EV-d (MAGEA4-EV-d) väga stabiilsed – 21 päeva pikkuse mõõtmisperioodi jooksul ei tuvastatud MAGE-A4 kontsentratsioonis ega EV omadustes muutusi. Kuid enam kui kaks sulatus-külmutustsüklit avaldavad MAGEA4-EV-dele kahjulikku mõju, millele viitas tuvastatud MAGE-A4 kontsentratsiooni langus. MAGEA4-EV-de keemiliste töötluste tulemused vihjasid MAGE-A4 seondumisele EV-de membraanile pigem hüdrofoobsete interaktsioonide toel, mitte aga elektrostaatiliste jõudude abil. Puhastatud MAGE-A4 valku ja EV-sid kokku segades järeldati, et MAGE-A4-l on biokeemilisi omadusi, mis võimaldavad sellel seonduda vesiikulite välismembraanile rakulisi mehhanisme kasutamata. MAGE-A4 ekspresseeriti ka liitvalguna rohelise fluorestsentse valguga EGFP-ga hiire fibroblastides, millest eraldati EV-d. Nende analüüsimisel selgus, et MAGE-A4 on võimeline suunama EV-desse täiendavat bioloogilist materjali, mis tavaliselt sinna ei pakita. Liitvalguna ekspresseerimine ei takistanud MAGE-A4 eksponeerumast ka EV-de pinnal nii rakust eraldatuna kui ka passiivse kontakti võimaldamisel. Nende tulemuste põhjal järeldati, et MAGE-A4 võib olla inkorporeeritud EV-desse ja VLP-desse ning paigutatud nende pinnale nii sihipäraste rakuliste radade kui ka MAGE-A4 biokeemiliste omaduste tõttu. Töös kirjeldatud omadused teevad MAGE-A4-st atraktiivse sihtmärgi EV-põhistes uudsetes vähivastastes teraapiates ning mitte-invasiivses diagnostikas.Cancer is a complex disease that still is a major cause of death worldwide. Therefore, cancer research has been one the highest priorities in science. Anti-cancer immune therapy is a promising novel technology that promotes the immune system of a patient to fight against the cancer more efficiently. One of the requirements of this technique is the presence of cancer specific biomarkers. Cancer-testis antigens (CTAs) are proteins that are normally expressed mainly in testis but aberrantly in various tumours. It has been shown that CTAs contribute to tumorigenic processes and, as antigens, these proteins are known to induce anticancer immune responses. MAGE-A4, a known CTA, is a soluble cytoplasmic or nuclear protein with a partially disordered structure. Although its exact cellular function has largely remained elusive, MAGE-A4 has been shown to have tumorigenic and antitumorigenic properties. One way how cancer may support is development and spreading in the body is the utilization of extracellular vesicles (EVs). EVs are membrane-bound nanoscale vesicles released into extracellular space by cells. EVs could be found in all body fluids like blood or urine, for example. EVs are the mediators of intercellular communication as they carry bioactive cargoes from one cell to another. EVs are essentially involved in most physiological processes and pathological conditions, including cancer. Virus-like particles (VLPs) are highly similar to EVs by their size and structure, differing only by the viral capsid inside the vesicle. Since the capsid in VLPs is void of viral genome, the particles are not contagious. VLPs can be induced if the capsid protein gene is introduced to a living cell. EVs and VLPs are considered potential candidates for novel anticancer therapy and diagnostic technologies. In this dissertation, MAGE-A4 was discovered to be incorporated into VLPs of Moloney murine leukaemia virus and native EVs released by cells that express it. The artificially induced VLPs and natively emerged EVs expose MAGE-A4 to their outer surface, which is considered a fascinating phenomenon considering the intracellular localization of MAGE-A4. MAGE-A4 was found in EVs of different sizes, indicating its incorporation into different EV subtypes. The MAGE-A4 carrying EVs (MAGE-A4-EVs) were found to be very stable in common storage conditions for at least 21 days, but subjecting them to more than two cycles of freeze-thawing damages the vesicles, as a decrease in the MAGE-A4 concentration was detected. Chemical manipulations of the MAGE-A4-EVs indicated that MAGE-A4 is not bound to the surface of EVs by electrostatic forces but rather by hydrophobic properties. MAGE-A4 was shown to have biochemical properties that allow it to bind to EVs and VLPs by passive incubation. This means that the cellular pathways for the MAGE-A4 incorporation into vesicles are not entirely essential. MAGE-A4 was also recombined with green fluorescent protein EGFP and expressed as a fusion protein that was found to be incorporated into the EVs, showing the ability of MAGE-A4 to populate EVs with additional cargo. Moreover, as a fusion protein, MAGE-A4 was still exposed on the outer surface of the vesicles, and it sustained its EV membrane binding ability in vitro conditions. The results indicated that MAGE-A4 might be incorporated into the EVs and VLPs and exposed to their surface by cellular mechanisms or its biochemical properties. The features of MAGE-A4 described in the dissertation make it appealing for EV-based noninvasive cancer diagnostics and anticancer therapeutic applications.https://www.ester.ee/record=b550627

A dual colour FISH method for routine validation of sexed Bos taurus semen

Background: Usage of sexed semen that allows to choose the gender of the calves, is commonly practiced in livestock industry as a profitable breeding alternative, especially in dairy farming. The flow cytometric cell sorting is the only commercially available method for bovine sperm sexing. For validation of the sexing procedure several methods have been developed including sperm fluorescence in situ hybridisation techniques. Latter usually include the use of pre-labelled nucleotides for probe synthesis which is relatively expensive approach compared to combined application of aminoallyl-dUTP and chemical binding of fluorescent dyes. Here a sex determining dual colour bovine sperm fluorescence in situ hybridisation method is presented which is considered more cost-effective technique than the previously reported approaches. Results: The reliability of sex chromosome identifying probes, designed in silico, was proven on bovine metaphase plate chromosomes and through comparison with a commercially available standard method. In the dual colour FISH experiments of unsexed and sexed bovine sperm samples the hybridisation efficiency was at least 98%, whereas the determined sex ratios were not statistically different from the expected. Very few cells carried both of the sex chromosome-specific signals (less than 0.2%). Conclusions: A protocol for a dual colour bovine sperm FISH method is provided which is cost-effective, simple and fast for sex determination of spermatozoa in bull semen samples.Peer reviewe

A dual colour FISH method for routine validation of sexed Bos taurus semen

Abstract Background Usage of sexed semen that allows to choose the gender of the calves, is commonly practiced in livestock industry as a profitable breeding alternative, especially in dairy farming. The flow cytometric cell sorting is the only commercially available method for bovine sperm sexing. For validation of the sexing procedure several methods have been developed including sperm fluorescence in situ hybridisation techniques. Latter usually include the use of pre-labelled nucleotides for probe synthesis which is relatively expensive approach compared to combined application of aminoallyl-dUTP and chemical binding of fluorescent dyes. Here a sex determining dual colour bovine sperm fluorescence in situ hybridisation method is presented which is considered more cost-effective technique than the previously reported approaches. Results The reliability of sex chromosome identifying probes, designed in silico, was proven on bovine metaphase plate chromosomes and through comparison with a commercially available standard method. In the dual colour FISH experiments of unsexed and sexed bovine sperm samples the hybridisation efficiency was at least 98%, whereas the determined sex ratios were not statistically different from the expected. Very few cells carried both of the sex chromosome-specific signals (less than 0.2%). Conclusions A protocol for a dual colour bovine sperm FISH method is provided which is cost-effective, simple and fast for sex determination of spermatozoa in bull semen samples

Animal reproduction, technology and welfare, December 3-4, 2018, Estonian University of Life Sciences, Tartu, Estonia : [presentations]

PresentationsPresentations of the final conference of the SEARMET "Animal reproduction, technology and welfare"