RIC8A roll hiire närvisüsteemis ja selle arengus

Väitekirja elektrooniline versioon ei sisalda publikatsiooneKesknärvisüsteemi ja perifeerse närvisüsteemi korrektne toimimine on ülioluline organismi täisväärtusliku elu tagamiseks. Seetõttu on närvisüsteemi arengus rangelt kontrollitud ja reguleeritud nii neuraalsete eellasrakkude ja neuronite teke, kui ka korrektne paigutumine ja võrgustike loomine. Neid protsesse koordineerivad mitmed signaalirajad, millest G-valkude (guaniin-nukleotiidi siduvad valgud) poolt vahendatud signaaliülekanne rakuvälise ja rakusisese keskkonna vahel on üks levinumaid mehhanisme närvisüsteemis. G-valkude aktiveerimisel osalevad nii G-valguga seonduvad retseptorid kui ka mitmed rakusisesed valgud, mis mõjutavad G-valkude aktiivsust retseptorist sõltumatult. Näiteks RIC8A, mis toimib G-valkude nukleotiidivahetusfaktorina ja tšaperonina. Imetajates on RIC8A avaldunud nii arenevas kui täiskasvanud närvisüsteemis osaledes tunnetuslike, õppimis- ning tajufunktsioonide kujunemisel. Käesoleva doktoritöö eesmärgiks oli uurida RIC8A rolli hiire närvisüsteemis ja selle arengus. RIC8A puudumine hiire kesk- ja perifeerse närvisüsteemi rakkudest põhjustab neuro-muskulaarset fenotüüpi, mida iseloomustavad lihaste atrofeerumine, motoorika-, tasakaalu- ja koordinatsiooni häired, tõmblused ning sünnijärgne suremus. Ric8A puudulikel hiirtel esinevad kõrvalekalded suurajukoore arengus (nt rakujagunemiste ja neuraalse migratsiooni häired, ajuvatsakeste laienemine, defektne kortikaalne basaalmembraan) ning südame ja silma arengus. Lisaks katsed RIC8A defitsiitsete rakkudega näitasid, et RIC8A on oluline β1-integriini vahendatud tsütoskeleti organiseerimisel ja rakkude migratsioonil. Kirjeldatud defektid on iseloomulikud kaasasündinud lihasdüstroofiatele (Fukuyama lihasdüstroofia, Walker-Warburgi sündroom ja lihase-silma-aju haigus). Seega, häired RIC8A funktsioonis koostöös G-valkude ja β1-integriinide vahendatud signalisatsiooniga võib olla seotud nende haiguste kujunemiselCorrect functioning of central- and peripheral nervous system is essential for normal functioning of the body. Therefore, it is fundamental to control the spatio-temporal development of neural precursor cell division into neurons and their final positioning where they can form functional network between each other. These processes are coordinated by different signalling pathways, where the G-protein mediated signal transduction is one of the prominent mechanism in cell signalling cascade. The activation of G proteins are accomplished via transmembrane G protein coupled receptors or via accessory proteins that influence G proteins receptor-independently. For example, RIC8A affects G protein activity as a guanine nucleotide exchange factor or as a chaperone. In mammals, RIC8A is expressed in the development of the nervous system and plays a role in the regulation of behavior, memory and learning in adults. Current thesis is focused on the role of RIC8A in the nervous system and its development. Ablation of RIC8A in central- and peripheral nervous system causes defects in neuromuscular signalling which is manifested by muscle atrophy and impairment of movements, occasional tremors and lethality after birth. Ric8A deficient mice also display malformations in the development of the cerebral cortex (e.g defects in cell division and cell migration, which affect cortical size and lamination, enlargement of the ventricles and defects in the integrity of cortical basement membrane) and in heart and eye development. In addition, studies on the RIC8A deficient cells showed that RIC8A is crucial for the β1-integrin mediated organisation of cytoskeleton and cell migration. These defects are characteristic to the congenital muscular dystrophies (Fukuyama congenital muscular dystropy, Walker Warbur g syndrome and Muscle-Eye-Brain disease) and that defects in RIC8A functioning in concert with G-protein and β1-integrin mediated signalling may be the cause of these diseases

A case report and follow-up of the first live birth after heterotopic transplantation of cryopreserved ovarian tissue in Eastern Europe

BackgroundOvarian insufficiency is a major concern for long-term cancer survivors. Although semen freezing is well established to preserve male fertility, the possibilities to secure post-cancer female fertility are mostly limited to oocyte or embryo freezing. These methods require time-consuming ovarian stimulation with or without in vitro fertilization (IVF) that evidently delays cancer therapy. Ovarian tissue cryopreservation and subsequent thawed tissue autotransplantation are considered the most promising alternative strategy for restoring the fertility of oncology patients, which has not yet received the full clinical acceptance. Therefore, all successful cases are needed to prove its reliability and safety.Case presentationHere we report a single case in Estonia, where a 28-year-old woman with malignant breast neoplasm had ovarian cortex cryopreserved before commencing gonadotoxic chemo- and radiotherapy. Two years after cancer therapy, the patient underwent heterotopic ovarian tissue transplantation into the lateral pelvic wall. The folliculogenesis was stimulated in the transplanted tissue by exogenous follicle-stimulating hormone and oocytes were collected under ultrasound guidance for IVF and embryo transfer. The healthy boy was born after full-term gestation in 2014, first in Eastern Europe.ConclusionDespite many countries have reported the first implementation of the ovarian tissue freezing and transplantation protocols, the data is still limited on the effectiveness of heterotopic ovarian transplant techniques. Thus, all case reports of heterotopic ovarian tissue transplantation and long-term follow-ups to describe the children's health are valuable source of clinical experience.Peer reviewe

The role of integrin β1 in the heterogeneity of human embryonic stem cells culture

The maintenance of the pluripotency of human embryonic stem (hES) cells requires special conditions for culturing. These conditions include specific growth factors containing media and extracellular matrix (ECM) or an appropriate substrate for adhesion. Interactions between the cells and ECM are mediated by integrins, which interact with the components of ECM in active conformation. This study focused on the characterisation of the role of integrin β1 in the adhesion, migration and differentiation of hES cells. Blocking integrin β1 abolished the adhesion of hES cells, decreasing their survival and pluripotency. This effect was in part rescued by the inhibition of RhoA signalling with Y-27632. The presence of Y-27632 increased the migration of hES cells and supported their differentiation into embryoid bodies. The differences in integrin β1 recycling in the phosphorylation of the myosin light chain and in the localisation of TSC2 were observed between the hES cells growing as a single-cell culture and in a colony. The hES cells at the centre and borders of the colony were found to have differences in their morphology, migration and signalling network activity. We concluded that the availability of integrin β1 was essential for the contraction, migration and differentiation ability of hES cells

Machine Learning Approaches to Classify Primary and Metastatic Cancers Using Tissue of Origin-Based DNA Methylation Profiles

Metastatic cancers account for up to 90% of cancer-related deaths. The clear differentiation of metastatic cancers from primary cancers is crucial for cancer type identification and developing targeted treatment for each cancer type. DNA methylation patterns are suggested to be an intriguing target for cancer prediction and are also considered to be an important mediator for the transition to metastatic cancer. In the present study, we used 24 cancer types and 9303 methylome samples downloaded from publicly available data repositories, including The Cancer Genome Atlas (TCGA) and the Gene Expression Omnibus (GEO). We constructed machine learning classifiers to discriminate metastatic, primary, and non-cancerous methylome samples. We applied support vector machines (SVM), Naive Bayes (NB), extreme gradient boosting (XGBoost), and random forest (RF) machine learning models to classify the cancer types based on their tissue of origin. RF outperformed the other classifiers, with an average accuracy of 99%. Moreover, we applied local interpretable model-agnostic explanations (LIME) to explain important methylation biomarkers to classify cancer types

Expression Pattern and Localization Dynamics of Guanine Nucleotide Exchange Factor RIC8 during Mouse Oogenesis.

Targeting of G proteins to the cell cortex and their activation is one of the triggers of both asymmetric and symmetric cell division. Resistance to inhibitors of cholinesterase 8 (RIC8), a guanine nucleotide exchange factor, activates a certain subgroup of G protein α-subunits in a receptor independent manner. RIC8 controls the asymmetric cell division in Caenorhabditis elegans and Drosophila melanogaster, and symmetric cell division in cultured mammalian cells, where it regulates the mitotic spindle orientation. Although intensely studied in mitosis, the function of RIC8 in mammalian meiosis has remained unknown. Here we demonstrate that the expression and subcellular localization of RIC8 changes profoundly during mouse oogenesis. Immunofluorescence studies revealed that RIC8 expression is dependent on oocyte growth and cell cycle phase. During oocyte growth, RIC8 is abundantly present in cytoplasm of oocytes at primordial, primary and secondary preantral follicle stages. Later, upon oocyte maturation RIC8 also populates the germinal vesicle, its localization becomes cell cycle dependent, and it associates with chromatin and the meiotic spindle. After fertilization, RIC8 protein converges to the pronuclei and is also detectable at high levels in the nucleolus precursor bodies of both maternal and paternal pronucleus. During first cleavage of zygote RIC8 localizes in the mitotic spindle and cell cortex of forming blastomeres. In addition, we demonstrate that RIC8 co-localizes with its interaction partners Gαi1/2:GDP and LGN in meiotic/mitotic spindle, cell cortex and polar bodies of maturing oocytes and zygotes. Downregulation of Ric8 by siRNA leads to interferred translocation of Gαi1/2 to cortical region of maturing oocytes and reduction of its levels. RIC8 is also expressed at high level in female reproductive organs e.g. oviduct. Therefore we suggest a regulatory function for RIC8 in mammalian gametogenesis and fertility

Dynamic changes in AI-based analysis of endometrial cellular composition: Analysis of PCOS and RIF endometrium

Background: The human endometrium undergoes a monthly cycle of tissue growth and degeneration. During the mid-secretory phase, the endometrium establishes an optimal niche for embryo implantation by regulating cellular composition (e.g., epithelial and stromal cells) and differentiation. Impaired endometrial development observed in conditions such as polycystic ovary syndrome (PCOS) and recurrent implantation failure (RIF) contributes to infertility. Surprisingly, despite the importance of the endometrial lining properly developing prior to pregnancy, precise measures of endometrial cellular composition in these two infertility-associated conditions are entirely lacking. Additionally, current methods for measuring the epithelial and stromal area have limitations, including intra- and inter-observer variability and efficiency. Methods: We utilized a deep-learning artificial intelligence (AI) model, created on a cloud-based platform and developed in our previous study. The AI model underwent training to segment both areas populated by epithelial and stromal endometrial cells. During the training step, a total of 28.36 mm2 areas were annotated, comprising 2.56 mm2 of epithelium and 24.87 mm2 of stroma. Two experienced pathologists validated the performance of the AI model. 73 endometrial samples from healthy control women were included in the sample set to establish cycle phase-dependent dynamics of the endometrial epithelial-to-stroma ratio from the proliferative (PE) to secretory (SE) phases. In addition, 91 samples from PCOS cases, accounting for the presence or absence of ovulation and representing all menstrual cycle phases, and 29 samples from RIF patients on day 5 after progesterone administration in the hormone replacement treatment cycle were also included and analyzed in terms of cellular composition. Results: Our AI model exhibited reliable and reproducible performance in delineating epithelial and stromal compartments, achieving an accuracy of 92.40% and 99.23%, respectively. Moreover, the performance of the AI model was comparable to the pathologists’ assessment, with F1 scores exceeding 82% for the epithelium and >96% for the stroma. Next, we compared the endometrial epithelial-to-stromal ratio during the menstrual cycle in women with PCOS and in relation to endometrial receptivity status in RIF patients. The ovulatory PCOS endometrium exhibited epithelial cell proportions similar to those of control and healthy women’s samples in every cycle phase, from the PE to the late SE, correlating with progesterone levels (control SE, r2 = 0.64, FDR < 0.001; PCOS SE, r2 = 0.52, FDR < 0.001). The mid-SE endometrium showed the highest epithelial percentage compared to both the early and late SE endometrium in both healthy women and PCOS patients. Anovulatory PCOS cases showed epithelial cellular fractions comparable to those of PCOS cases in the PE (Anovulatory, 14.54%; PCOS PE, 15.56%, p = 1.00). We did not observe significant differences in the epithelial-to-stroma ratio in the hormone-induced endometrium in RIF patients with different receptivity statuses. Conclusion: The AI model rapidly and accurately identifies endometrial histology features by calculating areas occupied by epithelial and stromal cells. The AI model demonstrates changes in epithelial cellular proportions according to the menstrual cycle phase and reveals no changes in epithelial cellular proportions based on PCOS and RIF conditions. In conclusion, the AI model can potentially improve endometrial histology assessment by accelerating the analysis of the cellular composition of the tissue and by ensuring maximal objectivity for research and clinical purposes

RIC8 in folliculogenesis and in the reproductive tract of adult mouse.

<p>RIC8 was visualized with RIC8 antibody (red), and cell nuclei were visualized with DAPI (blue). (<b>A-F</b>) Transversal cryosections of ovary with oocytes in different follicular stages starting from primordial follicle to Graafian follicle and (<b>G-I</b>) different regions of oviduct are shown. (<b>H</b>) Higher magnification of the region of ampulla and (<b>i</b>, indicated by white box) isthmus. Abbreviations: A, antrum; Amp, ampulla region of oviduct; Cb, basal layer of cilia; Cc, ciliated cell; Ci, cilia; Co, cumulus oophorus; Cr, corona radiata; Cx, cell cortex, Ec, epithelial cells; Fc, follicular cell; Gc, granulosa cells; Gf, Graafian follicle; GV, germinal vesicle; Ist, isthmus region of oviduct; Lp, lamina propria; Lu, lumen; Pc, peg cell; Pf, primary follicle; Pmf, primordial follicle; Po, primary oocyte; Sf, secondary follicle. Scale bars: 50 μm.</p

Neurospecific deletion of <i>Ric8a</i> in mice.

<p>(<b>A</b>) Schematic representation of neuron-specific deletion of the <i>Ric8a</i> gene. First four exons of floxed <i>Ric8a</i> are removed by Cre-recombinase which expression is under the control of <i>Synapsin I</i> promoter (SynCre). Numbered boxes represent <i>Ric8a</i> exons and arrows <i>loxP</i> sites. (<b>B</b>) PCR-based genotyping of mice using DNA from tail samples to detect <i>SynCre</i> transgene, floxed <i>Ric8a</i> allele and <i>LacZ</i> allele respectively. <i>Ric8a</i>^<i>CKO</i> genotype is emphasized with dotted line. (<b>C</b>) Representative PCR showing the deletion of floxed <i>Ric8a</i> in <i>Ric8a</i>^<i>CKO</i> mouse nervous system and no deletion in non-neural organs. (<b>D</b> and <b>E</b>) Comparison of Cre-recombinase (in <i>SynCre</i>^<i>+/-</i><i>R26R</i>) and <i>Ric8a</i> (in <i>Ric8a</i>^{<i>lacZ/+</i>}) expression in E12.5 embryos by X-gal staining. (<b>F</b>) Down regulation of <i>Ric8a</i> mRNA expression in <i>Ric8a</i>^<i>CKO</i> mice relative to littermate control in hippocampus (HIP), spinal cord (SC), cardiac muscle (CM) and liver (LIV). (<b>G</b>) Deficiency of RIC8A protein in <i>Ric8a</i>^<i>CKO</i> (CKO) mice compared to littermate control (LM) in hippocampus (HIP), spinal cord (SC), spinal ganglia (SG) and cardiac muscle (CM). GAPDH was used as a reference. Abbreviations: del, PCR fragment from the deleted allele Drg, dorsal root ganglia; F, floxed allele; Hb, hindbrain; Mb, midbrain; Nt, neural tube; SC, spinal cord; SG, spinal ganglia; Syt, sympathetic trunk; Vno, vomeronasal organ; wt, PCR fragment from the wild-type allele; V, trigeminal ganglion; X, vagus ganglion; ** <i>P</i> < 0,01. Error bars represent mean ± SEM scores. Scale bars: (D and E) 1 mm.</p