Diplomová práca je zameraná na mikrovlákna na báze biopolyméru poly(3-hydroxybutyrátu) a ich využitie v medicínskych aplikáciách. Teoretická časť práce sa zaoberá štúdiom procesu tvorby vláken pomocou technológie odstredivého zvlákňovania, jeho kinetikou a faktormi ovplyvňujúcimi vznik a vlastnosti vláken. Teoretická časť sa následne orientuje na krátky prehľad biopolymérov používaných v tejto technológii, charakteristiku materiálu poly(3-hydroxybutyrátu) a taktiež prezentuje návrh potenciálnej cieľovej aplikácie daných mikrovláken. Praktická časť sa koncentruje sa prípravu mikrovláken zo spomínaného poly(3-hydroxybutyrátu). Sledované a optimalizované sú viaceré parametre vedúce k lepšej zvlákniteľnosti materiálu. Praktická časť je rozšírená o modifikáciu polymérneho roztoku prídavkom iných biopolymérov a zmäkčovadiel a prípravu mikrovláken z takto modifikovanej polymérnej zmesi. Pozornosť je venovaná taktiež optimalizácii procesných parametrov. Pripravené mikrovlákna sú následne analyzované a charakterizované viacerými metódami a vzájomne porovnávané s cieľom vyvinúť alternatívu k súčasne používaným substrátom pre rast buniek v 3D.

Benign prostatic hyperplasia (BPH) and chronic prostatitis (CP) are common diseases in aging men. Though medications are available to alleviate these conditions, problems of possible side-effects of first-line synthetic drugs for prostatic conditions have allowed patients to switch to a safer plant-based medication. CernitinTM, a pollen extract, is used to alleviate these conditions. A recent in vitro study showed that CernitinTM inhibits cell proliferation and induce a regulatory effect on inflammatory parameters. To validate those results, the inter-batch variability of CernitinTM was assessed using the active ingredients CernitinTM T60 and CernitinTM GBX on the human prostatic cell lines BPH‐1 and WPMY‐ 1 and on human peripheral blood mononuclear cells (hPBMCs) in vitro. Cell proliferation assay was performed in prostatic cell lines, while inflammatory parameters were analyzed in hPBMCs. Results revealed that both CernitinTM active ingredients, regardless of batch production, significantly inhibited the proliferation of both prostatic cell lines after 48 and 72 hours, respectively (p < 0.05 to p < 0.001). Among the batches, there were no significant differences observed. Notably, the GBX batches 14164, 14548 and 14160 had a more pronounced effect on cell proliferation right after 48 hours on both cell lines. Whilst, T60 batches 11539 and 14144 had a pronounced effect right after 48 hours on BPH cells. In hPBMC, the production of the anti-inflammatory cytokine interleukin (IL)- 10 and its receptor IL-10 receptor subunit beta (RB), as well as pro-inflammatory cytokine IL-6 was significantly increased after treatment with the T60 formulation regardless of the batch, but not after treatment with the GBX batch. Moreover, IL-10 receptor subunit alpha (RA) and tumour necrosis factor‐related apoptosis‐inducing ligand (TRAIL) expression increased after the use of both formulations (p < 0.05 to p < 0.001). The pro-inflammatory cytokine IL-8 and chemokine CXCL-10 was significantly decreased using both batches of T60 (p < 0.05 to p < 0.001). Collectively, these results support the claim of the role of CernitinTM as an anti-proliferative agent and as a cytokine regulator.

Filip Lhota: Study of alternatively spliced variants of estrogen receptor alpha in breast cancer cell lines Abstract: Estrogen receptor α (ER-α) is a transcription factor responsible for mediation of the activities of its natural ligand 17-β-estradiol (E2), the hormone that together with progesterone belongs to the key regulators of mammary epithelial as well as breast cancer cells proliferation. Except to the major gene product consisting of all eight coding exons of ER-α, numerous qualitatively and quantitatively different spliced variants originated from primary transcript by activity of alternative splicing is expressed. Despite that some of these spliced variants have been functionally characterized, their precise role on final ER-α cellular activity remains to be elucidated. The functional characterization of individual alternative forms of ER-α and description of its participation on the overall ER-α activity is important for our understanding of their biogenesis and is also critical for the delineation of molecular bases for ER-α regulation during anti cancer chemotherapy. This work aimed to study the influence of alternatively spliced ER-α variants on the growth characteristics of clones constructed from stable mammary tissue cell lines in regulation to cultivation conditions and cellular...

Intracellular protein phosphorylation is a critical step in cellular activation stimulated by the binding of various ligands to cell surface receptors. This process is initiated by activation of specific protein-tyrosine kinases associated with intracellular domains of the respective ligand receptor. JAK-STATs pathway is one of the main pathways in the cell activation process and given their important role in various PIDs, STATs proteins have been extensively studied in immune function in health and disease. Therefore, our work has focused on investigating and evaluating STATs activation and establishing flow cytometric methods to assess their phosphorylation to be a surrogate marker as a fast and sensitive diagnostic tool to current methods such as WB. At the first, we studied STAT1 and STAT3 activation and established a flow cytometric procedure to analyze variations of INF-α- and IL-6-induced STAT1 and STAT3 phosphorylation in T cells from whole blood, respectively (publication ΙΙ). To examine whether our results were specific, the samples were also analyzed by WB in parallel. After that, we validated the normal values of pSTAT1 and pSTAT3 based on 21 healthy adult controls according to an appropriate validation process. We showed that, in contrast to the conventional methods like WB, our assay offers a diagnostic benefit by avoiding labor and time consumption, with the advantage of achieving an earlier diagnosis, which potentially leads to improve treatment decisions; hence, patient’s outcome (publication ΙΙ). Furthermore, we verified FCM-based pSTAT1 and pSTAT3 profiling established here in patients group suffering from CMC and HIES. Our results demonstrated that pSTAT1 and pSTAT3 assay is an effective tool to identify and characterize well-known PIDs such as CMC and HIES, respectively (publication ΙΙ). Next, we introduced a fast and straightforward flow cytometric assay for the assessment of T cell proliferation, based on the staining of phosphorylated STAT5A (publication ΙΙΙ). We showed that pSTAT5A represents an appropriate approach to predict the behavior of T cells upon activation by CD3/CD28 and PHA. FCM-based pSTAT5A profiling is an intracellular flow cytometric method, enabling the early and reliable detection of T cell proliferation without long time incubation (within 24 h instead to 5 days). Importantly, measurement of pSTAT5A represents a new principle to assess T cell proliferation. It reveals important information on T cell biology by using series of kinetics and different kinds of T cell stimulation. For instance: [1] after stimulation via CD3/CD28 and negative pSTAT5A and T cell proliferation, the immune defect could be occurred in the whole signaling cascade (TCR-IL-2 transcription-JAK3-STAT5), [2] After stimulation via external IL-2 and negative pSTAT5A, the immune defect could be localized in the signaling cascade (IL-2R – JAK3 – STAT5), [3] After stimulation via external IL-2 and positive pSTAT5A, the immune defect could be localized in the signaling cascade (TCR - IL-2 transcription) (publication ΙΙΙ). We showed a strong correlation between the STAT5A phosphorylation and the percentage of dividing cells (publication ΙΙΙ). Later on, we used the measurement established here to investigate whether the phosphorylation of STAT5A is an appropriate candidate for predicting CMV specific T cell proliferation. It is well-known that CMV specific T cells expand with CMV reactivation and are probably prerequisite for control and protection. We demonstrated that CMV specific pSTAT5A detection represents a fast and straightforward diagnostic tool to assess CMV specific T cell proliferation without requisite several days’ culture (publication ΙV). Furthermore, we showed a positive correlation between the percentage of pSTAT5A+ T cells vs. (1) CMV-IgG concentrations vs. (2) the percentage of expanded T cells and vs. (3) the percentage of initial CMV specific T cells (publication ΙV). Finally, we evaluated the diagnostic value of pSTAT5 assay and determined the percentage of pSTAT5A+ T cells cut-off value at which pSTAT5 assay has the greatest diagnostic potency. Our data showed that a cut-off value of 9.1 % could be used to assess CMV specific T cell proliferation with a specificity and sensitivity of 100% and 73%, respectively. We verified measurement established here by CMV specific T cells stimulation in three selected patients diagnosed with CARMIL2-mutation and suffering from chronic CMV infection. Our results showed that the complete and the partial deficiency of CMV and CD3/CD28 stimulated pSTAT5A correlated with the complete and the partial deficiency of CMV and CD3/CD28 stimulated T cell proliferation, respectively (publication ΙV). In conclusion, disorders in JAKs-STATs signal pathways in T cells may result in insufficient response to stimulants. Therefore, FCM-based pSTATs profiling is an effective tool for clinical laboratory diagnostics [1] to understand the susceptibility to recurrent opportunistic infections [2] to rapidly identify T cell proliferation [3] to investigate tumor-specific responses of CD8 T effector and memory cells (56) and finally [4] to identify and distinguish well-known PIDs like CARMIL-2 mutations, CMC, AD-HIES or AR-HIES.

No presenta resumen. / Revisión por pares

The NAD-dependent deacetylase SIRT1 regulates several factors involved in stress response and cell survival but its function in cancer is largely unknown. Research suggests that SIRT1 influences several transcription factors and molecules that are important components of pathways often deregulated in cancer. Our experiments have shown that SIRT1 knock down by short hairpin RNA accelerates tumor xenograft formation by HCT116 colon cancer cells, whereas SIRT1 overexpression inhibits tumor formation. We have also found that, pharmacological inhibition of SIRT1 stimulates cell proliferation under conditions of growth factor deprivation suggesting a tumor suppressive function of SIRT1. Paradoxically, SIRT1 inhibition sensitizes the same cells to apoptosis by chemotherapeutic drugs. Immunohistochemical staining of a colon tumor microarray revealed high SIRT1 expression levels in normal colon mucosa and benign adenomas. SIRT1 overexpression was observed in nearly 25% of stage I/II/III colorectal adenocarcinomas but rarely found in advanced stage IV tumors. Furthermore, about 30% of carcinomas showed lower than normal SIRT1 expression. These results suggest a pleiotropic effect of SIRT1 in cancer, i.e., anti-proliferative as well as anti-apoptotic. Further experiments along these lines and examination of a larger patient cohort could provide a rationale for the use of SIRT1 activators and inhibitors in the prevention and treatment of cancer.

L’hématopoïèse est initiée à partir de cellules souches hématopoïétiques et aboutit à la production continue et contrôlée des cellules sanguines matures. Runt-related transcription factor 1 (RUNX1) code pour un facteur de transcription qui joue un rôle clé dans l'hématopoïèse. Des dérégulations de RUNX1 sont fréquemment associées à des cancers hématologiques, en particulier dans les leucémies aiguës lymphoblastiques à précurseurs B chez l'enfant (LAL-B). De plus, son activité transcriptionnelle est contrôlée par le recrutement de cofacteurs. Pour comprendre le mécanisme impliqué dans le contrôle de l’activité transcriptionnelle de RUNX1 nous avons réalisé des immunoprécipitations de RUNX1 couplées à de la spectrométrie de masse et identifié Far Upstream Element binding protein 1 (FUBP1) comme un partenaire protéique potentiel de RUNX1. FUBP1 est un régulateur multifonctionnel impliqué dans divers processus cellulaires. Il a notamment été décrit récemment comme essentiel à l’expansion et à l’auto-renouvellement des cellules souches hématopoïétiques. Par ailleurs, une surexpression du gène et des mutations potentiellement oncogéniques ont été décrits dans des cas de leucémies lymphoblastiques. Nous avons montré, grâce à la technique de PLA (Proximity Ligation Assay) que nous avons optimisé sur cellules non-adhérentes, que FUBP1 est un nouveau cofacteur de RUNX1 et qu’ils peuvent faire partie d’un même complexe régulateur dans les lymphoblastes pré-B humains. Par des expériences de ChIP couplées à du séquençage, nous avons localisé les régions de la chromatine sur lesquelles étaient fixées RUNX1 et FUBP1 de façon commune. Nous avons identifié l’oncogène c-KIT comme un gène cible commun et nous avons caractérisé deux régions régulatrices fixées par RUNX1, FUBP1 et des marques d’activation de la chromatine, au niveau du premier intron de c-KIT : une à +700 pb et l’autre au niveau d’un enhancer à +30 kb. De plus, nous avons déterminé les motifs de liaison de RUNX1 et FUBP1 essentiels pour l'activation de l’enhancer. Enfin nous avons démontré que la surexpression de FUBP1 et RUNX1 conduit à une augmentation de l’expression de c-KIT en ARNm et en protéine, augmente une des voies activées par c-KIT en présence de son ligand, favorise la prolifération cellulaire in vitro et in vivo et rend les cellules moins sensibles à un inhibiteur de c-KIT. En conclusion, nous avons démontré que FUBP1 est un nouveau partenaire de RUNX1 et qu’ensembles, ils activent la transcription de l’oncogène c-KIT en se fixant sur un enhancer commun, favorisant ainsi la prolifération cellulaire. Par conséquent, puisque FUBP1 et RUNX1 sont surexprimés dans certains types de leucémie, des altérations de cette régulation pourraient participer à l'apparition ou au maintien de leucémies. Nos résultats ouvrent donc de nouvelles perspectives sur la compréhension du contrôle de l’activité transcriptionnelle de RUNX1 et sur les hémopathies malignes associées à des dérégulations de RUNX1, FUBP1 ou c-KIT. / Hematopoiesis is initiated from hematopoietic stem cells and results in the continuous and controlled production of mature blood cells. RUNX1 (Runt-related transcription factor 1) encodes a transcription factor playing a key role in hematopoiesis. Abnormal functions of this protein are implicated in blood cancer, notably in pediatric B-cell precursor acute lymphoblastic leukemia (BCP-ALL). Moreover, its transcriptional activity is controlled by the recruitment of cofactors. To unravel the mechanisms behind the regulation of RUNX1 transcriptional activity, we performed RUNX1 specific immunoprecipitation experiments followed by mass spectrometry and identified the Far Upstream Element Binding Protein 1 (FUBP1) as a potential cofactor of RUNX1. FUBP1 is a multifunctional regulator involved in diverse cellular processes. FUBP1 has recently been described to be essential for expansion and self-renewal of hematopoietic stem cells and to function as a potential cancer driver gene in lymphoblastic leukemia. We have shown, with a proximity ligation assay that we have optimized in non-adherent cells, that FUBP1 is a new cofactor of RUNX1 and that these two proteins can be part of the same regulatory complex in human pre-B lymphoblasts. By chromatin immunoprecipitation combined with sequencing, we have localized common chromatin regions bound by RUNX1 and FUBP1. We have identified the oncogene c-KIT as a common target gene, and characterized two regulatory regions bound by both RUNX1, FUBP1 and active histone marks, within the first c-KIT intron: one at +700 bp and the other on an enhancer at +30 kb. Moreover, we have determined RUNX1 and FUBP1 binding sites essential for the enhancer activation. Finally, we have demonstrated that RUNX1 and FUBP1 overexpression in a pre-B cell line increases the expression of c-KIT both at mRNA and protein levels, exacerbates one of the c-KIT downstream pathways, promotes cell proliferation in vitro and in vivo and renders cells more resistant to a c-KIT inhibitor. In conclusion, we have demonstrated that FUBP1 is a new cofactor of RUNX1 and that they activate the transcription of the c-KIT oncogene by binding on a common enhancer, thus promoting cell proliferation. Therefore, since FUBP1 and RUNX1 are overexpressed in some types of leukemia, alterations in this regulation may contribute to the onset or maintenance of leukemias. These new findings open new perspectives on understanding the control of RUNX1 transcriptional activity, and on leukemias related to RUNX1, FUBP1 or c-KIT deregulations.

Understanding cancer biomarkers for diagnosis and prognosis leads to improved patient treatments and care. This thesis addresses the relevance of thymidine kinase 1 (TK1) as a cancer biomarker and the role of TK1 in cancer progression. Worldwide, cancer leads to more than 12 million deaths annually. In the United States alone, each year over 1.5 million cases will be diagnosed and over half a million persons will die. The most prevalent cancer types include skin, lung, breast, prostate, and colon. TK1 is known to be present in the serum of patients with multiple cancer types, including lung, breast, colon and prostate. In fact, it is shown to be detectable in cancer patients even before they manifest clinical symptoms. Additionally, the levels of TK1 increase progressively with increasing tumor grade; meaning that levels of TK1 can indicate tumor grade. Cellular proliferation markers such as p53 and Ki-67 have been compared to TK1 in cancer diagnosis and prognosis. TK1 has potential as both a prognostic and diagnostic biomarker in various cancer types including breast. Breast cancer is one of the most aggressive cancer types with 20-30% of diagnosed tumors becoming metastatic. Recent findings have identified additional involvement of TK1 downstream of cellular proliferation in cancer progression, including cellular invasion which is a part of cancer metastasis. These findings while efficacious, fail to identify the individual contribution of TK1 in downstream processes that aid in cancer progression. As mentioned previously, TK1 is upregulated in several different cancer types. We propose that there is an advantage to upregulated levels of TK1 in cancer progression and seek to explore its role specifically in cell invasion and survival. Based on our current understanding of TK1, we first wanted to review the history of TK1 and show the importance of understanding this crucial enzyme. Finally, we report our results from experiments exploring the influence of TK1 in vitro on breast cancer cell invasion and survival.

The cyclin-dependent kinase (Cdk) inhibitor p27Kip1 contributes to the timing of cell cycle withdrawal during development and, consequently, in organogenesis. Within the retina, this effector protein is up-regulated during the birth of neuronal and glial cells [Dev. Biol. (2000) 299]. However, its expression within the retinal pigment epithelium (RPE), a supporting cell layer that is essential for neural retina development and function, has not previously been reported. We show that p27Kip1 protein expression in the RPE occurs in two phases: an up-regulation during mid-to late embryonic stages and a down-regulation during the subsequent postnatal period. In the early phase of up-regulation, an inverse relationship is seen between expression of p27Kip1 and PCNA, an indicator of cycling cells. During both up-and down-regulation, the change in spatial pattern of expression proceeds in a central to peripheral manner, with p27Kip1 up-regulation paralleling retinal maturation. These data suggest that this cell cycle regulator may be an important factor controlling the timing of RPE cell cycle withdrawal.

Interleukin-7 is an essential cytokine that plays a major role in the development and homeostatic maintenance of T-cells. The presence of soluble forms of various cytokine receptors have been proposed to be involved in the endogenous regulation of cytokine activity. Due to the natural ability of soluble CD127 (sCD127) to bind to IL-7, there is an interest in its potential application as an immunotherapeutic agent in diseases, where IL-7 has been found to be relevant, including HIV infection. In this study, I hypothesize that by administering sCD127 to healthy mice, IL-7 activity should be enhanced, thus enhancing T cell proliferation in vivo. The work presented here focuses on three main objectives: 1) evaluating the effect of IL-7 with or without sCD127 on T cell proliferation in healthy mice; 2) validating a mouse model of T cell depletion using anti-CD4 and CD8 antibodies; and 3) determining the effect of sCD127 treatment with or without IL-7 on T cell reconstitution and proliferation in the T cell depletion model. To assess the effect of administering exogenous sCD127, IL-7 or the combination on T cell proliferation, peripheral blood mononuclear cells and spleen were isolated, and stained to characterize T cell number, proliferation, and surface CD127 expression by flow cytometry. For the T cell depletion model, wild type C57BL/6 mice were injected intra-peritoneally with 150 μg single dose of anti-CD4 and anti-CD8 depleting antibodies. Consequently, mice were bled weekly to demonstrate the kinetics of T cell reconstitution following depletion (from d7 to d63). Our results demonstrated that in healthy mice daily treatment with murine IL-7 significantly stimulated T cell proliferation and consequently increased cell number. This observation was further boosted by pre-complexing IL-7 with sCD127. For T cell depletion experiments, the kinetics of T-cell reconstitution was different between the CD4+ and CD8+ T cells. CD4+ T cell reconstitution was almost complete 6 weeks following T cell depletion, while CD8+ T cells were only partially reconstituted at this time point. Treatment with IL-7 or combined therapy had a transient and significant effect on T cell proliferation and reconstitution, and this influence was abrogated after treatment discontinuation. Interestingly, CD8+ T cells exert greater responses to our treatments in that a more pronounced proliferation and significant increase in cell number was observed relative to the effect seen on CD4+ T cells in both healthy and depleted mice. In conclusion, antibody-mediated T cell depletion is a potentially valuable tool to investigate lymphopenia-induced proliferation and potential therapies thereof. This study suggests that combining sCD127 and IL-7 therapies enhances IL-7-mediated T cell proliferation, and provides important information for the potential therapeutic use of sCD127 and its impact on IL-7 function.