STAT5 drives abnormal proliferation in autosomal dominant polycystic kidney disease.

Autosomal dominant polycystic kidney disease (ADPKD) leads to renal failure. The hallmark of ADPKD is increased epithelial proliferation, which has been proposed to be due to atypical signaling including abnormal JAK-STAT activity. However, the relative contribution of JAK-STAT family members in promoting proliferation in ADPKD is unknown. Here, we present siRNA JAK-STAT-focused screens discovering a previously unknown proliferative role for multiple JAK-STAT components (including STAT1, STAT2, STAT4, STAT5a, and STAT5b). Amongst these, we selected to study the growth hormone/growth hormone receptor/STAT5-axis because of its known role as a regulator of growth in nonrenal tissues. Loss of STAT5 function, facilitated by pharmacological inhibition or siRNAs, significantly reduced proliferation with an associated reduction in cyst growth in vitro. To study whether STAT5 is abnormally activated in vivo, we analyzed its expression using two independent mouse models of ADPKD. STAT5 was nuclear, thus activated, in renal epithelial cyst lining cells in both models. To test whether forced activation of STAT5 can modulate proliferation of renal cells in vivo, irrespective of the Pkd1 status, we overexpressed growth hormone. These mice showed increased STAT5 activity in renal epithelial cells, which correlated with de novo expression of cyclin D1, a STAT5 target gene. Chromatin immunoprecipitation experiments revealed that STAT5 transcriptionally activated cyclin D1 in a growth hormone-dependent fashion, thus providing a mechanism into how STAT5 enhances proliferation. Finally, we provide evidence of elevated serum growth hormone in Pkd1 mutant mice. Thus, the growth hormone/STAT5 signaling axis is a novel therapeutic target in ADPKD

Transcriptional regulation of the COL1A2 gene in fibroblasts.

Renal tubulointerstitial fibrosis is a major predictor of progressive glomerular disease. It occurs as a result of persistent inflammation and is characterised by excessive deposition of extracellular matrix (ECM) proteins, including accumulation of type I collagen, the most abundant protein of the ECM. Type I collagen is encoded by two genes, COL1A1 and COL1A2, that are tightly regulated at a transcriptional level. A key aim of this study was to use the previously identified COL1A2 promoter and enhancer sequences in order to identify novel regulatory cis-acting elements and the relevant transcription factors that regulate collagen transcription in cells derived from healthy or diseased kidneys. Moreover, the effects of hypoxia and transforming growth factor beta (TGFβ), which are both profibrotic stimuli, on collagen transcription were studied. TGFβ is known to activate CDP/cux transcription factors which can suppress gene activation; based on this finding the role of CDP/cux in COL1A2 transcriptional regulation was assessed. In conclusion,the work presented in this thesis provides an insight into the complex control mechanisms that regulate collagen transcription in both physiological and pathological conditions

Design and pharmacological investigation newly synthesized oxytocin analogues with antagonistic activity: structure - biological activity relationships

In the present study we present the synthesis and pharmacological investigation of twenty seven newly synthesized oxytocin analogues. Basic modification at positions 7, 8 and 9 (introduction of α,α-dialkyl amino acid, α-aminoisobutyric acid [Aib] or the unnatural imino acid D-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid [D-Tic] or tert-butyl-glycine [(But)Gly]) was combined with D-Cys6/Pen6, D-Tyr(Et)2, and D-Nal(1)2, Mpa1/Pen1 modifications and their various combinations.The new analogues (following table) were synthesized by Fmoc solid phase methodology utilizing a 2-chlorotrityl chloride resin as solid support bearing a Rink-Bernatowitz linker to provide the peptidic amide and diisopropylcarbodiimide/1-hydroxy-benzotriazol (DIC/HOBt) as coupling agent. The cyclization was performed in DMSO/H2O (2:8, v/v) for 24-36h Final purification was achieved by preparative HPLC (Pharmacia LKB-2250) on reversed-phase support C-18 with a linear gradient from 25 to 75% acetonitrile (0.1% TFA) for 35 min. The appropriate fractions were pooled and lyophilized. Electron-spray MS were in agreement with the expected results.The analogues were tested for their potency in two pharmacological tests, i.e. uterotonic in vitro test in the absence of magnesium ions on an isolated strip of rat uterus and in the pressοr test on phenoxybenzamine treated male rats. Parallel determination of binding affinity of the analogues to cloned human oxytocin recep-tors on HEK cell membranes using tritiated oxytocin from NEN Life Science, Boston, MA, USA was performed. We obtained a satisfactory number of quite potent and selective (as it concerns the binding with the vasopresor receptor V1a) oxytocin antagonists. Ten of these analogues showed higher antagonistic affinity than Atosiban (pA2=8.29) and they were completely inactive as far as it concerns the rat pressor test. Three of them (MOPACIN I, II and III) are under patent procedure. On the other hand, as it concerns the binding affinity and the biological evaluation, we observed a paradox phenomenon. Despite the high anta-gonistic potencies the affinity to the receptor was lower than oxytocin. This may be attri-buted to the species difference.Furthermore, some of the analogues were tested as far as it concerns their ability to in-crease or decrease the growth of MCF-7 breast cancer cells. The results indicate the ability of some analogues to decrease the growth of the MCF-7 breast cancer cells, but these re-sults need further investigation due to extract safe conclusions.Finally, the results from the presence study may aid in the design of novel and selective antagonists of oxytocin with pharmacological/pharmaceutical use. Furthermore, the evi-dence that some analogues have the ability to decrease the growth of MCF-7 cells, may find future applications in the radioimaging of different neoplasms, as wel as in their therapy, using oxytocin analogues that conserve high oxytocin receptor affinity as either radiotracers or chemiotherapeutic vectors.Στην παρούσα διατριβή παρουσιάζεται η σύνθεση εικοσιεπτά νέων αναλόγων της ωκυτοκίνης (ΟΤ), τα οποία περιέχουν πέραν της Gly(But)8 ή/και Gly(But)9 και Gly(But)3 ή/και Gly(But)7, D-Cys6;ή Pen6, το μη φυσικό αμινοξύ α-αμινοϊσοβουτυρικό οξύ (Aib), το ιμινοξύ 1,2,3,4- τετραϋδροκινολινο-3-καρβοξυλικό οξύ (Tic) στη D- μορφή του στις θέσεις 7 ή/και 9 και τέλος τα παράγωγα D-Tyr(Et) και D-Nal(1) στη θέση 2. Τα νέα αυτά ανάλογα παρουσιάζονται στον πίνακα που ακολουθεί.H σύνθεση των νέων αναλόγων έγινε σύμφωνα με την Fmoc/But μεθοδολογία σύνθεσης επί στερεάς φάσεως σε στερεό υπόστρωμα Rink Bernatowitz 2-χλωροτριτυλο-ρητίνη. H απομάκρυνση της Fmoc-ομάδας επιτυγχάνεται με επίδραση διαλύματος 20% πιπεριδίνης σε DMF και οι συζεύξεις πραγματοποιήθηκαν με τη μέθοδο του διϊσοπροπυλοκαρβοδιϊμιδίου/1-υδροξυ-βενζοτριαζολίου (DIC/HOBt) για 2 ώρες.Η απομάκρυνση του πεπτιδίου από το στερεό υπόστρωμα επετεύχθη με κατεργασία της ρητίνης με διάλυμα τριφθοροξικού οξέος-διχλωρομεθανίου (80:15,v/v) παρουσία δεσμευτών κατιόντων για 4 ώρες. Η δημιουργία του δισουλφιδικού δεσμού πραγματοποιήθηκε μετά από παραμονή των απροστάτευτων πεπτιδίων σε διάλυμα DMSO/H2O (20:80, v/v) για 36-48h. Η απομόνωση σε καθαρή κατάσταση έγινε με υγρή χρωματογραφία υψηλής απόδοσης ανάστροφης φάσης (RP-HPLC) σε σύστημα διαλυτών CH3CN/H2O μεταβαλλόμενης σύστασης (από 25% CH3CN έως 75% CH3CN για 30 min σε 0,05% CF3COOH/H2O). Τα παρασκευασθέντα πεπτίδια ταυτοποιήθηκαν με ES-MS. Τα πεπτίδια δοκιμάστηκαν όσον αφορά στην ωκυτόκειο δράση in vitro, σε απομονωμένο ιστό μήτρας επίμυος ενώ η δοκιμή επί της πιέσεως πραγματοποιήθηκε σε επίμυες οι οποίοι είχαν επεξεργαστεί με φαινοξυβενζαμίνη. Από τα προκαταρκτικά βιολογικά αποτελέσματα φαίνεται ότι τα συντεθέντα ανάλογα παρουσιάζουν στο σύνολό τους ανταγωνιστικές ιδιότητες, 10 εκ των οποίων παρουσίασαν ιδιαίτερα ισχυρή ανταγωνιστική δράση και εκλεκτικότητα εφ’ όσον στη δοκιμή επί της πιέσεως δεν εμφάνισαν δράση. Τα 10 αυτά ανάλογα εμφανίζουν ανταγωνιστική ισχυρότερη από αυτήν του Atosiban (pA2=8,29±0,05) και είναι εκλεκτικά. Επιπλέον, τρία από τα νέα ανάλογα (MOPACIN I,II και III) βρίσκονται σε διαδικασία κατοχύρωσης.Τα νέα ανάλογα δοκιμάστηκαν και όσον αφορά στη συγγένεια τους με τον ανθρώπινο ωκυτόκειο υποδοχέα, η δοκιμή πραγματοποιήθηκε σε ανθρώπινα εμβρυϊκά νεφρικά κύτταρα (HEK) τα οποία υπερεκφράζουν τον υποδοχέα. Υπήρξε μια διαφοροποίηση μεταξύ των αποτελεσμάτων από τη βιολογική δράση και την συγγένεια με τον υποδοχέα, αφού ανάλογα με ισχυρή ανταγωνιστική δράση (π.χ. ανάλογο 23 με τιμή pA2 =8,31 και συγγένεια με τον υποδοχέα περισσότερο από 100 φορές χαμηλότερη σε σύγκριση με αυτή της φυσικής ορμόνης) εμφάνισαν χαμηλή συγγένεια με τον υποδοχέα. Η διαφοροποίση αυτή ίσως οφείλεται στις διαφορές μεταξύ των ειδών.Επιπρόσθετα, κάποια από τα ανάλογα αυτά δοκιμάστηκαν όσον αφορά στην επίδρασή τους στον πολλαπλασιασμό των καρκινικών κυττάρων του μαστού (MCF-7). Τα αποτελέσματα αυτών αποτελούν ένδειξη της βιολογικής δράσης των συνθετικών πεπτιδίων. Όμως χρειάζεται περισσότερη διερεύνηση ο ρόλος τους για την εξαγωγή ασφαλών συμπερασμάτων.Εν κατακλείδι, τα συμπεράσματα στα οποία καταλήγουμε, όσον αφορά στην παρούσα διατριβή προσφέρουν νέα δεδομένα όσον αφορά στη σύνθεση και τον σχεδιασμό νέων αναλόγων της ωκυτοκίνης τα οποία πιθανότατα θα αποτελέσουν και καλούς ανταγωνιστές με ενδεχόμενη θεραπευτική/φαρμακευτική εφαρμογή. Επιπλέον, το εύρημα ότι η ωκυτοκίνη καθώς και τα ανάλογα αυτής μπορεί να εμπλέκονται στον πολλαπλασιασμό των νεοπλασματικών κυττάρων μπορεί να βρει εφαρμογή στην ραδιοαπεικόνιση διαφόρων νεοπλασμάτων, καθώς και στη θεραπεία αυτών. Επίσης ανάλογα της ωκυτοκίνης τα οποία παρουσιάζουν υψηλή συγγένεια με τον υποδοχέα μπορούν να χρησιμοποιηθούν ως ραδιοϊχνηθέτες ή ως χημειοθεραπευτικοί φορείς

Transcriptional regulation of the COL1A2 gene in fibroblasts

Renal tubulointerstitial fibrosis is a major predictor of progressive glomerular disease. It occurs as a result of persistent inflammation and is characterised by excessive deposition of extracellular matrix (ECM) proteins, including accumulation of type I collagen, the most abundant protein of the ECM. Type I collagen is encoded by two genes, COL1A1 and COL1A2, that are tightly regulated at a transcriptional level. A key aim of this study was to use the previously identified COL1A2 promoter and enhancer sequences in order to identify novel regulatory cis-acting elements and the relevant transcription factors that regulate collagen transcription in cells derived from healthy or diseased kidneys. Moreover, the effects of hypoxia and transforming growth factor beta (TGFβ), which are both profibrotic stimuli, on collagen transcription were studied. TGFβ is known to activate CDP/cux transcription factors which can suppress gene activation; based on this finding the role of CDP/cux in COL1A2 transcriptional regulation was assessed. In conclusion,the work presented in this thesis provides an insight into the complex control mechanisms that regulate collagen transcription in both physiological and pathological conditions.EThOS - Electronic Theses Online ServiceKidney Research UKGBUnited Kingdo

Some criteria for selecting best subsets of regressor variables

Some criteria for selecting best subsets of regressor variable

Evaluating the Molecular Properties and Function of ANKHD1, and Its Role in Cancer

Ankyrin repeat and single KH domain-containing protein 1 (ANKHD1) is a large, scaffolding protein composed of two stretches of ankyrin repeat domains that mediate protein–protein interactions and a KH domain that mediates RNA or single-stranded DNA binding. ANKHD1 interacts with proteins in several crucial signalling pathways, including receptor tyrosine kinase, JAK/STAT, mechanosensitive Hippo (YAP/TAZ), and p21. Studies into the role of ANKHD1 in cancer cell lines demonstrate a crucial role in driving uncontrolled cellular proliferation and growth, enhanced tumorigenicity, cell cycle progression through the S phase, and increased epithelial-to-mesenchymal transition. Furthermore, at a clinical level, the increased expression of ANKHD1 has been associated with greater tumour infiltration, increased metastasis, and larger tumours. Elevated ANKHD1 resulted in poorer prognosis, more aggressive growth, and a decrease in patient survival in numerous cancer types. This review aims to gather the current knowledge about ANKHD1 and explore its molecular properties and functions, focusing on the protein’s role in cancer at both a cellular and clinical level

Sleep Disturbances and Interleukin 6 Receptor Inhibition in Rheumatoid Arthritis

Objective. Interleukin 6 (IL-6)-mediated interactions have been associated with sleep disturbances in healthy subjects. In this pilot study we examined whether administration of the IL-6 receptor antagonist tocilizumab in patients with rheumatoid arthritis (RA) affects sleep disturbances. Methods. Fifteen patients (13 women) with sleep disturbances at baseline received 6 monthly infusions of tocilizumab 8 mg/kg for moderately or severely active RA. Sleep quality was assessed by Pittsburgh Sleep Quality Index (PSQI), daytime sleepiness by Epworth Sleepiness Scale, disease activity by the 28-joint Disease Activity Score-erythrocyte sedimentation rate, functional disability by Health Assessment Questionnaire Disability Index (HAQ-DI), and fatigue by the Functional Assessment of Chronic Illness Therapy (FACIT-Fatigue Scale; FFS) at baseline and first, second, third, and sixth month of treatment. Medications used before enrollment remained unchanged during followup. Results. Sleep quality improved and daytime sleepiness decreased significantly at first-month assessment (p < 0.00001 and p < 0.004, respectively, by repeated measurement analysis) compared to baseline, and these changes became more evident through 6 months. Disease activity decreased, fatigue decreased, and functional status improved significantly. Changes in PSQI score over time were not associated with the corresponding changes in DAS28-ESR (r = 0.37, p = 0.17), but correlated significantly with HAQ-DI changes (r = 0.60, p = 0.02) and marginally with changes in FFS scores (r = -0.46, p = 0.08). Conclusion. Improvement of sleep quality after tocilizumab treatment in patients with RA does not appear to directly result from decreased disease activity, further suggesting that aberrant IL-6 regulation is associated with sleep disturbances. (First Release Dec I 2011; J Rheumatol 2012;39:60-2; doi:10.3899/jrheum.110617

Quality of Life and Psychological Burden of Parents of Children, Adolescents, and Young Adults with Type 1 Diabetes: A Cross-Sectional Study during the Lockdown Period of COVID-19

The current study aimed to investigate how parents of children, adolescents, and young adults with DM1 perceived quality of life and psychological burden during the lockdown period of COVID-19. A cross-sectional study was carried out on 110 parents in Greece in spring 2021. Perceived quality of life was measured using the Parent Diabetes Distress Scale, and psychological burden was measured using the Spielberger State/Trait Anxiety Inventory, and both were assessed with correlational analysis. Overall, 79.1% of the parents were females ,while the mean age of all was 44.4 years (±5.8). PDDS was found to be moderate (mean 2.42 ± 0.76): 63.6% of respondents had moderate/high distress. The highest mean score was for Teen Management Distress and the lowest for Healthcare Team (3.02 vs. 1.49, p < 0.001). STAI was found to be moderate to high, with a higher mean score for state versus trait anxiety (49.8 vs. 48.0, p = 0.006). Increased distress or poorer parents’ quality of life was related with the highest number of hyperglycemic episodes (β = 0.25, p = 0.002), the fewest hypoglycemic episodes (β = −0.18, p = 0.024), and the highest parental trait anxiety (β = 0.04, p < 0.001). Parents were found with moderate-to-high distress and anxiety, and their correlation also shows that there is an urgent need for suitable education of parents on managing the disease to improve quality of life and eliminate health risks to all involved

Investigation of Perception of Quality of Life and Psychological Burden of Patients Undergoing Hemodialysis—Quality of Life of Hemodialysis Patients

Chronic kidney disease (CKD) has a significant impact on the life of patients undergoing chronic periodic hemodialysis. It negatively affects their social, economic and family status, and particularly their psychological well-being. The aim of this study was to investigate the perception of the quality of life (QoL) and psychological burden of patients undergoing hemodialysis. A cross-sectional study was conducted with 63 patients. Τhe majority were men (63.5%), and the mean age of the patients was 66.7 years (±12.9) with 61.9% aged 65–89 years. Data collection was performed in 2021 using the Hospital Anxiety and Depression Scale (HADS) and the Kidney Disease and Quality of Life-Short Form (KDQOL-SF™) research tools, and their relationships were assessed using parametric and non-parametric methods. Moderate to mild levels of Anxiety and Depression were found. Physical and Mental Composite Scores were mild to moderate, with the Mental Composite Score being significantly higher (p p p p p p < 0.05). Patients with moderate or severe levels of Anxiety and Depression had a lower QoL in the Physical and Mental Composite Scores, indicating their dependence on the appropriate medical, nursing and social environment in order to attain higher levels of well-being, leading to the improvement of patients’ health. This study was not registered