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1
TURAZZI, NICE. "BAFF RECEPTOR (BAFF-R) CAR-REDIRECTED T CELLS: A NOVEL TOOL TO TREAT HIGH RISK B -CELL ACUTE LYMPHOBLASTIC LEUKEMIA (B-ALL)." Doctoral thesis, Università degli Studi di Milano-Bicocca, 2017. http://hdl.handle.net/10281/153238.
Full textAbstract:
La leucemia linfoblastica acuta B (LLA-B) è la leucemia più comune nei bambini (80%), ma ha anche un picco di incidenza in età adulta. Recentemente, gli approcci di immunoterapia diretti contro la molecola CD19 hanno dimostrato un notevole successo nel trattamento della LLA-B recidiva e refrattaria, che rimane una delle principali necessità cliniche. Svantaggi importanti di tali strategie sono la comparsa di recidive CD19-negative e aplasia delle cellule B come risultato della persistenza delle cellule anti-CD19 CAR. In questo contesto, abbiamo ipotizzato che il recettore per il fattore di attivazione delle cellule B (BAFF-R), una proteina transmembrana fondamentale nella maturazione delle cellule B e nella loro sopravvivenza, potrebbe essere una molecola interessante per strategie di targeting, avendo come vantaggio il fatto che questo recettore non è rilevabile nelle cellule B precursori del midollo osseo.
Nel nostro lavoro abbiamo dimostrato che BAFF-R è altamente espresso in tutti i campioni primari di LLA-B sia all'insorgenza che alla ricaduta. Al fine di sviluppare un approccio mediato da un recettore chimerico (CAR) specifico per l’antigene BAFF-R, sono state sviluppate sei tipologie di CAR anti-BAFFR che si differenziano per l'inversione della VH e VL e la lunghezza del dominio cerniera. Cellule killer indotte da citochine (CIK), ingegnerizzate utilizzando un sistema non virale che sfrutta trasposoni Sleeping Beauty (SB), esprimono stabilmente anti-BAFFR.CARs e mantengono il loro caratteristico fenotipo. Tra i CAR generati, il CAR più breve VHVL esercita la massima attività anti-leucemica verso le cellule bersaglio, come NALM-6, con un'attività citotossica in vitro pari al 60%. Abbiamo valutato anche le funzioni effettrici a lungo termine di rilascio di citochine mediante colorazione intracellulare (8,9 ± 2% di cellule producesti IFN-γ e 16,4 ± 5,5% di cellule producesti IL-2). Inoltre, abbiamo anche rilevato una specifica attività citotossica nei confronti di blasti primari di LLA-B (media 65,6 ± 4,5%, n = 9). Combinando le cellule trasdotte Invsh.CAR con le cellule trasdotte CD19.CAR abbiamo rilevato una attività antitumorale superiore nei confronti di tutti le linee target. Infine, utilizzando un campione raccolto da un paziente con recidiva di malattia negativa per l’antigene CD19, abbiamo dimostrato la capacità del INVsh.CAR di lisare blasti CD19-negativi.
Concludendo, questi risultati dimostrano come questo recettore sia un bersaglio sicuro e attraente per un trattamento immunoterapeutico di seconda linea per la LLA-B in caso di recidiva dopo terapia con approcci di targeting dell’antigene CD19 o per un approccio di targeting doppio. Essendo BAFF-R ristretto a cellule B mature e assente in precursori e plasmablast, la nostra strategia potrebbe avere una tossicità inferiore, per quanto riguarda l'emergere di aplasia delle cellule B osservata nei pazienti trattati con le cellule T anti-CD19 CAR.B-cell Acute Lymphoblastic Leukemia (B-ALL) is most common in children (80%), but it has also a peak of incidence in adult age. Recently, immunotherapeutic approaches targeting the CD19 molecule have demonstrated remarkable success in the treatment of relapsed and refractory B-ALL, which remains a major clinical need. Important downsides of these strategies are the emergence of CD19-negative relapses and B-cell aplasia as a result of anti-CD19 CAR T-cell persistence. In this context, we hypothesized that the receptor for B-cell activating factor (BAFF-R), a transmembrane protein fundamental in B-cell maturation and survival, could be an interesting molecule to be targeted, taking the advantage that this receptor is undetectable on bone marrow B-cell precursors. Here we showed that BAFF-R is highly expressed in B-ALL primary samples at the onset and relapse In order to develop a chimeric antigen receptor (CAR) approach targeting BAFF-R molecule, six anti-BAFFR CAR genes that differ for the inversion of the VH and VL and the length of the spacer domain have been generated. Cytokine-induced Killer (CIK) cells, engineered using an improved Sleeping Beauty (SB) transposon system, stably expressed anti-BAFFR.CARs, and maintained their characteristic phenotype. Among the newly constructed CARs, the shortest VHVL CAR exerted the highest anti-leukemic activity towards target cells, such as NALM-6, with an in vitro killing activity of 60%. We also evaluated later effector functions in terms of cytokine release by intracellular staining (8,9±2% of IFN-γ and 16,4±5,5% of IL-2 producing cells). Importantly, we also detected a specific cytotoxic activity towards primary B-ALL blasts (average 65,6±4,5%, n=9). Combining the Invsh.CAR with CD19.CAR we detected a superior antitumor activity towards ALL targets. Furthermore, by using a sample collected from a patient relapsed with CD19 negative disease, we demonstrated the ability of the INVsh.CAR to lysate CD19-negative blasts. Taken together, these findings make this receptor a safe and attractive target for a second line B-ALL immunotherapy in case of relapse after CD19-targeting therapies or for a double targeted approach. Being restricted to mature B cells, but absent in precursors and plasmablasts, our strategy could have an inferior toxicity concerning the emergence of B-cell aplasia observed in patients treated with anti-CD19 CAR-modified T cells.
2
ALBERTI, GAIA. "Evaluation of a Tandem CD33-CD146 Chimeric Antigen Receptor (CAR) for the simultaneous targeting of Acute Myeloid Leukemia (AML) blasts and stromal cells in the niche." Doctoral thesis, Università degli Studi di Milano-Bicocca, 2022. http://hdl.handle.net/10281/382304.
Full textAbstract:
La leucemia mieloide acuta (LMA) è la neoplasia ematologica maggiormente diagnosticata nei pazienti adulti (25%) e mentre rappresenta il 15-20% dei casi nei pazienti pediatrici. La chemioterapia convenzionale, che impiega antraciclina e citarabina, rappresenta il trattamento standard per l’LMA, con tassi di remissione completa dal 60% all'80% nei bambini e dal 40% al 60% negli adulti (>60 anni). Sfortunatamente, la ricaduta dopo tale terapia è comune e la sopravvivenza dei pazienti stimata a 5 anni è ancora inferiore al 30%. Risulta quindi di primaria importanza trovare alternative terapeutiche per i pazienti recidivanti e refrattari. Il recente successo clinico, ottenuto nelle leucemie di tipo B, dell'immunoterapia con cellule CAR (chimeric antigen receptor) T ha portato allo sviluppo di nuove strategie terapeutiche nell’ambito dell’LMA. Tuttavia, lo sviluppo del trattamento con cellule CAR T nel contesto dell'LMA è ancora agli albori a causa dell'eterogeneità della malattia, della mancanza di un antigene bersaglio adatto e del ruolo protettivo del microambiente tumorale (TME). Infatti, non esiste ancora un protocollo clinico approvato per il trattamento della leucemia mieloide. Per creare le cellule CAR T abbiamo scelto di utilizzare la piattaforma non virale Sleeping-Beauty (SB) per ingegnerizzare le cellule CIK (cytokine-induced killer). In primo luogo, abbiamo scelto di utilizzare come potenziale strumento per il targeting del TME le cellule CIK ingegnerizzate con anti-CD146.CAR. Di conseguenza, abbiamo ottimizzato 6 diverse molecole CAR aventi un design differente, ottenendo un'espressione ottimale di CD146 nella variante VLVH Long. Abbiamo quindi testato le cellule CD146.CAR-CIK in vitro, ottenendo l’attivazione specifica delle funzioni effettrici (in termini di capacità di killing, produzione di citochine e proliferazione) contro cellule target CD146+. In seguito, abbiamo progettato un Tandem CAR bispecifico (CD33xCD146.CAR-CIKs) che ha mostrato una significativa attività antileucemica in vitro. È stato ampiamente dimostrato che la nicchia midollare contribuisce al supporto e alla protezione delle cellule staminali leucemiche (CSLs). Quindi, per mimare al meglio l’azione del CAR nella nicchia midollare umana, abbiamo testato le cellule CD33xCD146.CAR-CIK contro le linee cellulari stromali CD146+ e le cellule mesenchimali (MSC) primarie sane (HD-) e di derivazione mieloide (LMA-). I dati mostrano una inibizione delle funzioni effettrici delle cellule CAR-CIK e una drastica diminuzione della produzione di citochine e della proliferazione. Inoltre, l'equilibrio tra citochine pro e antinfiammatorie è risultato alterato, infatti la produzione di citochine Th1/Tc1 da parte delle cellule CD146.CAR-CIK è stata inibita dalla co-coltura con cellule stromali, mentre è stato rilevato un aumento delle citochine Th2/Tc2. Questi risultati suggeriscono un potenziale ruolo immunosoppressivo del compartimento stromale nei confronti delle cellule CAR-CIK. Sulla base dell’ effetto immunomodulatorio delle MSC sui linfociti T, abbiamo ipotizzato che la nicchia midollare possa influenzare le funzioni effettrici delle cellule CAR T. Di conseguenza, il targeting del CD146 rappresenta una "proof-of-principles" del fatto che aggredire il microambiente leucemico possa migliorare la terapia CAR T nell’ambito dell’LMA. Per ridurre al minimo la tossicità "off-target ", stiamo cercando di selezionare un antigene bersaglio specifico ed overespresso sulle cellule stromali dell'LMA, che abbia un'espressione minima nello stroma sano e che sia coinvolto nelle interazioni leucemia/nicchia. Il nuovo marker di interesse sarà accoppiato al CD33.CAR nella creazione di un CAR bispecifico, che verrà confrontato con il costrutto CD33xCD146.CAR, valutandone i profili di efficacia e sicurezza sia in vitro che in vivo.Acute myeloid leukemia (AML) is the most frequently diagnosed leukemia in adults (25%) and accounts for 15-20% cases in pediatric patients. Conventional chemotherapy employing anthracycline and cytarabine represents the gold standard treatment for AML, with rates of complete remission from 60% to 80% in children and from 40% to 60% in adults (>60 years). Despite these high rates, relapse after conventional therapy is common and the estimated five-year survival of AML patients is still below 30%. Indeed, there is an urgency to find alternative therapeutic strategies for relapsed and refractory patients. The recent clinical success of chimeric antigen receptor (CAR) T cell immunotherapy in the context of B-cell malignancies has opened a new route of investigation also towards AML. However, the development of CAR T cell therapy in the context of AML is still in its infancy due to heterogeneity of the disease, the lack of a suitable target antigen and the leukemia protective role of the tumor microenvironment (TME) and no approved CAR T cells study exists for AML treatment yet. Non-viral Sleeping-Beauty (SB) transposon platform was employed to redirect cytokine-induce killer (CIK) cell. In this scenario, we firstly characterize non-viral SB engineered CIK cells with anti-CD146.CAR as a potential tool for the targeting of the bone marrow (BM) microenvironment. We optimized the CAR design structure by testing 6 different CAR molecules, achieving a specific and efficient CD146 expression in the VLVH Long variant. CD146.CAR-CIK cells were subsequently tested in vitro, showing an optimal activation of effector functions (in terms of killing activity, cytokines production and proliferation) when they were engaged against CD146+ target cells. Consequently, we developed a bispecific Tandem CAR (CD33xCD146.CAR-CIKs), which displayed anti-leukemic activity in vitro. It has been extensively proven that BM niche contribute to establish a sanctuary in which leukemic stem cells (LSCs) are able to acquire drug-resistant phenotype, therefore, to better mimicking the human BM niche we tested CD33xCD146.CAR-CIK cells against CD146+ stromal cell lines (HS-27A and HS-5) and primary derived healthy (HD-) and patient-derived (AML-) mesenchymal stromal cells (MSCs). Results showed inhibition of the redirected CAR-CIK cells effector functions, resulting in a drastic decrease of cytokines production and proliferation. The balance between pro- and anti- inflammatory cytokines showed that Th1/Tc1 cytokines production by CD146.CAR-CIK cells was inhibited by the co-culture with stromal cells, while increase Th2/Tc2 cytokines was detected when CD146.CAR-CIK cells were co-cultured with stromal target cells. These results suggest a potential immunosuppressive role of the stromal compartment against CAR-CIK cells. According to these results, we hypothesized that BM stromal cells can potentially exert an immunomodulatory effect on T cells, suggesting that the niche microenvironment may be involved in the regulation of CAR T cells therapy effectiveness. Indeed, the targeting of CD146 on stroma represents a “proof-of-principle” that stromal components of leukemic microenvironment may be attractive targets for CAR T based immunotherapy. To minimize “off-tumor” toxicity, we are looking for a specific surface target antigen selectively overexpressed on AML stromal cells, with minimal expression in healthy stroma and possibly involved in leukemia/niche interactions. The newly marker of interest will be coupled to the CD33.CAR and this bispecific CAR will be compared with CD33xCD146.CAR construct, evaluating their efficacy and safety profiles both in vitro and in vivo.
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PIEVANI, ALICE SILVIA. "Cytokine-induced killer (cik) cell cultures for the adoptive immunotherapy of hematological malignancies: characterization and new therapeutic strategies for clinical application." Doctoral thesis, Università degli Studi di Milano-Bicocca, 2011. http://hdl.handle.net/10281/20178.
Full textAbstract:
Cytokine-induced killer (CIK) cells are a heterogeneous population of lymphocytes obtained in vitro within 21 days from mononuclear cells under the influence of cytokines. CIK cells show potent MHC-unrestricted cytotoxicity against a variety of tumor cells, in particular hematological malignancies, and minimal tendency to induce graft-versus-host disease.
The expanded bulk CIK culture consists of over 90% CD3+ cells, of which the majority coexpress CD56 and the remaining cells are CD56-. CD3+CD56+ “true” CIK cells are terminally differentiated non dividing lymphocytes which could deliver potent MHC-unrestricted cytotoxicity for the immediate destruction of tumor cells. The other less cytotoxic CD3+CD56- cell subset represents a progenitor reservoir that could proliferate and differentiate into CD3+CD56+ CIK cells. CD3+CD56+ CIK cells express activating NK receptors including NKG2D, DNAM-1 and low levels of NKp30. Cell signalling not only through TCR/CD3, but also through NKG2D, DNAM-1 and NKp30, leads to CIK cell activation resulting in granule exocytosis and cytotoxicity. Antibody blocking experiments revealed that NKG2D, DNAM-1 and NKp30 are actually involved in tumor cell recognition and killing. Anti-CMV specific CIK cells could be expanded in standard CIK conditions and mediate both specific, MHC-restricted recognition of a CMV-pulsed autologous target and NK-like non specific cytolytic activity against leukemic cell targets. Antibody blocking of NKG2D and NKp30 only inhibited NK-like cytotoxicity. Their dual effector function suggests that CIK cells, when used in a clinical setting, may control both neoplastic relapses and viral infections, two frequently associated complications in transplanted patients.
B-cell non-Hodgkin lymphoma is only partially susceptible to CIK-mediated lysis. The addition of anti-CD20 monoclonal antibodies GA101 or rituximab increased cytotoxicity mediated by CIK cell cultures by 35% and 15%, respectively. This enhancement was mainly due to antibody-dependent cytotoxicity mediated by the 1%-10% NK cells contaminating CIK cultures. The addition of human serum inhibited NK-cell activation induced by rituximab, but not activation induced by GA101. Overall lysis in presence of serum, even of a resistant B-NHL cell line, was significantly increased by 100 mcg/mL of rituximab, but even more so by GA101, with respect to CIK cultures alone. The combined use of CIK cells with anti-CD20 mAbs could represent a novel immunotherapy protocol for the treatment of B lymphoma patients with resistant disease.
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Bach, Martin. "Der Einfluss muriner mesenchymaler Stammzellen auf murine zytokin induzierte Killerzellen in der Kokultur." Doctoral thesis, Universitätsbibliothek Leipzig, 2014. http://nbn-resolving.de/urn:nbn:de:bsz:15-qucosa-149957.
Full textAbstract:
Stimulating lymphocytes with Ifn-γ, anti-CD3, and interleukin-2 promotes the proliferation of a cell population coexpressing T-lymphocyte surface antigens such as CD3, CD8a, and CD25 as well as natural killer cell markers such as NK1.1, CD49, and CD69. These cells, referred to as cytokine-induced killer cells (CIKs), display cytotoxic activity against tumour cells, even without prior antigen presentation, and offer a new cell-based approach to the treatment of malignant diseases. Because CIKs are limited in vivo, strategies to optimize in vitro culture yield are required.
In the last 10 years, mesenchymal stem cells (MSCs) have gathered considerable attention. Aside from their uses in tissue engineering and as support in haematopoietic stem cell transplantations, MSCs show notable immunomodulatory characteristics, providing further possibilities for therapeutic applications. In this study, we investigated the influence of murine MSCs on proliferation, phenotype, vitality, and cytotoxicity of murine CIKs in a coculture system. We found that CIKs in coculture proliferated within 7 days, with an average growth factor of 18.84, whereas controls grew with an average factor of 3.7 in the same period. Furthermore, higher vitality was noted in cocultured CIKs than in controls. Cell phenotype was unaffected by coculture with MSCs and, notably, coculture did not impact cytotoxicity against the tumour cells analysed. The findings suggest that cell–cell contact is primarily responsible for these effects. Humoral interactions play only a minor role. Furthermore, no phenotypical MSCs were detected after coculture for 4 h, suggesting the occurrence of immune reactions between CIKs and MSCs. Further investigations with DiD-labelled MSCs revealed that the observed disappearance of MSCs appears not to be due to differentiation processes.
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Cappuzzello, Elisa. "A DONOR-DEPENDENT SUBSET OF CYTOKINE-INDUCED KILLER (CIK) CELLS EXPRESS CD16 AND CAN BE RETARGETED TO EXERT A POTENT ANTIBODY-DEPENDENT CELL-MEDIATED CYTOTOXICITY (ADCC)." Doctoral thesis, Università degli studi di Padova, 2016. http://hdl.handle.net/11577/3424343.
Full textAbstract:
Cancer adoptive cell therapy (ACT) relies on the infusion of immune cell populations mediating direct antitumor effects, such as cytotoxic CD8+ T lymphocytes (CTL), natural killer (NK) cells and Cytokine-Induced Killer (CIK) cells. In this study, we aimed at improving CIK cell potential for adoptive immunotherapy strategies. CIK cells are a heterogeneous population of ex vivo expanded lymphocytes, which share phenotypic and functional features with both NK and T cells. They exert a potent MHC-independent antitumor activity against both hematological and solid malignancies, but not normal tissues and hematopoietic precursors. Several clinical trials have demonstrated the feasibility and the therapeutic efficacy together with low toxicity of CIK cells infusion, supporting CIK cells as a very promising cell population for adoptive immunotherapy. In this work, CIK cells were obtained from PBMCs of healthy donors by the timed addition of IFN-γ, anti-CD3 antibody and IL-2. Analyzing their phenotype, we demonstrated for the first time a relevant expression of CD16 in a donor-dependent manner and, based on this observation, we proved the ability of CIK cells to kill tumors by an Antibody-Dependent Cell-mediated Cytotoxicity (ADCC) mechanism. Indeed, the concurrent administration of clinically therapeutic mAbs, such as trastuzumab or cetuximab, led to a significant improvement of their antitumor activity in vitro against both ovarian and breast cancer cell lines. To formally prove that the CD16 receptor is functional and directly involved in the ADCC, an anti-CD16 blocking antibody was added to the assays. NK cell depletion from bulk cultures confirmed that the ADCC activity is accountable to the CIK CD16+ subpopulation. This novel function of CIK cells, never exploited before, was assessed for therapeutic efficacy in mouse models of human ovarian carcinoma xenografted in NOD/SCID common γ chain knockout (NSG) mice. Co-administration of CIK cells and mAbs significantly increased the survival of tumor-bearing mice, as compared to animals receiving CIK cells alone. CIK cell antitumor activity in vitro was also enhanced by the combination with bispecific antibodies and immunoligands, which are able to target both a tumor-associated antigen and activating receptors expressed by effector cells. Taken together, these data envisage new perspectives for adoptive immunotherapy where antigen-specific retargeting of T cells can be achieved by a combination therapy with clinical-grade monoclonal antibodies already widely used in cancer therapy, and CIK cell populations that are easily expandable in very large numbers, inexpensive, safe and do not require genetic manipulations. In conclusion, this new therapeutic strategy for the ACT treatment of different types of tumors could find wide implementation and application, and be expanded to the use of additional therapeutic antibodies.La terapia cellulare adottiva (Adoptive Cell Therapy, ACT) si basa sulla somministrazione di popolazioni di cellule immunitarie in grado di mediare un effetto antitumorale in modo diretto, ad esempio linfociti T CD8+ citotossici (CTL), cellule natural killer (NK) e cellule killer indotte da citochine (Cytokine-Induced Killer cells, CIK). Lo scopo di questo lavoro è stato quello di incrementare il potenziale delle cellule CIK nelle strategie di immunoterapia adottiva. Le cellule CIK sono una popolazione eterogenea di linfociti espansi ex vivo che condividono caratteristiche fenotipiche e funzionali sia con le cellule NK sia con le cellule T. Queste cellule esercitano una potente citotossicità MHC-indipendente nei confronti di tumori sia ematologici sia solidi, ma non di tessuti normali e precursori ematopoietici. Diversi trial clinici hanno dimostrato l’attuabilità, l’efficacia terapeutica e la bassa tossicità delle infusioni di cellule CIK, supportandole come popolazione cellulare molto promettente per l’immunoterapia adottiva. In questo lavoro, le cellule CIK sono state ottenute da cellule mononucleate del sangue periferico (Pheripheral Blood Mononuclear Cells, PBMCs) di donatori sani mediante l’aggiunta di interferone gamma (Interferon-γ, IFN-γ), anticorpi anti-CD3 e interleuchina 2 (Interleukin-2, IL-2). Analizzando il fenotipo, abbiamo dimostrato per la prima volta una rilevante espressione donatore-dipendente del recettore CD16 e, basandoci su questa osservazione, abbiamo analizzato la capacità delle cellule CIK di uccidere cellule tumorali mediante citotossicità cellulo-mediata anticorpo-dipendente (Antibody-Dependent Cell-mediated Cytotoxicity, ADCC). Infatti, abbiamo osservato che la simultanea somministrazione di anticorpi monoclonali terapeutici, come il trastuzumab e il cetuximab, portano ad un significativo incremento dell’attività antitumorale in vitro delle CIK nei confronti di linee cellulari di tumore ovarico e mammario. Per dimostrare che il CD16 è funzionale ed è direttamente coinvolto nell’ADCC, è stato aggiunto al saggio un anticorpo bloccante anti-CD16. La deplezione delle cellule NK ha confermato che l’ADCC è attribuibile alla sottopopolazione CD16+ delle cellule CIK. Questa nuova funzione delle cellule CIK, descritta qui per la prima volta, è stata valutata per la sua efficacia terapeutica in un modello murino di carcinoma ovarico umano trapiantato in topi NOD/SCID knockout per la catena comune γ (topi NSG). La co-somministrazione di cellule CIK e anticorpi monoclonali ha aumentato significativamente la sopravvivenza dei topi con tumore, in confronto ai topi trattati soltanto con le CIK. Inoltre, l’attività antitumorale in vitro delle cellule CIK è stata incrementata mediante la combinazione con anticorpi bispecifici e immunoligandi, in grado di legare contemporaneamente un antigene associato al tumore e un recettore attivatore espresso dalle cellule effettrici. Complessivamente, questi dati prospettano nuove possibilità per l’immunoterapia adottiva, in cui il reindirizzamento antigene-specifico dei linfociti T può essere ottenuto mediante la combinazione di anticorpi monoclonali di utilizzo clinico, già ampiamente utilizzati per la terapia antitumorale, con popolazioni di cellule CIK, che sono facilmente espandibili, economiche, sicure e non richiedono manipolazioni genetiche. In conclusione, questa nuova strategia terapeutica per trattamento di diversi tipi di tumori mediante terapia cellulare adottiva potrà trovare ampie possibilità di implementazione e applicazione, e potrà essere estesa all’utilizzo di ulteriori anticorpi terapeutici.
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BIONDI, MARTA. "Enhancing AML CAR CIK therapeutic potency increasing the localization of engineered cells in the malignant niche and its selectivity by LSCs specific targeting." Doctoral thesis, Università degli Studi di Milano-Bicocca, 2022. http://hdl.handle.net/10281/365153.
Full textAbstract:
La terapia CAR-T rappresenta un approccio promettente, ma ha riportato una ridotta efficacia nella leucemia mieloide acuta (AML), a causa dell’eterogeneità del tumore, dell’assenza di antigeni target AML-specifici e del ruolo del microambiente leucemico nella protezione dei blasti e delle cellule staminali leucemiche (LSC). La nicchia midollare, nella quale risiedono le LSC, è coinvolta in attività che promuovono la progressione leucemica e sopprimono l’ematopoiesi sana. Quindi ipotizziamo che bersagliare le LSC nascoste nella nicchia potesse migliorare l’efficacia delle CAR-T. Per testare la nostra ipotesi, abbiamo agito su due fronti: 1) promuovere una migrazione efficiente delle CAR-T nella nicchia midollare, 2) selezionare un antigene target ristretto ai blasti leucemici e alle LSC. Prima, abbiamo proposto una strategia per guidare le cellule CD33.CAR CIK (Cytokine-Induced Killer), una sottopopolazione di cellule T effettrici, verso la nicchia leucemica. La chemochina CXCL12, rilasciata dalle cellule mesenchimali stromali (MSC), nella nicchia midollare, e il suo recettore CXCR4, sono coinvolti nella regolazione della migrazione dei leucociti all’interno della nicchia. Quindi, abbiamo ipotizzato che sfruttare questo asse potesse migliorare la capacità di homing delle CD33.CAR-CIK nella nicchia e favorire l’eradicazione della leucemia. Tuttavia i protocolli di manipolazione ex vivo delle CD33.CAR-CIK riducono l’espressione di CXCR4, compromettendo la capacità delle cellule infuse di raggiungere la nicchia. Quindi per implementare la capacità di homing delle CD33.CAR-CIK nel microambiente midollare, abbiamo sviluppato delle CD33.CAR-CIK overesprimenti CXCR4, nella sua forma wild-type o iperattiva mutata. Le CIK ingegnerizzate con i costrutti CD33.CAR-CXCR4 hanno mostrato un consistente aumento dell’espressione di CXCR4, senza riportare alterazioni fenotipiche e nelle funzioni effettrici CAR-associate. Inoltre, rispetto alle CD33.CAR-CIK, le cellule CD33.CAR-CXCR4WT -CIK ed in particolare le CD33.CAR-CXCR4MUT-CIK hanno dimostrato non solo una superiore risposta chemotattica in vitro verso il CXCL12 ed i surnatanti delle MSC, ma anche un aumentato homing in vivo. In seguito, per promuovere lo sviluppo di un approccio CAR-T più efficace e sicuro, abbiamo proposto di re-indirizzare il CAR verso un antigene espresso selettivamente dalle cellule AML, ma assente sulle cellule staminali ematopoietiche (HSC). TIM-3 è un immune checkpoint, svolge un ruolo centrale nella regolazione delle risposte immunitarie nell’AML e costituisce un marcatore selettivo per le LSC, senza essere espresso dalle HSC. Abbiamo disegnato un CAR di terza generazione diretto contro TIM-3, utilizzando la porzione scFv derivante da un anticorpo monoclonale anti-TIM-3. In vitro, le TIM-3.CAR-CIK hanno dimostrato di eliminare sia le linee AML che i blasti primari, senza dare tossicità verso le cellule TIM-3+ sane, come le CIK attivate, i monociti e le cellule NK. Inoltre, le TIM-3.CAR-CIK hanno eliminato in maniera selettiva le LSC (CD34+ CD38-). Infine, le TIM-3.CAR-CIK hanno mantenuto le loro capacità effettrici nonostante multiple ristimolazioni in vitro, gettando le basi per lo studio di questo costrutto in vivo. Complessivamente, entrambi gli approcci, uno implementando l’homing delle CAR-CIK alla nicchia midollare e l’altro conferendo una superiore selettività, potrebbero migliorare l’efficacia della terapia CAR-T nel contesto dell’AML.Chimeric Antigen Receptor (CAR) T-cell therapy has produced remarkable clinical responses in patients affected by acute lymphoblastic leukemia. Unfortunately, CAR T-cells have not been equally successful in acute myeloid leukemia (AML) due to tumor heterogeneity, lack of truly AML-restricted target antigens and the role of leukemia microenvironment in blasts protection and leukemia stem cells (LSCs) maintenance. Specifically, the bone marrow (BM) niche, where LSCs reside, is involved in leukemia promoting activities whilst suppressing normal hematopoiesis. Therefore, we hypothesized that targeting LSCs at their location may enhance the potency and selectivity of CAR-T cells. To address this issue, we have designed two aims: 1) promote rapid and efficient localization of CAR T-cells within the BM niche, 2) select a leukemia-restricted antigen to specifically target AML blasts and LSCs. First, we proposed to harness CD33.CAR-redirected Cytokine-Induced Killer (CIK) cells, an alternative effector T-cell population with acquired NK-like cytotoxic activity as well as minimal alloreactivity, to selectively route their activity to leukemia transformed niche. The chemokine ligand 12 (CXCL12), released by mesenchymal stromal cells (MSCs) within the medullary niche, and its chemokine receptor 4 (CXCR4) are two pivotal players regulating leukocytes trafficking to the BM. In AML, CXCL12 interacts with CXCR4 overexpressed on blasts, promoting their migration and homing in the niche. Hence, taking advantage of this axis might facilitate CD33.CAR-CIK cells homing to the BM and therefore leukemia eradication. However, ex vivo manipulation protocols of CD33.CAR-CIK cells consistently downregulate CXCR4 expression and may affect the capacity of adoptively infused cells to migrate to BM and exert their anti-leukemic action. Therefore, to improve CD33.CAR-CIKs homing in the BM microenvironment we have developed CD33.CAR-CIK cells overexpressing CXCR4, in its wild-type or hyperactive mutant form. Notably, CIK cells engineering with CD33.CAR-CXCR4 constructs led to a consistent increase in CXCR4 expression, without altering CIK cells phenotype and CAR-related effector functions. Interestingly, compared to conventional CD33.CAR-CIK cells, CD33.CAR-CXCR4WT and especially CD33.CAR-CXCR4MUT-CIK cells demonstrated significantly superior in vitro chemotactic response toward CXCL12 and MSC-derived supernatants, and greater in vivo BM homing ability and persistence. Furthermore, to develop an effective anti-AML CAR T-cell therapy, it is fundamental to identify a LSC-specific marker, sparing the normal counterpart of hematopoietic stem cells (HSCs). T-cell immunoglobulin and mucin protein 3 (TIM-3) is an immune checkpoint molecule, it plays a central role in immune responses in AML and it is an LSC-specific marker, lacking expression on HSCs. Therefore, we designed a third-generation anti-TIM-3.CAR using the single-chain fragment variable (scFv) derived from an antagonistic ligand-blocking anti-TIM-3 antibody. In vitro, TIM-3.CAR-CIK cells efficiently killed both AML cell lines and primary AML blasts, but not normal TIM-3+ activated CIK cells, monocytes and NK-cells. Notably, we observed selective elimination of primary LSC-enriched population (CD34+ CD38-). Furthermore, TIM-3.CAR-CIK cells maintained their effector functions despite multiple in vitro restimulations, setting the basis for further exploration in in vivo models. Overall, both approaches, one improving CAR-CIK cells homing to the transformed niche and the other conferring superior safety and selectivity, might improve the efficacy of anti-AML CAR-CIK therapy.
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Weylandt, Karsten-Henrich. "Towards a functional role for human CIC-3 and human CIC-4, two members of the CIC chloride channel family." Thesis, University of Oxford, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.341009.
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Zeid, Rhamy. "Characterization and Disruption of Cis Regulatory Elements in Cancer." Thesis, Harvard University, 2016. http://nrs.harvard.edu/urn-3:HUL.InstRepos:33493536.
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Enhancers are cis regulatory elements that play key roles in the control of cell-type specific gene expression programs. In cancer, enhancer deregulation plays a key role in maintaining gene regulatory programs that underlie an oncogenic state. This dissertation focuses on understanding and modulating aberrant enhancer activity to identify potential vulnerabilities in human cancers. These studies were empowered by evolving technologies in genome-wide measurements of enhancer factors, computational approaches, and chemical and genetic tools to disrupt enhancer function.
In high-risk pediatric neuroblastoma, the transcription factor MYCN is frequently amplified and treatment options for these patients are largely ineffective thus establishing the need for improved therapeutic options. To identify previously unrecognized dependencies in neuroblastoma, we generated genome-wide maps of the active enhancer gene regulatory landscape leading to the identification of ID1 as an uncharacterized dependency in neuroblastoma. These results outline a strategy to identify alternative therapeutic avenues based on a holistic understanding of aberrant enhancer activity.
While MYCN amplification is the defining feature of high-risk neuroblastoma, a detailed mechanistic understanding of oncogenic transcriptional rewiring has been stalled by a lack of genome-wide binding data.
Here we present the dynamic and temporally resolved landscape of genome-wide MYCN occupancy in neuroblastoma. We find that deregulated MYCN binding at enhancers (termed enhancer invasion) is critical to maintaining the oncogenic station and identify the lineage specific transcription factor TWIST1 as a key collaborator and synthetic lethality of oncogenic MYCN. These data suggest that MYCN enhancer invasion shapes transcriptional amplification in neuroblastoma to promote tumorigenesis.
The development of small molecule inhibitors of the bromodomain and extra-terminal (BET) family of proteins provides a pharmacological strategy to inhibit enhancer activity. The efficacy of BET inhibition in several cancers has prompted efforts to predict and understand mechanisms of resistance to BET inhibition. Here, we use a newly developed class of small molecules to pharmacologically induce targeted degradation of the BET family. In triple negative breast cancer, we demonstrate that targeted BET family degradation effectively overcomes BET inhibitor resistance. These studies suggest BET degradation as a strategy to overcome BET inhibitor resistance and further disrupt and dissect enhancer activity in cancer.Medical Sciences
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Ganakammal, Satishkumar Ranganathan. "CIS REGULATORY MODULE DISCOVERY IN TH1 CELL DEVELOPMENT." Thesis, Proceeding ISB '10 Proceedings of the International Symposium on Biocomputing ACM New York, NY, USA ©2010 table of contents ISBN: 978-1-60558-722-6 doi>10.1145/1722024.1722039, 2010. http://hdl.handle.net/1805/2678.
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Indiana University-Purdue University Indianapolis (IUPUI)Immune response enables the body to resist foreign invasions. The Inflammatory response is an important aspect in the immune response which is articulated by elements such as cytokines, APC, T-cell and B-cell, effector cell or natural killer. Of these elements, T-cells especially T-helper cells; a sub class of T-cells plays a pivotal role in stimulating the immune response by participating in various biological reactions such as, the transcription regulatory network. Transcriptional regulatory mechanisms are mediated by a set of transcription factors (TFs), that bind to a specific region (motifs or transcription factor binding sites, TFBS), on the target gene(s) controlling the expression of genes that are involved in T-helper cell mediated immune response. Eukaryotic regulatory motifs, referred to as cis regulatory modules (CRMs) or cistrome, co-occur with the regulated gene’s transcription start site (TSS) thus, providing all the essential components for building the transcriptional regulatory networks that depends on the relevant TF-TFBS interactions. Here, we study IL-12 stimulated transcriptional regulators in STAT4 mediated T helper 1 (Th1) cell development by focusing on the identification of TFBS and CRMs using a set of Stat4 ChIP-on-chip target genes. A region containing 2000 bases of Mus musculus sequences with the Stat4 binding site, derived from the ChIP-on-chip data, has been characterized for enrichment of other motifs and, thus CRMs. Our experiments identify some potential motifs, (such as NF-κB and PPARγ/RXR) being enriched in the Stat4 binding sequences compared to neighboring background sequences. Furthermore, these predicted CRMs were observed to be associated with biologically relevant target genes in the ChIP-on-chip data set by meaningful gene ontology annotations. These analyses will enable us to comprehend the complicated transcription regulatory network and at the same time categorically analyze the IL-12 stimulated Stat4 mediated Th1 cell differentiation.
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Calzone, Laurence. "Mathematical Modeling of the Budding Yeast Cell Cycle." Thesis, Virginia Tech, 2000. http://hdl.handle.net/10919/31988.
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The cell cycle of the budding yeast, Saccharomyces cerevisiae, is regulated by a complex network of chemical reactions controlling the activity of the cyclin-dependent kinases (CDKs), a family of protein kinases that drive the major events of the cell cycle. A previous mathematical model by Chen et al. (2000) described a molecular mechanism for the Start transition (passage from G1 phase to S/M phase) in budding yeast. In this thesis, my main goal is to extend Chen's model to include new information about the mechanism controlling Finish (passage from S/M phase to G1 phase). Using laws of biochemical kinetics, I transcribed the hypothetical molecular mechanism into a set of differential equations. Simulations of the wild-type cell cycle and the phenotypes of more than 60 mutants provide a thorough understanding of how budding yeast cells exit mitosis.Master of Science
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Lindqvist, Arne. "Regulation of CDK dephosphorylation in mitotic entry /." Stockholm, 2005. http://diss.kib.ki.se/2005/91-7140-362-0/.
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Rindler, Paul Michael. "Eukaryotic replication, cis-acting elements, and instability of trinucleotide repeats." Oklahoma City : [s.n.], 2009.
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Auckland, Ian. "Quantitative Analysis of a Cell Cycle Checkpoint in Xenopus laevis Cell-Free Egg Extracts." Thesis, Virginia Tech, 2005. http://hdl.handle.net/10919/34734.
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In somatic cells, checkpoint pathways trigger cell cycle arrest in response to
unreplicated or damaged DNA by inhibiting the activity of cyclin-dependent kinases
(Cdks). In the Xenopus laevis embryo, checkpoints are not operational until the
midblastula transition (MBT). Studies in cell-free egg extracts indicate that a threshold
concentration of nuclei, which approximates the MBT concentration, is required to elicit
a checkpoint. The checkpoint response to unreplicated DNA in the extract prevents
transition into mitosis by inhibiting Cdk1/cyclin B, causing an increase in the minimum
amount of cyclin B necessary to enter mitosis, termed the cyclin threshold. Once the
threshold of cyclin is maintained or exceeded, the system will proceed into mitosis after a
lag time. We have investigated the relationship between nuclear concentration and cell
cycle regulation in the extract. By precisely regulating the concentration of cyclin B and
nuclear content in extract samples, we have found 1) the concentration of nuclei affects
cyclin B thresholds and lag time of entry into mitosis, 2) elevated cyclin thresholds
caused by DNA replication blocks are further increased by increasing the concentration
of nuclei, and 3) double-stranded DNA breaks in the extract system do not affect cyclin
thresholds or lag time of entry into mitosis within the range of nuclear concentrations that
can be efficiently replicated. This data provides evidence of the importance of the
nucleocytoplasmic ratio in normal cell cycle progression and its importance for
checkpoint acquisition during early Xenopus laevis development.Master of Science
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Birot, Adrien. "Regulation of fission yeast cohesin by the Cyclin Dependent Kinase PeF1." Thesis, Bordeaux, 2016. http://www.theses.fr/2016BORD0386/document.
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Le complexe cohésine est un complexe protéique en forme d'anneau composé de quatre sous-unités essentielles très conservées: Smc1, Smc3, Rad21 et Scc3. Par sa capacité à encercler les molécules d’ADN, les cohésines participent à de nombreux processus cellulaires tels que la ségrégation des chromosomes, la signalisation et la réparation des dommages à l’ADN, la régulation de la transcription et l'organisation du génome. Pour assurer ces différentes fonctions biologiques les cohésines doivent être finement régulées à la fois dans le temps et l’espace. Ces régulations reposent en partie sur le contrôle de leur association à la chromatine (capture de l’ADN). Cela nécessite l'action d'un «facteur de chargement » composé de deux protéines conservées et essentielles, Mis4 et Ssl3 chez la levure S. pombe. Comment ce complexe régule la capture de l’ADN par l’anneau de cohésine dans l'espace et le temps demeure à ce jour très mal compris. Afin d’identifier des régulateurs de l’association des cohésines à la chromatine, nous avons réalisé un crible génétique visant à rechercher des suppresseurs de la mutation thermosensible mis4-367. Ce crible a conduit à l’identification de la Cyclin-Dependent Kinase Pef1 qui agit comme un régulateur négatif de la cohésion des chromatides soeurs en contrôlant vraisemblablement négativement l’association des cohésines à la chromatine. De forts arguments expérimentaux indiquent que Pef1 exerce sa fonction en régulant directement la phosphorylation de la sous-unité Rad21 du complexe cohésine. De façon intéressante, via un autre crible génétique, nous avons identifié la phosphatase Pph3/Psy2 qui joue un rôle dans l’établissement de la cohésion des chromatides soeurs en contrôlant la déphosphorylation de Rad21.Ensemble, ces données suggèrent que le contrôle de l’état de phosphorylation de la sous-unité Rad21 du complexe cohésine joue un rôle central dans le processus de cohésion chez la levure S. PombeCohesin is a highly conserved ring-shaped protein complex made of four essential subunits: Psm1, Psm3, Rad21 and Psc3. By its ability to capture DNA molecules within its ring-like structure, cohesion plays a key role in many cellular processes such as chromosome segregation, DNA damage signalling and repair, transcriptional gene regulation and nuclear organization. To ensure all of its biological functions, cohesin must be tightly regulated in space and time. This regulation relies in part on the control of cohesin binding to chromatin (DNA capture). Cohesin recruitment to chromatin requires the action of a “loading complex” made of two conserved and essential proteins named Mis4 and Ssl3 in the fission yeast. How this complex regulates where and when DNA capture by the cohesin ring must occur remains poorly understood. To identify regulators of cohesin binding to chromatin we have performed a genetic screen for suppressors of the thermosensitive mutation mis4-367. This genetic screen has led to the identification of the cyclin-dependent-kinase Pef1 that acts as a negative regulator of sister chromatids cohesion may be bynegatively controlling cohesin binding to chromatin. Strong experimental evidences indicate that Pef1 exerts its function at least in part by directly phosphorylating the Rad21 subunit of the cohesin complex. Interestingly, a genetic screen made in parallel identified the Pph3/Psy2 phosphatase as implicated in the establishment of sister chromatid cohesion by regulating Rad21 dephosphorylation. Strikingly, the control of Rad21 phosphorylation status appears central to the cohesion process in the fission yeast S. pombe
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Paik, Jisun. "CIS-retinol dehydrogenase : characterization and biochemical analysis of 9-cis-retinol metabolism in two model systems /." Thesis, Connect to this title online; UW restricted, 2000. http://hdl.handle.net/1773/6600.
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Zhang, Hong. "Involvement of Cdk/cyclin motif in ciliate cell cycle regulation." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 2000. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape2/PQDD_0015/NQ56653.pdf.
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He, Tao. "Post-translational modifications of intermediate filament proteins : implications for cell signaling /." Åbo : Åbo Akademi University, 2003. http://catalogue.bnf.fr/ark:/12148/cb399327811.
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Liku, Muluye E. "CDK regulation of replication proteins: Mcm2-7 and DNA polymerase alpha primase." Diss., Search in ProQuest Dissertations & Theses. UC Only, 2008. http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&res_dat=xri:pqdiss&rft_dat=xri:pqdiss:3324598.
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Profant, Deborah Ann. "Defining the cis-acting requirements in the HMG-CoA reductase gene for karmellae biogenesis /." Thesis, Connect to this title online; UW restricted, 1999. http://hdl.handle.net/1773/5137.
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Blosco, John. "Using Receiver Squelch Techniques to Create Scalable Cellular Networks in Capacity Oriented IEEE 802.11 Deployments." University of Akron / OhioLINK, 2006. http://rave.ohiolink.edu/etdc/view?acc_num=akron1159976343.
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Rogers, David. "CIS-REGULATORY ANALYSIS OF THE PIGMENT CELL DIFFERENTIATION GENE POLYKETIDE SYNTHASE." Master's thesis, University of Central Florida, 2008. http://digital.library.ucf.edu/cdm/ref/collection/ETD/id/2701.
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The analysis of Gene Regulatory Networks (GRNs) is essential to understanding the complete process of embryo development. Elucidating every gene regulatory circuit from maternal regulatory inputs all the way to the activation of differentiation gene batteries is an important step in increasing our understanding of developmental biology. In this work I study the cis-regulatory architecture of a pigment cell differentiation gene, polyketide synthase (SpPks) in the sea urchin Strongylocentrotus purpuratus. SpPks encodes an enzyme that is responsible for the biosynthesis of the sea urchin pigment echinochrome in larval pigment cells. The analysis of the promoter of a differentiation gene will lead to identifying the direct upstream regulators and ultimately to elucidating the structure of the upstream gene regulatory network, which is mostly uncharacterized. From previous studies the transcription factors SpGcm and SpGatae are predicted to be positive regulators of SpPks. Here, I identify a minimal 1kb promoter region containing putative DNA-binding sites for both GCM and GATAE that is able to recapitulate the expression of SpPks. I further show by mutagenesis that a putative DNA-binding site for GCM located 1,179 base pairs upstream of the start of transcription is a direct target for the positive cis-regulation of SpPks. Quantitative analysis of the transcriptional regulatory function of the GCM-mutagenized construct suggests that GCM is not necessary for the start of SpPks transcription but is required for its maintenance. Several GATA E binding sites have been identified within the minimal promoter for SpPks by means of consensus sequence. My analysis suggests that GATA E may be a direct positive regulator and could potentially be required for the onset of transcription of SpPks, though further experimentation will be necessary to characterize the exact regulatory function of GATA E.M.S.
Department of Biology
Sciences
Biology MS
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Cot, Emilie. "Inhibition chimique des Cdk : mécanisme biochimiques et conséquences cellulaires." Thesis, Montpellier 2, 2010. http://www.theses.fr/2010MON20054.
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Les kinases dépendantes des Cyclines (Cdk) contrôlent le déroulement du cycle cellulaire, mais leur étude est difficile car des mécanismes de compensation se développent lorsqu'une Cdk est absente. Cdk2 est principalement impliquée dans la phase de réplication de l'ADN, cependant l'ablation génétique de Cdk2 chez la souris n'a pas d'effet sur leur développement: les fonctions de Cdk2 sont compensées par d'autres Cdk. L'inhibition chimique permet de bloquer une Cdk et de limiter les compensations. Pour étudier les rôles de Cdk2, nous l'avons inhibé avec NU6102 qui sélectif pour Cdk2 dans le modèle xénope. Nous avons aussi développé des mutants de Cdk2 résistants à NU6102 pour vérifier sa sélectivité et avons cherché à mieux comprendre les paramètres qui déterminent l'affinité entre Cdk2 et un ligand. D'autre part, nous déterminons in vitro que NU6102 serait plus sélectif pour Cdk2 que pour les autres Cdk chez l'humain, et avons décrit les phénotypes induits par cet inhibiteur dans les cellules humaines en cultures. Ces résultats ne permettent pas de confirmer la sélectivité de NU6102 mais montrent que NU6102 a des caractéristiques intéressantes pour être utilisé dans le traitement contre le cancer. L'activité des Cdk est essentielle à l'initiation de la réplication de l'ADN, mais aucun substrat essentiel n'a été identifié chez les métazoaires. Nous avons réalisé un crible des protéines chargées sur la chromatine en présence et en absence d'activité Cdk dans le modèle xénope, afin d'identifier des substrats qui pourraient être impliqués dans la réplication. Ces résultats suggèrent que l'activité Cdk, qui initie la réplication au niveau des origines de réplication de l'ADN, pourrait être impliquée dans d'autres fonctions cellulairesCycline Dependant Kinases (Cdk) control cell cycle progression. The study of their roles is often difficult because of functional redundancy; when a given Cdk is absent, others may compensate. The main role of Cdk2 in the cell cycle is in the initiation of DNA replication, but absence of Cdk2 is compensated for by Cdk1. For example, mice with a genetic knockout of Cdk2 are viable. The chemical inhibition of Cdks may limit compensation by other Cdks. Therefore, to study Cdk2 roles, we have studied chemical inhibition by NU6102, which seems to be selective for Cdk2 in the Xenopus model. To verify the selectivity and study parameters that determine selectivity, we have designed and produced mutants of Cdk2 which are resistant to NU6102, allowing restoration of function in the presence of inhibitor. Moreover, we demonstrate in vitro that NU6102 is selective for Cdk2 compared to other human Cdks, and we describe phenotypes induced by NU6102 in cultured cells, which are interesting in the light of potential applications of NU6102 in cancer chemotherapy. Cdk activity is essential for initiation of DNA replication, but in metazoans no essential substrates are known. To identify potential Cdk substrates during DNA replication, we have performed a proteomics screen of the proteins loaded onto chromatin in the presence or absence of Cdk activity, in the Xenopus model. The results suggest that Cdk activity is not only required for assembling DNA replication complexes onto origins of replication, but may also be implicated in other cellular functions
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Menoyo, Molins Alexandra. "Nutrient availability regulates cell cycle through a Pho85 CDK-dependent control of Cln3 cyclin stability." Doctoral thesis, Universitat Internacional de Catalunya, 2012. http://hdl.handle.net/10803/101414.
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Cell cycle control by trophic factors has a key role in regulation of cell proliferation in all organisms. Nutrients are one of these important factors needed by cells to reproduce, so very well regulated mechanisms must exist that connect nutrient availability to cell cycle. Hence the importance on studying how exactly nutrient-dependent signaling pathways work.
Cln3, the most upstream G1 cyclin in Saccharomyces cerevisiae, is one well demonstrated common effector of multiple nutrient-dependent signaling pathways. Moreover, its role in cell cycle is crucial. So it is a good candidate to regulate cell cycle progression in response to nutrient availability.
One important question is to find the protein that could directly modulate Cln3 levels in response to nutrient availability. This protein could play as a nutrient sensor and as a cell cycle regulator at the same time.
In the present thesis, Pho85 is founded to be the protein that could run these two highly different tasks, because of its well-characterized properties on sensing phosphate availability and the well-known functions on modulating cell cycle as CDK.
The results of the present work clearly demonstrate that when phosphate is present, Pho85 regulates Cln3 levels by increasing the stability of the cyclin through specific phosphorylations, promoting cell cycle progression. Contrary, under phosphate depletion conditions, Pho85 become inactive and Cln3 is rapidly degraded, leading to a cell cycle arrest in order to maintain cell chronological lifespan.El control del cicle cel•lular per factors tròfics té un paper important en la proliferació cel•lular de tots els organismes. Els nutrients són uns d’aquests factors importants requerits per les cèl•lules per reproduir-se, per tant deuen existir mecanismes molt ben regulats que connecten la disponibilitat de nutrients amb el cicle cel•lular. Per això, l’estudi de com funciona la senyalització cel•lular de nutrients i com afecta a la progressió del cicle és altament rellevant. Cln3, la ciclina de G1 més primerenca a Saccharomyces cerevisiae, és un efector comú de múltiples vies de senyalització de nutrients. A més, el seu paper en el cicle cel•lular és crucial. Per tant aquesta proteïna és una bona candidata per regular la progressió del cicle cel•lular en resposta a la disponibilitat de nutrients. Una qüestió important a resoldre és trobar la proteïna que podria modular directament els nivells de Cln3 depenent de la presència de nutrients. Aquesta proteïna actuaria com a sensor de nutrients i com a reguladora del cicle cel•lular alhora. A la present tesi, es mostra a Pho85 com la proteïna que pot fer aquestes dues tasques, tant per les seves propietats ben conegudes en la detecció de fosfat, com per les seves funcions de CDK modulant el cicle cel•lular. Els resultats d’aquesta tesi demostren clarament que quan el fosfat és present, Pho85 modula els nivells de Cln3 incrementant l’estabilitat de la ciclina mitjançant fosforilacions específiques, promovent la progressió del cicle cel•lular. Per altra banda, sota condicions de manca de fosfat, Pho85 esdevé inactiva i Cln3 és degradada ràpidament, conduint a un arrest del cicle cel•lular per mantenir la longevitat de la cèl•lula.
El control del ciclo celular por factores tróficos tiene un papel importante en la proliferación celular de todos los organismos. Los nutrientes son uno de estos factores importantes requeridos por las células para reproducirse, por lo tanto deben existir mecanismos muy bien regulados que conecten la disponibilidad de nutrientes con el ciclo celular. Por ello, el estudio de cómo funciona la señalización celular de nutrientes y cómo afecta a la progresión del cicle es altamente relevante. Cln3, la ciclina de G1 más temprana en Saccharomyces cerevisia, es un efector común de múltiples vías de señalización de nutrientes. Además, su papel en el ciclo celular es crucial. Por lo tanto esta proteína es una buena candidata para regular la progresión del ciclo celular en respuesta a la disponibilidad de nutrientes. Un tema importante a resolver es encontrar la proteína que podría modular directamente los niveles de Cln3 dependiendo de la presencia de nutrientes. Esta proteína actuaría como sensor de nutrientes y como reguladora del ciclo celular. En la presente tesis, se muestra a Pho85 como la proteína que puede hacer estas dos tareas, tanto por sus propiedades bien conocidas en la detección de fosfato, como por sus funciones de CDK modulando el ciclo celular. Los resultados de esta tesis demuestran claramente que cuando el fosfato está presente, Pho85 modula los niveles de Cln3 incrementando la estabilidad de la ciclina mediante fosforilaciones específicas, promoviendo la progresión del ciclo celular. Por otro lado, bajo condiciones de ausencia de fosfato, Pho85 es inactivada y Cln3 se degrada rápidamente, conduciendo a una parada del ciclo celular para mantener la longevidad de la célula.
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Kraus, Lea-Franziska [Verfasser]. "9-cis-retinoic acid modulates dendritic cell differentiation towards a regulatory T cell inducing phenotype / Lea-Franziska Kraus." Ulm : Universität Ulm, 2018. http://d-nb.info/1152324411/34.
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Petersen, Birgit Otzen. "Regulation of mammalian CDC6 by CDK phosphorylation and proteasome dependent degradation." Thesis, Open University, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.298212.
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Lefebvre, Calvin. "Cis-regulatory somatic mutations and gene-expression alteration in B cell lymphomas." Thesis, University of British Columbia, 2015. http://hdl.handle.net/2429/54666.
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Substantial progress has been achieved in characterizing protein coding (PC) regions for cancer genomes, with large contributions coming from The Cancer Genome Atlas (TCGA) and the International Cancer Genome Consortium (ICGC). In order to obtain a complete mutational profile of cancer genomes, the whole genome must be analyzed for two reasons: a large proportion of somatic mutations are within the non-coding region and 80% of the human genome is estimated to have some biological functionality. The dramatic cost reduction afforded by next generation sequencing has now made it tractable to sequence entire cancer genomes, allowing mutational profiling of the functional loci in the non-coding regions, such as cis-regulatory elements. Recent cancer genomic studies observed somatic mutations within cis-regulatory elements have the capacity to deregulate gene expression, but their impact remains underexplored. Initial attempts to prioritize cis-regulatory mutations did not incorporate RNASeq. We used 84 B cell lymphoma samples to address this limitation by prioritizing disruptive cis-regulatory mutations based on their potential to be the cause of observable cascading expression changes throughout biological networks. BCL6, ROBO1, GNA13, HAS2 and MYC were dysregulated genes targeted with somatic mutations through different mechanisms. Mutations either targeted the genes directly (PC mutations), indirectly (cis-regulatory mutations) or both. Our analyses demonstrates the importance of identifying genomically altered cis-regulatory elements, along with gene expression data, to interpret the mutational landscapes of cancers.Science, Faculty of
Graduate
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Bollu, Lakshmi Reddy. "The Effect of Endothelin-1 on the expression of CDK Inhibitors p21 & p27 in Bovine Corneal Endothelial Cells." TopSCHOLAR®, 2009. http://digitalcommons.wku.edu/theses/112.
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Mammalian corneal endothelial cells are considered to be non-proliferative due to the arrest of cells at the G1 phase of the cell cycle. The purpose of this study was to determine whether the down regulation of cyclin dependant kinase inhibitors (p21cip1 and p27kip1) levels by Endothelin-1 (ET-1), would overcome the G1 phase arrest and promote cell cycle progression and proliferation in cultured BCECs (Bovine corneal endothelial cells). BCECs were isolated from bovine corneas and cultured in DMEM supplemented with 10% serum. 5-Bromo 2-deoxy Uridine (BrdU) incorporation was determined in serum starved cultures in 24-well plates as a measure of cell proliferation. Confluent serum starved cells grown in T-25 flasks were treated with 100nM Endothelin-1 in DMEM. The control cells were left untreated in serum free medium. Total cellular protein was isolated using RIPA buffer and quantified according to the Peterson modification of the Lowry method. The level of expression of p21cip1 and p27kip1 proteins relative to β-actin was determined by western blotting technique. Immuno fluorescent localization of p27kip1 was performed using polyclonal anti-p27kip1 and anti-p21cip1 antibodies in confluent and growing cells. An increase in cell proliferation was observed in sub-confluent cultures with Endothelin-1 treatment. This evidence was supported by an increase (~18%) in BrdU incorporation in response to Endothelin-1. Densitometry analysis of immunoblots revealed an increase in the expression of p27kip1 in confluent cell cultures when compared to sub-confluent, dividing cells. p21cip1 was almost undetectable in sub-confluent, actively dividing cultures. Immuno fluorescent analysis revealed that the nuclear staining of p27kip1 was apparently decreased with ET-1 treatment. In conclusion, Endothelin-1 treatment resulted in decrease in p27kip1 and p21cip1 expression in confluent cultures that was greatest at 30 hr of post incubation with Endothelin-1. Endothelin-1 appears to promote cell proliferation. Expression of p27kip1 and p21cip1 was greatly reduced in actively dividing BCECs. Endothelin-1 treatment down-regulated these cyclin dependent kinase inhibitors and may promote cell cycle progression via this mechanism.
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Ramos, Rodríguez Mireia. "β-cells cis-regulatory networks and type 1 diabetes." Doctoral thesis, Universitat de Barcelona, 2020. http://hdl.handle.net/10803/672192.
Full textAbstract:
Type 1 Diabetes (T1D) is a celltargeted autoimmune disease, leading to a reduction in pancreatic cell mass that renders patients insulindependent for life. In early stages of the disease, cells from the immune system infiltrate pancreatic islets in a process called insulitis. During this stage, a crosstalk is established between cells in the pancreatic islets and the infiltrating immune cells, mediated by the release of cytokines and chemokines. Studying the gene regulatory networks driving cell responses during insulitis, will allow us to pinpoint key gene pathways leading to cell lossoffunction and apoptosis, and also to understand the role cells have in their own demise. In the present thesis, we used two different cytokine cocktails, IFN and IFN + IL1, to model early and late insulitis, respectively. After exposing cells and pancreatic islets to such proinflammatory cytokines, we characterized the changes in their chromatin landscape, gene networks and protein profiles. Using both models, we observed dramatic chromatin remodeling in terms of accessibility and/or H3K27ac histone modification enrichment, coupled with upregulation of the nearby genes and increased abundance of the corresponding protein. Mining gene regulatory networks of cells exposed to IFN revealed two potential therapeutic interventions which were able to reduce interferon signature in cells: 1) Inhibition of bromodomain proteins, which resulted in a downregulation of IFNinduced HLAI and CXCL10 expression; 2) Baricitnib, a JAK1/2 inhibitor, which was able to reduce both IFNinduced HLAI and CXCL10 expression levels and cell apoptosis. In
cells exposed to IFN + IL1, we were able to identify a subset of novel
regulatory elements uncovered upon the exposure, which we named Induced Regulatory Elements (IREs). Such regions were enriched for T1Dassociated risk variants, suggesting that cells might carry a portion of T1D genetic risk. Interestingly, we identified two T1D lead variants overlapping IREs, in which the risk allele modulated the IRE enhancer activity, exposing a potential T1D mechanism acting through cells. To facilitate the access to these genomic data, together with other datasets relevant for the pancreatic islet community, we developed the Islet Regulome Browser (http://www.isletregulome.org/), a free web application that allows exploration and integration of pancreatic islet genomic data.
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Hauck, Fabian. "Primary T cell immunodeficiencies associated with disturbed proximal T cell receptor signalling caused by human autosomal recessive LCK, ZAP-70 and ITK-mutations." Phd thesis, Université René Descartes - Paris V, 2013. http://tel.archives-ouvertes.fr/tel-00914375.
Full textAbstract:
T lymphocytes express either a preTCR, or a clonotyoic γδ TCR or αβ TCR together with the CD3-complex and the associated ζ-chain. TCR:CD3:ζ-signalling is crucial for T cell development and antigen-specific activation including proliferation, differentiation, effector functions and apoptosis of mature T cells. Protein tyrosine kinase (PTK) cascades lie at the heart of proximal TCR:CD3:ζ-signalling. The CSK-, SRC-, SYK- and TEC-family members C-terminal SRC kinase (CSK), lymphocyte-specific protein tyrosine kinase (LCK), ζ-chain associated protein tyrosine kinase of 70 kDa (ZAP-70) and interleukin-2-inducible T cell kinase (ITK), respectively, are the major T cell players. After TCR:CD3:ζ-complex triggering, activation of PTKs result in tyrosine phosphorylation signals. These include phosphorylation of immunoreceptor tyrosine-based activation motifs (ITAMs) of the CD3 and ζ-chains, adaptor proteins that nucleate the proximal LAT:SLP-76-signalosome controlling almost all TCR:CD3:ζ-induced signalling events. These events initiate Ca2+-flux, activation of mitogen-activated protein kinases (MAPKs), activation of nuclear factor of kappa light polypeptide gene enhancer in B-cells (NF-κB), activation of nuclear factor of activated T cells (NFAT) and activator protein 1 (AP-1) as well as actin reorganization, cell-adhesion and motility.Througout the last five decades, the immune system has been extensively investigated in vitro and in animal models such as the murine system. Additionally, studying and taking care of human primary immunodeficiency diseases (PIDs) has been seminal for our understanding of the human immune system as animal models not always recapitulates the subtleties found in men.In my doctoral thesis I report the first case of autosomal recessive human LCK-deficiency, a novel autosomal recessive mutation leading to human ZAP-70-deficiency and a novel autosomal recessive mutation leading to human ITK-deficiency. I provide detailed clinical, immunological and biochemical analyses especially of TCR:CD3:ζ-signalling and compare my findings to the well-established Lck-/-, Zap-70-/- and Itk-/- murine models.
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Northen, Julian S. "Design of novel pyrimido[5,4-d]pyrimidine cyclin dependent kinase (cdk) inhibitors." Thesis, University of Newcastle Upon Tyne, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.391320.
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Samuelsson, Magnus. "p57Kip2, a glucocorticoid-induced CDK inhibitor, involved in cell proliferation, apoptosis and differentiation /." Stockholm, 2003. http://diss.kib.ki.se/2003/91-7349-382-1/.
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Ofori-Acquah, Solomon Fiifi. "Molecular basis for CIS regulation of fetal haemoglobin expression in sickle cell disease." Thesis, King's College London (University of London), 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.324882.
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Stark, Azadeh T. "Cis linoleic acid, a potential biomarker for squamous cell carcinoma of the skin." Diss., The University of Arizona, 1994. http://hdl.handle.net/10150/186976.
Full textAbstract:
A population based case-control study was designed to test the hypothesis that cases of squamous cell carcinoma (SCC) had lower concentration of cis linoleic (LA) in their red blood cell membranes and it would remain a predictor of SCC after adjusting for other risk factors. The probability of LA as a potential biomarker for SCC was investigated because it is the precursor of arachidonic acid (AA), a secondary messenger for proteins involved in cell growth and proliferation. Ras oncogene perpetually activates the two phospholipases A-2 and C, causing release of AA from the membrane phospholipids. Red blood cell membranes were used as the medium for assessing the concentration of cis linoleic acid because donation of blood was well accepted by the participants. Tissue availability, and the low cost of operation were the other factors to utilize red blood cell membranes. Cases who had confirmed pathological diagnosis of SCC between December 1991-October 1994 were identified and consecutively sampled from the Arizona Skin Cancer Registry. Controls were recruited from the same geographic area using a random digit dialing technique. Subjects, limited to White-Anglo European descendent, were frequency matched by their age (± 5 years). Participants were screened for use of steroids, prescribed non-steroid anti-inflammatory drugs and history of chronic illness. Data on sun exposure habits, medication usage, lifestyle habits, and family history of SCC were collected during a personal interview. A 15 cc blood sample was collected. The composition of fatty acids in the red blood cell membranes was analyzed using gas-liquid chromatography techniques. Statistical analysis revealed that concentration of LA was significantly lower in the red blood cell membranes of the cases than the controls. However, cases had higher concentration of linolenic and trans linoleic acids. The concentration of 17 other fatty acids were equal, indicating that the general dietary pattern of cases and controls were similar. Use of logistic regression statistics showed that LA remained a significant independent risk factor for SCC (OR = 2.83, 95% Cl = 1.38-5.97) after adjusting for other risk factors. Based on this preliminary research, it is cautiously suggested that LA could be used as a potential biomarker of SCC.
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Yabuta, Paula Barbim Donate. "O papel dos microRNAs de células T na susceptibilidade/resistência a artrite reumatóide experimental." Universidade de São Paulo, 2012. http://www.teses.usp.br/teses/disponiveis/17/17147/tde-16022012-150623/.
Full textAbstract:
Os microRNAs são pequenos RNAs, não-codificantes que funcionam como reguladores a nível pós-transcricional da expressão gênica. Nos últimos anos, novas evidências demonstram o papel importante dos microRNAs na regulação e desenvolvimento do sistema imune. Apesar da função de poucos microRNAs ser conhecida, a sua expressão alterada vêm sendo associada a patogênese de diversas doenças autoimunes, incluindo a artrite reumatóide (AR). Recentemente a expressão desregulada de uma série de microRNAs está sendo descrita em pacientes com AR, e o papel patogênico de apenas uma parte deles foi investigada em modelos animais. A artrite reumatóide é uma doença autoimune sistêmica caracterizada por um intenso processo inflamatório na sinóvia, podendo causar destruição óssea e articular. Os linfócitos T apresentam papel importante na indução, manutenção e progressão da doença. A artrite induzida por colágeno é um modelo animal amplamente utilizado por suas características fisiopatológicas muito similares à doença em humanos. A linhagem de camundongos DBA-1/J desenvolve a doença após imunização e booster com colágeno do tipo II, enquanto que a linhagem DBA-2/J se mostra refratária. Isso confere um sistema modelo de susceptibilidade/resistência à artrite, que pode ser estudado em diferentes abordagens. O objetivo do nosso estudo foi identificar o perfil transcricional e as redes de interação entre um grupo de microRNAs e seus respectivos alvos nos timócitos e linfócitos T CD3+ periféricos nos camundongos da linhagem DBA-1/J e DBA-2/J. Para a avaliação da expressão gênica, utilizou-se a tecnologia de microarrays. O uso de programas de análise e para a construção das redes foi imprescindível. Os resultados encontrados evidenciam uma expressão diferenciada de mRNAs e microRNAs em timócitos e linfócitos T CD3+ periféricos entre as duas linhagens utilizadas. Novos microRNAs foram encontrados nos diferentes estágios de desenvolvimento do linfócito T. Nas redes de interação microRNA-RNAm obtidas, genes importantes associados aos processos de sistema imune, adesão e diferenciação celular, apoptose, recombinação, ativação de linfócitos T e resposta inflamatória, foram encontrados como potenciais alvos. Além disso, em uma perspectiva clínica, baseados nos resultados obtidos em camundongo, nos encontramos a expressão do miR-505 nos linfócitos T de pacientes com AR. Nossos resultados contribuem para a melhor compreensão dos mecanismos molecular envolvidos na resistência/susceptibilidade a CIA.MicroRNAs (miRNAs) are small non-coding RNA molecules that modulate the expression of multiple protein-encoding genes at the post-transcriptional level. During the last several years, evidence has emerged to show their critical role for the regulation and development of immune system. Although the function of most mammalian miRNAs has yet to be determined, their aberrant expression has been associated with several autoimmune conditions, including rheumatoid arthritis (RA). Recently, the deregulated expression of a dozen miRNAs has been reported in patients with RA, and the pathogenic role of only a few of these has been investigated in experimental mouse models. RA is a systemic autoimmune disorder mainly characterized by the inflammation of synovial tissue that can lead to destruction of bone and cartilage. The role of effectors T cells in induction, maintenance and progression of the disease is now becoming better understood. Collagen-induced arthritis is an animal model widely studied due to its similarities to human disease. The DBA-1/J mouse strain develops arthritis after immunization process and booster with Type II collagen, and the DBA-2/J strain is refractory to the disease induction. This offers an useful susceptibility/resistance model-system to study RA. The aim of this study was to identify the expression profiles and interaction networks between a set of microRNAs and their mRNA targets in thymocytes and peripheral CD3+ T lymphocytes in DBA-1/J and DBA-2/J mice strain. For this purpose we used the microarray technology to evaluate the expression of the miRNAs and mRNAs as possible targets involved in this process. The use of bioinformatics software to reconstruct the networks was essential. The results show differential expression of mRNAs and miRNAs in thymocytes and peripheral CD3+ T lymphocytes between both strains. New miRNAs were found during all the stages of T cells development. The microRNA-mRNA interaction networks obtained in this study showed that important genes related to apoptose, immune system, recombination, cell adhesion and differentiation, inflammatory process and T cell activation were found as potential targets. In addition, in a clinical prospects, based on the results obtained in mice, we found the expression of the miRNA miR-505 in T cells of RA patients. Our results contribute to a better understand of the molecular mechanisms evolved in the resistance/susceptibility to CIA.
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Bockstaele, Laurence. "Réévaluation de la régulation de l'activité de la CDK4, kinase dépendante des cyclines D, clé de l'engagement dans le cycle cellulaire: rôle de l'inhibiteur p27." Doctoral thesis, Universite Libre de Bruxelles, 2006. http://hdl.handle.net/2013/ULB-DIPOT:oai:dipot.ulb.ac.be:2013/210626.
Full textAbstract:
La progression à travers le cycle cellulaire est assurée par l’activation séquentielle d’une série de complexes cycline-CDK. Les complexes cycline D-CDK4 assurent la progression au cours de la phase G1 du cycle cellulaire en phosphorylant les protéines « antioncogéniques » de la famille Rb. L’activation de la CDK4 nécessite son association à une cycline D et sa phosphorylation sur Thr172 par la CAK nucléaire (CDK activating kinase). Le rôle essentiel des protéines de la famille Cip/Kip dans la régulation de l’activité de ces complexes reste controversé. Les protéines de cette famille (comprenant p27 et p21) ont initialement été identifiées comme des inhibiteurs puissants des complexes cycline-CDK et comme les intermédiaires de l’action antimitogénique de différents signaux intra ou extra-cellulaires. Il a été proposé que la liaison de la p27 ou de la p21 aux complexes cycline D-CDK4 empêche leur phosphorylation activatrice par la CAK et leur activité pRb kinase. Cependant, l’observation que des complexes cycline D-CDK4 associés à p21/p27 possèdent une activité pRb kinase a donné naissance à une seconde hypothèse sur la régulation de ces complexes par la p27 ou la p21. Ces « inhibiteurs » ont été paradoxalement proposés comme facteurs nécessaires et suffisants d’assemblage et de localisation nucléaire des complexes cycline D-CDK4. Dans le modèle physiologiquement relevant des thyrocytes de chien en culture primaire, la mitogénèse dépendante de l’AMPc activée par la TSH diffère des cascades des facteurs de croissance puisqu’elle n’induit pas les cyclines D mais au contraire augmente l’accumulation de l’«inhibiteur» de CDK p27. L’AMPc stimule l’assemblage des complexes cycline D3-CDK4, leur translocation nucléaire et leur association à p27. Dans ce modèle, le TGF&61538; inhibe la mitogénèse dépendante de l’AMPc en inhibant la translocation nucléaire des complexes cycline D3-CDK4 et leur association à la p27.Nous avons étudié l’activité catalytique et l’activation des complexes cycline D3-CDK4-p27 issus des thyrocytes de chien en culture primaire ou produits en cellules d’eucaryotes supérieurs (CHO et Sf9). Nous avons pu montrer que les complexes cycline D3-CDK4-p27 issus des thyrocytes de chien stimulés par la TSH présentent une activité pRb-kinase qui est inhibée par le TGF&61538; En outre, la production des complexes cycline D3-CDK4-p27 en cellules CHO ou Sf9 nous a permis de montrer que l’impact de la p27 sur l’activité catalytique des complexes cycline D3-CDK4 dépend de sa stoechiométrie de liaison à ces complexes. L’analyse du profil de séparation par électrophorèse bidimensionnelle de la CDK4 issue de ces trois systèmes montre que la p27 n’empêche pas la phosphorylation activatrice de la CDK4, même aux concentrations de p27 qui empêchent l’activité pRb kinase du complexe cycline D3-CDK4. Nous avons également montré dans les cellules CHO que la p27 détermine la localisation nucléaire des complexes cycline D3-CDK4, ceux-ci étant relocalisés dans le cytoplasme par la transfection d’un mutant de la p27 dépourvu de son signal de localisation nucléaire. Ces résultats valident les observations réalisées par immunofluorescence dans les thyrocytes de chien dans lesquels nous avons mis en évidence une étroite corrélation au niveau des cellules individuelles stimulées par la TSH entre la translocation nucléaire de la CDK4 et l’apparition de la p27 nucléaire. Cette colocalisation est partiellement inhibée par le TGF&61538; Ces observations renforcent l’hypothèse d’un rôle de la p27 dans l’ancrage nucléaire des complexes cycline D3-CDK4.
Alors que la localisation de la CAK est considérée comme exclusivement nucléaire et son activité catalytique constitutive, nous avons pu montrer que la phosphorylation activatrice de la CDK4 associée à la cycline D3 n’est pas affectée par sa localisation sub-cellulaire et qu’elle est régulée par le TGF&61538; dans les thyrocytes de chien et par le sérum dans les cellules T98G indépendamment de l’association de la CDK4 à la p27. De plus, la phosphorylation de la CDK4 sur Thr172 dans les cellules T98G est stimulée par le sérum, alors que la phosphorylation activatrice de la CDK6, son homologue fonctionnel, ne l’est pas. La comparaison de la séquence de ces deux CDKs à proximité des Thr phosphorylées (Thr177 pour la CDK6) révèle, outre une forte similarité de séquence, une différence au niveau de l’acide aminé situé en aval de la thréonine :il s’agit d’une proline dans la CDK4 et d’une sérine dans la CDK6. La mutation P173S de la CDK4 abolit la phosphorylation sur Thr172 et l’activité de la CDK4 associée à la cycline D3 dans les cellules CHO, mais n’affecte pas la phosphorylation et l’activation de la CDK4 par la CAK recombinante in vitro. L’ensemble de ces résultats suggère que la/les CAKs régulée(s) responsables de l’activation de la CDK4 n’ont pas encore été identifiées et que la proline située en aval de la Thr172 de la CDK4 est essentielle pour sa phosphorylation activatrice et son activité pRb kinase.
Doctorat en sciences biomédicales
info:eu-repo/semantics/nonPublished
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Chen, Jian 1969. "Regulation of CAK activity of Cdk7 in Drosophila melanogaster." Thesis, McGill University, 2003. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=82842.
Full textAbstract:
Cdk7 (Cyclin-dependent kinase 7) is conserved from yeast to human and involved in multiple functions. Cdk7 acts as a CAK (Cdk activating kinase) in a trimeric complex with Cyclin H and Mat1. The CAK activity is required for the full activation of the Cdks that directly regulate the cell cycle transitions. In addition, Cdk7 is the kinase subunit of TFIIH, the general transcription/DNA repair factor IIH. TFIIH is required for the general transcription of messenger RNAs by RNA polymerase II and for the transcription-coupled nucleotide excision repair functions. As in other systems, Drosophila Cdk7 has multiple functions. In order to understand how different functions of Cdk7 are regulated, I performed genetic screens to identify the regulators or downstream factors of multiple functions of Cdk7. Several candidate dominant suppressors and enhancers were identified in these screens. One strong suppressor of cdk7, xpd, encodes another subunit of TFIIH. The genetic suppression by xpd attracted me to further characterize the biological significance of this interaction. I showed that Xpd does have a novel function in regulating CAK activity of cdk7 , it down-regulates mitotic CAK activity. Furthermore, I found that Xpd protein levels are cell cycle dependent, being down-regulated at the beginning of the mitosis. Based on these data, I propose a model that mitotic down-regulation of Xpd results in increased CAK activity, positively regulating mitotic progression. Simultaneously, this down-regulation can be expected to contribute to the mechanisms of mitotic silencing of basal transcription.
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McClain, David Alan. "Increasing IFN-[gamma] productivity in CHO cells through CDK inhibition." Thesis, Massachusetts Institute of Technology, 2010. http://hdl.handle.net/1721.1/60138.
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Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemical Engineering, 2010.In title on title-page, "[gamma]" appears as the lower case Greek letter. Cataloged from PDF version of thesis.
Includes bibliographical references (p. 179-190).
Approximately 60-70% of all recombinant human glycoproteins are produced in Chinese Hamster Ovary (CHO) cells. Production in CHO cells, however, is often plagued by low productivity when compared with other host cell lines, including bacteria and yeast. For this reason, investigating ways of improving the productivity of CHO cells producing recombinant proteins has been an active area of research for many decades. The induction of growth arrest is one such area that shows particular promise. Through the use of siRNA and chemical cyclin dependent kinase (CDK) inhibitors, we have developed new methods to improve and better understand recombinant protein production during growth arrest. In this study, we have shown that the specific inhibition of the CDK2-CcnE complex through chemical inhibition leads to growth arrest and a subsequent increase in specific productivity. In addition, we have shown that the knockdown of CcnEl alone leads to increases in specific productivity. With the advent of improved shRNA expression systems, we believe that the targeted knockdown of CcnE1 has the potential to induce growth arrest and improve total recombinant protein production The relationship between growth-arrested cell cycle phase and productivity is very poorly understood. In this work, we have used various CDK inhibitors to better understand the relationship between growth-arrested cell cycle phase, specific growth rate, and productivity. We have shown that increases in specific productivity are cell-cycle independent following growth arrest induced by CDK inhibition. Instead, specific productivity increases correlate strongly with a decreasing specific growth rate. Lastly, in this work, we have identified an interesting CDK2 inhibitor that inhibits mitosis and induces a subsequent growth arrest. Following its addition, we observe a decrease in specific growth rate, an increase in DNA content, and a drastic increase in the specific productivity of a recombinant protein (IFN-[gamma]). We used this inhibitor to increase total IFN-[gamma] productivity by 73% in a modified batch culture. With the development of an optimized feed medium, we believe that this CDK2 inhibitor could also be used to increase recombinant protein production in fed-batch cultures.
by David Alan McClain.
Ph.D.
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Richard, Stéphane. "Identification of Cis-acting elements in the human oxytocin gene promoter mediating cell-specificity, estrogen stimulation, and retinoic acid induction." Thesis, McGill University, 1991. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=70214.
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Oxytocin (OT) is a nonapeptide produced in hypothalamic neurons. This neuropeptide hormone plays important roles in the physiology of reproduction. The gene encoding OT has been cloned in several species including human, rat, cow and, most recently, mouse. Although the gene sequences were known for some time, no information about the mechanisms regulating the OT gene promoter in any of the species has become available. This has been due, in part, to the absence of a cellular system expressing the endogenous OT gene. As a first step, the present thesis research was oriented towards the identification of a suitable cellular system for the investigation of OT gene expression. The cell line which responded to these needs was the mouse neuroblastoma cell line, Neuro-2a. In these cells, the transfected human OT gene promoter was highly active as opposed to all the other neuronal and non-neuronal cell lines tested. Using this system, we began investigating the molecular mechanisms underlying hormone induced and cell-specific expression of the OT gene. First, we determined that the OT gene promoter is regulated by estrogens. By 5$ sp prime$ and 3$ sp prime$ deletion analyses, we identified an estrogen response element (ERE) at position $-$164 to $-$152 which bears resemblance to the ERE consensus sequence. This ERE is necessary and sufficient for mediating the estrogen induction of the human OT gene promoter. The sequence of the ERE is GGTGACCTTGACC, and the underlined nucleotide represents the deviation from the perfectly palindromic ERE consensus sequence.By testing the effect of other members of the steroid hormone receptor family on the OT gene promoter, we found that the promoter responded strongly to retinoic acid (RA). Mapping of the RA response element (RARE) showed that four direct repeats of TGACC motifs were required for optimal RA induction. Interestingly, two of these TGACC repeats are contained within the ERE, whereas the other TGACC repeats are located downstream of the ERE. When the estrogen and RA treatments were performed simultaneously, additive and interfering interactions were observed. Additive effects resulted when the ERE and RARE were intact in the human OT promoter. By contrast, RA repressed estrogen induction by 50% when one of the two downstream TGACC repeats was mutated. Therefore, we speculate that in the latter case, the RA receptor forms in the presence of RA, an inactive complex with the two TGACC motifs that overlap the ERE and thus, prevents the estrogen receptor from forming a transcriptionally active complex with the ERE.
The specific expression of the human OT promoter in Neuro-2a cells was shown to involve three cell-specific DNA elements located in the proximal promoter region between $-$49 to +36. These cell-specific elements, referred to as PPE 1, 2 and 3, are crucial for the elevated baseline expression observed in Neuro-2a cells. All these three elements contain highly conserved regions among species. Of note, PPE 2 contains the purine rich sequence GAGAGA which resembles the binding site for the GAGA factor and for the transcription factor PU.1. Each of three proximal promoter elements bound a specific factor(s) from Neuro-2a nuclear extracts, as assayed by gel-retardation. Moreover, PPE 1, 2 and 3 could synergize with the ERE or with the human metallothionein II$ sb{ rm A}$ glucocorticoid response element.
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González, Pérez Laura. "Role of the atypical CDK activator RINGO beyond meiosis." Doctoral thesis, Universitat de Barcelona, 2019. http://hdl.handle.net/10803/668658.
Full textAbstract:
The cell cycle is orchestrated by the periodic activation of Cyclin-dependent kinases (CDKs). The enzymatic activity of CDKs depends on their association with cyclins, however, in some cases these kinases can be activated by non-cyclin proteins. RINGO is an atypical CDK activator which regulates the meiotic maturation of Xenopus oocytes and has been recently described as essential for meiotic prophase in mouse. As an activator of CDKs, RINGO has a potential role in cell cycle regulation. CDK regulation by RINGO has been extensively studied in vitro. However, the implication of RINGO in particular cellular processes is poorly understood. Moreover, very little is known about RINGO functions in vivo beyond meiosis.
This thesis has addressed the functional relevance of mammalian RINGO proteins in somatic cells, both during homeostasis and in cancer. We have characterized the effects of RingoA knock-down in human cells and found changes in cell cycle progression as well as reduced cell viability. In an attempt to reveal the RingoA interactome, an unbiased proteomic approach was used, which allowed the identification of the cohesin complex and ANKRD11 as new RingoA interactors. Moreover, we describe the expression of endogenous RingoA during the cell cycle of human cells and show that it is present in nuclear speckles. The study of RingoA expression in vivo using a reporter system and gene expression analysis pointed to the brain as the somatic tissue where RingoA is most expressed. We have also analyzed genetically modified mice and found that RingoA and RingoB are not essential for somatic tissue homeostasis. Nevertheless, RingoA is expressed in the sub-ventricular zone of the adult mouse brain and is important for neural stem cell self-renewal ex vivo. Finally, using the Polyoma middle T mammary tumorigenesis model, we showed that RingoA and RingoB are required for tumor growth.El cicle cel·lular és orquestrat per l’activació periòdica de les quinases dependents de ciclines (CDKs). L’activitat enzimàtica de les CDKs depèn de la seva associació amb ciclines, no obstant hi ha excepcions on aquestes quinases poden ser activades per proteïnes no englobades en la família de les ciclines. RINGO n’és un exemple; aquesta proteïna és un activador atípic de CDKs que regula la maduració meiòtica dels oòcits de Xenopus. A més, recentment també s’ha descrit com a essencial en la profase meiòtica i progrés meiòtic en ratolins. RINGO, com a activador de CDKs, té un rol potencial en la regulació del cicle cel·lular. La regulació d’aquestes quinases per RINGO s’ha estudiat en detall in vitro però poc se sap de la implicació de RINGO en processos cel·lulars. A més no se sap gairebé res de la funció de RINGO in vivo més enllà de la meiosi. En aquesta tesi s’estudia la rellevància funcional de les proteïnes RINGO de mamífers en cèl·lules somàtiques, durant condicions homeostàtiques i càncer. S’han caracteritzat els efectes del knock-down de RingoA en cèl·lules humanes i trobat canvis en la viabilitat i cicle cel·lular d’aquestes. Amb l’objectiu de revelar l’interactoma de RINGO, s’ha utilitzat un cribratge de proteòmica que ha permès la identificació del complex de cohesines i la proteïna ANKRD11 com interactors de RingoA. A més, s’ha descrit l’expressió de RingoA durant el cicle cel·lular de cèl·lules humanes i descobert que està present en nuclear speckles. L’estudi de l’expressió de RingoA utilitzant un sistema reporter i l’anàlisis de l’expressió gènica ha permès la identificació del cervell com el teixit somàtic amb més expressió de RingoA. Mitjançant l’estudi de models de ratolí modificats genèticament s’ha demostrat que RingoA i RingoB no són essencials per la homeòstasi de teixits somàtics. No obstant, RingoA s’expressa en la zona sub-ventricular del cervell adult i és important per la renovació de cèl·lules mare ex vivo. Per últim, utilitzant el model tumoral Polyoma middle T, que permet la generació de tumors mamaris en ratolí, s’ha demostrat que RingoA i RingoB són importants en el creixement tumoral.
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Prahalad, Priya. "9-cis-retinoic acided mediated endothelial transdifferentiation in breast cancer cells." Connect to Electronic Thesis (CONTENTdm), 2008. http://worldcat.org/oclc/645463979/viewonline.
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Gibson, Kathryn. "The pathogenic potential of endogenous-self reactive CD4 T cells in collagen-induced arthritis." Thesis, King's College London (University of London), 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.313722.
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Carballar, Ruiz María de los Reyes. "CDK-mediated phosphorylation of Yku80 and its role in DNA repair." Doctoral thesis, Universitat Internacional de Catalunya, 2021. http://hdl.handle.net/10803/671096.
Full textAbstract:
DNA double-strand breaks (DSBs) are considered the most deleterious lesions of DNA and they are
repaired by means of two main mechanisms: homologous recombination (HR) and non-homologous endjoining (NHEJ). These mechanisms are regulated throughout the cell cycle being HR restricted to the S/G2
phases while NHEJ is active during the different phases of the cell cycle. Here I presented evidence of
NHEJ regulation by CDK phosphorylation of one of the key proteins of the NHEJ repair pathway, Yku80.
Yku80 is phosphorylated both in vitro and in vivo by the CDK Pho85 in association with the G1 cyclin Pcl1.
A non-phosphorylatable version of Yku80 (yku80-S623A) shows increased NHEJ activity, reduced HR
events and higher sensitivity upon bleomycin treatment, a DSB-inducing agent, specifically when DNA
damage was induced during the G2 phase of the cell cycle. Interestingly, the overexpression of the nonphosphorylatable version of human Ku80 (Ku80T629A) increased bleomycin sensitivity in different cancer
linessuggesting Ku80 phosphorylation and its role in DSB repair regulation might be conserved. Thus, the
results presented in this work provide evidence of a new mechanism to regulate DSB repair pathway
choice by CDK-mediated phosphorylation of Yku80.
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Gunaicha, Purnaansh Prakash. "Optical Modeling of Solar Cells." University of Toledo / OhioLINK, 2012. http://rave.ohiolink.edu/etdc/view?acc_num=toledo1344815193.
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Kurzawa, Laetitia. "Développement de biosenseurs peptidiques fluorescents pour la détection des Cdk-cyclines dans les cellules vivantes." Thesis, Montpellier 2, 2011. http://www.theses.fr/2011MON20086.
Full textAbstract:
Chez les eucaryotes supérieurs, la progression ordonnée du cycle cellulaire est régie par une dizaine de kinases Cdk-cyclines. Les altérations génétiques ou épigénétiques impliquant des oncogènes ou des gènes codant pour des suppresseurs de tumeurs sont souvent associées à l'expression ou l'activation aberrante des Cdks, favorisant ainsi la prolifération cellulaire incontrôlée et notamment le développement de cancers. Malgré la pertinence oncogénique et thérapeutique de ces protéines, leur détection est restée jusqu'à présent limitée à des méthodes indirectes et invasives. Dans ce contexte, mes travaux de thèse ont permis de développer un biosenseur peptidique fluorescent permettant de reconnaître spécifiquement les Cdk-cyclines. Associé à une stratégie de vectorisation non invasive basée sur l'utilisation de peptides vecteurs pénétrants, le biosenseur a été délivré efficacement dans les cellules. La mise au point d'une quantification ratiométrique du signal a par ailleurs permis d'évaluer l'abondance relative des Cdk-cyclines endogènes. Deux variants plus spécifiques de certains complexes ont pu être développés. Enfin, d'autres versions du biosenseur ont quant à elles permis d'évaluer sa biodistribution in vivo et de mettre au point un essai cellulaire en vue d'un criblage de petites molécules ayant un effet sur l'abondance relative des Cdk-cyclinesCdk-cyclins represent key regulators of cell cycle progression among superior eukaryotes. Genetic and epigenetic alterations involving oncogenes or tumor suppressor genes are often associated with aberrant expression or activation of Cdks, leading to the sustained proliferation of cells and by the way to the development of cancers. Despite the oncogenic and therapeutic relevance of these proteins, their detection has so far remained limited to indirect and invasive methods. My Ph.D. thesis work aimed in this context at developing peptidic fluorescent biosensors that specifically recognize Cdk-cyclins. Combined to cell-penetrating peptides, the biosensor was efficiently delivered into cells. Following the development of the signal ratiometric quantification, the relative abundance of endogenous Cdk-cyclins was directly evaluated in living cells. Two other variants, that are more specific towards specific Cdk-cyclin complexes, were also designed. Finally, the development of novel versions of the biosensor allowed us to evaluate its biodistribution in vivo and to set up a cell-based assay to screen small molecules having an effect on Cdk-cyclin relative abundance
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Ghazzaui, Nour. "Eléments cis-régulateurs du locus IgH et lymphomagenèse B." Thesis, Limoges, 2018. http://www.theses.fr/2018LIMO0058/document.
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Le locus des chaînes lourdes d’immunoglobulines (IgH) subit trois processus de remaniements géniques durant la lymphopoïèse B. Ces événements induisent des cassures de l’ADN potentiellement oncogéniques, d’où la nécessité d’une régulation extrêmement stricte. Ceci est dû aux deux principaux éléments cis-régulateurs du locus IgH. L’enhancer 5’Eµ régule les recombinaisons VHDJH qui établissent un répertoire antigénique fonctionnel lors des phases précoces. La région régulatrice en 3’ (3’RR) est essentielle aux hypermutations somatiques (SHM) et à la recombinaison de classe (CSR) aux stades tardifs, modifiant respectivement, l’affinité et les fonctions effectrices de l’Ig. La plupart des lymphomes B matures portent les stigmates de translocations d’oncogènes au locus IgH. Le but de ma thèse a été de mieux comprendre les interactions transcriptionelles entre les enhancers Eµ et 3’RR et évaluer si le ciblage de cette dernière pourrait se révéler une approche thérapeutique potentielle. Nous avons démontré que la 3’RR est l’élément essentiel qui contrôle la transcription du locus IgH dans les lymphocytes B matures. Elle est dispensable lors des phases initiales (recombinaisons VHDJH), mais agit comme silencer sur l’expression des segments DJH. L’analyse de la lymphomagenèse dans trois modèles murins porteurs d’une insertion de Myc en trois points du locus IgH a montré des différences dans les cinétiques d’émergence des lymphomes, leurs phénotypes et index de prolifération. L’effet de la 3’RR sur l’oncogène est suffisant pour l’émergence de lymphomes B. Son absence ne semble pas être préjudiciable au développement de réactions inflammatoires/immunes. Son ciblage pourrait donc se révéler une approche thérapeutique intéressante pour diminuer son activité transcriptionelle sur l’oncogène transloqué. Un rôle potentiel des inhibiteurs des histones désacétylases est à l’étudeThe immunoglobulin heavy chain locus (IgH) undergoes several changes along B-cell differentiation. VHDJH recombinations during the early stages give the diversity of the antigenic repertoire. Somatic hypermutation (SHM) and class switch recombination (CSR) during late stages allow affinity maturation and the acquisition of new effectors functions. These rearrangements are highly regulated and are under the control of the IgH locus cis-regulatory elements. The 5’ Eµ enhancer is important for VHDJH recombination. The 3’ regulatory region (3’ RR) is essential for both CSR and SHM. These events induce breaks into the IgH locus, making it a hotspot for oncogenic translocations. The aim of my thesis was to understand the transcriptional interactions between Eμ and 3'RR enhancers and to evaluate whether the targeting of the latter could be of a potential therapeutic approach. We have demonstrated that 3'RR is essential to control IgH transcription in mature B cells. It is dispensable during the initial stages of developement (VHDJH recombinations). At the pro-B cell stage, it has a silencer effect rather than a transcriptional one on the DJH segments expression. The analysis of lymphomagenesis in three mice models carrying an insertion of Myc in different locations at the IgH locus showed significant differences in lymphoma kinetics, phenotypes and proliferation index. 3'RR alone, as a major transcriptional activator of the IgH locus, is capable of leading to B-cell lymphomas. Its absence is not detrimental for the development of classical inflammatory/immune reactions. Its targeting may be of a potentially interesting therapeutic approach to decrease its transcriptional activity on the translocated oncogene. A potential role for histone deacetylase inhibitors is under study
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Kato, Tomoko. "Gene expression of nutrient-sensing molecules in I cells of CCK reporter male mice." Doctoral thesis, Kyoto University, 2021. http://hdl.handle.net/2433/263549.
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BRADEN, WESLEY A. "EMERGING ROLES FOR THE RB-PATHWAY IN DNA REPLICATION CONTROL." University of Cincinnati / OhioLINK, 2007. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1195228828.
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Kurtyka, Courtney A. "Novel Therapeutic Strategies in Lung Cancer." Scholar Commons, 2014. https://scholarcommons.usf.edu/etd/5363.
Full textAbstract:
Lung cancer is the leading cause of cancer-related death and the second most diagnosed cancer in the United States. Unfortunately, many patients either do not have any common mutations for which there are already targetable agents, or they eventually become resistant to these compounds. As such, there is a high demand for new, effective methods of treating this disease as well as predicting patient prognosis and potential benefit from chemotherapy. In this work, numerous strategies for treating lung cancer are explored.
The first method described here is through the use of a pan-early 2 factor (E2F) inhibitor, HLM006474, which is shown to synergize with paclitaxel in non-small cell lung cancer (NSCLC). Next, we explored the creation and utilization of an E2F signature that is prognostic and predictive of early-stage NSCLC patient benefit from adjuvant chemotherapy (ACT). The third project examined possible targets to enhance sensitivity to cisplatin in NSCLC lacking Kirsten rat sarcoma viral oncogene homolog (KRAS) and epidermal growth factor receptor (EGFR) mutations and anaplastic lymphoma receptor tyrosine kinase (ALK) fusions (triple-negative), for which cisplatin is one of the few treatment options. These studies led to the identification of a kinase that is overexpressed in NSCLC and whose knockdown sensitizes cells to platinum agents.
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Adhikari, Deepak. "Signaling pathways in the development of female germ cells." Doctoral thesis, Umeå universitet, Institutionen för medicinsk kemi och biofysik, 2014. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-88309.
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Primordial follicles are the first small follicles to appear in the mammalian ovary. Women are born with a fixed number of primordial follicles in the ovaries. Once formed, the pool of primordial follicles serves as a source of developing follicles and oocytes. The first aim of this thesis was to investigate the functional role of the intra-oocyte signaling pathways, especially the phosphatidylinositol-3 kinase (PI3K) and mammalian target of rapamycin complex 1 (mTORC1) pathways in the regulation of primordial follicle activation and survival. We found that a primordial follicle remains dormant when the PI3K and mTORC1 signaling in its oocyte is activated to an appropriate level, which is just sufficient to maintain its survival, but not sufficient for its growth initiation. Hyperactivation of either of these signaling pathways causes global activation of the entire pool of primordial follicles leading to the exhaustion of all the follicles in young adulthood in mice. Mammalian oocytes, while growing within the follicles, remain arrested at prophase I of meiosis. Oocytes within the fully-grown antral follicles resume meiosis upon a preovulatory surge of leutinizing hormone (LH), which indicates that LH mediates the resumption of meiosis. The prophase I arrest in the follicle-enclosed oocyte is the result of low maturation promoting factor (MPF) activity, and resumption of meiosis upon the arrival of hormonal signals is mediated by activation of MPF. MPF is a complex of cyclin dependent kinase 1 (Cdk1) and cyclin B1, which is essential and sufficient for entry into mitosis. Although much of the mitotic cell cycle machinery is shared during meiosis, lack of Cdk2 in mice leads to a postnatal loss of all oocytes, indicating that Cdk2 is important for oocyte survival, and probably oocyte meiosis also. There have been conflicting results earlier about the role of Cdk2 in metaphase II arrest of Xenopus oocytes. Thus the second aim of the thesis was to identify the specific Cdk that is essential for mouse oocyte meiotic maturation. We generated mouse models with oocytespecific deletion of Cdk1 or Cdk2 and studied the specific requirements of Cdk1 and Cdk2 during resumption of oocyte meiosis. We found that only Cdk1 is essential and sufficient for the oocyte meiotic maturation. Cdk1 does not only phosphorylate the meiotic phosphoproteins during meiosis resumption but also phosphorylates and suppresses the downstream protein phosphatase 1, which is essential for protecting the Cdk1 substrates from dephosphorylation.
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Gao, Yanzhe. "Regulation of The DNA Unwinding Element Binding Protein DUE-B in The Cell." Wright State University / OhioLINK, 2012. http://rave.ohiolink.edu/etdc/view?acc_num=wright1355250568.
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