Studies on pancreatic cell physiology rely on the investigation of exocrine and endocrine cells in vitro. Particularly, in the case of the exocrine tissue these studies have suffered from a reduced functional viability of acinar cells in culture. As a result not only investigations on dispersed acinar cells and isolated acini were limited in their potential, but also prolonged studies on pancreatic exocrine and endocrine cells in an intact pancreatic tissue environment were unfeasible. To overcome these limitations, we aimed to establish a pancreas tissue slice culture platform to allow long-term studies on exocrine and endocrine cells in the intact pancreatic environment. Mouse pancreas tissue slice morphology was assessed to determine optimal long-term culture settings for intact pancreatic tissue. Utilizing optimized culture conditions, cell specificity and function of exocrine acinar cells and endocrine beta cells were characterized over a culture period of 7 days. We found pancreas tissue slices cultured under optimized conditions to have intact tissue specific morphology for the entire culture period. Amylase positive intact acini were present at all time points of culture and acinar cells displayed a typical strong cell polarity. Amylase release from pancreas tissue slices decreased during culture, but maintained the characteristic bell-shaped dose-response curve to increasing caerulein concentrations and a ca. 4-fold maximal over basal release. Additionally, endocrine beta cell viability and function was well preserved until the end of the observation period. Our results show that the tissue slice culture platform provides unprecedented maintenance of pancreatic tissue specific morphology and function over a culture period for at least 4 days and in part even up to 1 week. This analytical advancement now allows mid -to long-term studies on the cell biology of pancreatic disorder pathogenesis and therapy in an intact surrounding in situ.

<div> <p>Pancreatic ductal adenocarcinoma (PDAC) is a poorly immune responsive, treatment refractory disease, representing the fourth leading cause of cancer deaths in the United States. A lack of significant improvements in patient prognoses over the last few decades highlights the necessity for a more basic understanding of how PDAC develops and progresses. To this end, the research outlined here investigates the contributions of SOX9 and PAR1 in PDAC initiation and tumor immune evasion, respectively. </p> <p>SOX9 is a developmental transcription factor important for proper pancreas development that is restricted to only a small subset of cells in the adult organ. However, SOX9 is aberrantly expressed in precancerous lesions of the pancreas and throughout PDAC development. Using genetically engineered mouse models we demonstrated that PDAC precursor lesions cannot form in the absence of SOX9 and conversely formed at an accelerated rate when SOX9 was ectopically expressed. Surprisingly deletion of SOX9 in primary mouse PDAC cell lines had no impact on tumor growth in subcutaneous allograft experiments, indicating that although SOX9 expression is necessary for PDAC initiation, it is dispensable in many cases for tumor maintenance and growth. Research investigating the transcriptional changes induced by SOX9 prior to lesion formation is ongoing to identify additional downstream factors critical for disease initiation. </p> <p>Previous research has shown that PDAC tumors frequently display low levels of immune infiltration, which is a major limitation for the use of immune-based therapeutics and is generally an unfavorable prognostic factor. We show that in primary mouse tumor cells ablation of the thrombin receptor PAR1 caused a significant increase in the infiltration of tumor targeting CD8a⁺T cells which in turn were found to eliminate PAR1 knockout tumors. When PAR1^KO and PAR1 expressing PDAC tumor cells were co-injected into wild type mice, cells lacking PAR1 were preferentially targeted and eliminated by the immune system, indicating that PAR1 provides cell autonomous protection during an active anti-tumor adaptive immune response. Furthermore, we identified a previously underappreciated association between PAR1-mediated expression of <i>Csf2</i> and <i>Ptgs2</i>, and PDAC tumor immune evasion. Together these findings provide novel insights into the mechanisms and drivers of PDAC initiation and immune evasion.</p> </div> <br>

L’ischémie est une des problématiques majeurs de la transplantation, elle intervient au moment de la déconnexion d’un organe du système vasculaire et dure jusqu’à sa reconnexion chez le receveur. Elle provoque une diminution de la qualité des greffons et seulement un-tiers des pancréas éligibles aboutissent à une transplantation. Ce travail de thèse avait pour objectif d’améliorer la qualité des greffons en préparant les pancréas à subir l’ischémie par le préconditionnement et à améliorer leur préservation après avoir identifié les voies impliquées dans l’ischémie. Des changements métaboliques majeurs au niveau de l’organe ont été mis en évidence dépendant du temps d’ischémie, impliquant des dommages sur la partie endocrine du pancréas, les îlots. Ceci a été empêché par le préconditionnement du pancréas à l’ischémie activant les défenses antioxydantes et par l’utilisation de l’hémoglobine M101 issue de ver marin apportant de l’oxygène au cours de la préservation des pancréas de rat et humains. Ainsi, il est possible d’améliorer la qualité des greffons en agissant en amont du prélèvement et au cours de la préservation, ceci permettant une meilleure fonction. / Ischemia is one of the major problems of transplantation, it occurs at the time of disconnection of an organ of the vascular system and lasts until its reconnection in the recipient. It causes a decrease in graft quality and only one-third of the eligible pancreases results in a transplant. This work aimed to improve the quality of the grafts in preparing the pancreas to undergo ischemia preconditioning and improve their preservation after identifying the pathways involved in ischemia. Major metabolic changes in the organ have been demonstrated that appeared to ischemia time dependent enhancing damages to the endocrine part of the pancreas, the islets. This was prevented by the preconditioning of the pancreas to ischemia activating the antioxidant defenses and by the use of the marine worm M101 hemoglobin providing oxygen during the preservation of rat pancreas and humans. Thus, it is possible to improve the quality of the grafts by acting upstream of the sample and during the preservation, this allowing a better function.

Background: Pancreatic cancer is one of the leading causes of cancer-related death. Using DNA gene expression analysis based on a custom made Affymetrix cancer array, we investigated the expression pattern of both primary and established pancreatic carcinoma cell lines. Methods: We analyzed the gene expression of 5 established pancreatic cancer cell lines (AsPC-1, BxPC-3, Capan-1, Capan-2 and HPAF II) and 5 primary isolates, 1 of them derived from benign pancreatic duct cells. Results: Out of 1,540 genes which were expressed in at least 3 experiments, we found 122 genes upregulated and 18 downregulated in tumor cell lines compared to benign cells with a fold change > 3. Several of the upregulated genes (like Prefoldin 5, ADAM9 and E-cadherin) have been associated with pancreatic cancer before. The other differentially regulated genes, however, play a so far unknown role in the course of human pancreatic carcinoma. By means of immunohistochemistry we could show that thymosin [β-10 (TMSB10), upregulated in tumor cell lines, is expressed in human pancreatic carcinoma, but not in non-neoplastic pancreatic tissue, suggesting a role for TMSB10 in the carcinogenesis of pancreatic carcinoma. Conclusion: Using gene expression profiling of pancreatic cell lines we were able to identify genes differentially expressed in pancreatic adenocarcinoma, which might contribute to pancreatic cancer development. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

Pancreatic ductal adenocarcinoma (PDAC) has an extremely poor prognosis. To improve the prognosis, novel molecular markers and targets for earlier diagnosis and adjuvant and/or neoadjuvant treatment are needed. Recent advances in human genome research and high-throughput molecular technologies make it possible to cope with the molecular complexity of malignant tumors. With DNA array technology, mRNA expression levels of thousand of genes can be measured simultaneously in a single assay. As several studies using microarrays in PDAC have already been published, this review attempts to compare the published data and therefore to validate the results. In addition, the applied techniques are discussed in the context of pancreatic malignancies. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

<p>Akrilamid&nbsp; je toksična hemijska supst anca koja je već dugi niz godina prisutna u životnoj sredini,&nbsp; jer se kao važan monomer koristi u različite industrijske i laboratorijske svrhe. U poslednjih petnaest godina, akrilamid je postao posebno zanimljiv za &scaron;ire naučne krugove jer&nbsp; se pokazalo da&nbsp; se&nbsp; nalazi&nbsp; i u&nbsp; hrani&nbsp; biljnog porekla, posebno hrani bogatoj skrobom, koja se priprema pečenjem ili prženjem na temperaturama vi&scaron;im od 120&deg;C.&nbsp; Do sada ustanovljeni negativni zdravstveni efekti akrilamida su veoma raznovrsni i mogu biti rezultat delovanja samog&nbsp; akrilamida ili delovanja njegovog metabolita glicidamida koji nastaje&nbsp; in vivo&nbsp; kada se jedan deo molekula akrilamida metaboli&scaron;e oksigenacijom dvostruke veze pomoću enzima citohrom P450 2E1 (CYP2E1). Akrilamid je supstanca koja ima dokazan negativan efekat&nbsp; na organske sisteme kod ljudi i životinja, i koja je svrstana u moguće humane karcinogene. Negativan efekat akrilamida na egzokrini pankreas je poznat, ali o mogućim efektima akrilamida na endokrini pankreas se i dalje veoma malo zna. Ima puno dokaza koji&nbsp; ukazuju na to da akrilamid ima citotoksični efekat koji se&nbsp; manifestuje kroz uticaj na redoks-status ćelija i dovodi do promena u vrednostima biomarkera oksidativnog i nitrozativnog stresa, kao i u aktivnosti antioksidativnih enzima. Pankreas&nbsp; je&nbsp; jedan od ciljnih&nbsp; organa za delovanje akrilamida te je&nbsp; glavni predmet istraživanja&nbsp; doktorske teze&nbsp; bio proučavanje potencijalnog efekta akrilamida na endokrini pankreas pacova.&nbsp; Ispitivanje je vr&scaron;eno na 3&nbsp; eksperimentalne grupe&nbsp; juvenilnih&nbsp; mužjaka pacova soja Wistar,&nbsp; od kojih je&nbsp; jedna grupa bila kontrolna, dok su dve bile tretirane&nbsp; sa akrilamidom u dozama od 25 mg/kg tm i 50 mg/kg tm,&nbsp; 5 dana nedeljno,&nbsp; tokom 3 nedelje. Po isteku tretmana,&nbsp; nakon dekapitacije, kompletno tkivo pankreasa&nbsp; je&nbsp; fiksirano u 10% rastvoru formalina&nbsp; tokom&nbsp; 24&nbsp; h i obrađeno prema&nbsp; standardnoj proceduri za kalupljenje u parafinu.&nbsp; Parafinski kalupi su sečeni na serijske preseke debljine 5 &micro;m, nakon čega su bojeni&nbsp; histohemijskom i imunohistohemijskim metodama.&nbsp; Kod eksperimentalnih grupa posmatrane&nbsp; su&nbsp; histolo&scaron;ke promene na endokrinom pankreasu, sa akcentom na &alpha;-&nbsp; i &beta;-ćelije.&nbsp; Takođe, posmatrana je&nbsp; i&nbsp; ekspresija&nbsp; hormona insulina i glukagona, enzima inducibilne azot -oksi d&nbsp; sintetaze (iNOS) i&nbsp; CYP2E1,&nbsp; kao&nbsp; i ekspresija&nbsp;&nbsp; antioksidativnih enzima&nbsp; katalaza&nbsp; (CAT) i superoksid dismut aza 1 i 2&nbsp; (SOD1 i SOD2)&nbsp; u ćelijama Langerhansovih ostrvaca. Potencijalna promena u funkcionalnosti &beta;-ćelija je ispitana i kroz analizu nivoa glukoze u serumu pacova tretiranih sa akrilamidom.<br />Budući da &beta;-ćelije čine 80% ćelija koje grade Langerhansova ostrvca pankreasa,&nbsp; pored in vivo&nbsp; eksperimenata, ispitana&nbsp; je&nbsp; i toksičnost akrilamida na&nbsp; Rin-5F ćelijsku liniju insulinoma &beta;-ćelija pacova u in vitro uslovima. Glavni cilj in vitro&nbsp; istraživanja je bio&nbsp; da se&nbsp; ispita&nbsp; uticaj&nbsp; rastućih&nbsp; koncentracija akrilamida na preživljavanje tretiranih&nbsp; Rin-5F&nbsp; ćelija, ali i efekat IC₅₀&nbsp; koncentracije ove supstance primenjene&nbsp; tokom&nbsp; različitih vremenskih intervala&nbsp; (0,5, 1, 3, 6, 12 i 24 h)&nbsp; na pojavu oksidativnog i nitrozativnog stresa. Redoks-status Rin-5F ćelija tretiranih&nbsp; sa akrilamidom je ispitan preko analize prisustva biomarkera oksidativnog i nitrozativnog stresa, akrivnosti CAT i ukupne SOD, kao i promene u ekspresiji gena za CAT, SOD1, SOD2&nbsp;&nbsp; i iNOS.&nbsp; Pored toga, analiziran je i efekat istog tretmana na&nbsp; ekspresiju gena za insulin, CYP2E1, Bax i Bcl-2. U okviru teze je pokazano da akrilamid ne dovodi do&nbsp; značajnih promena u histolo&scaron;koj građi, dijametru i broju Langerhansovih ostrvaca&nbsp; kod&nbsp; tretiranih životinja.&nbsp; Primena stereolo&scaron;kih metoda&nbsp; je&nbsp; ukazala&nbsp; na mikrostrukturne promene na&nbsp; endokrinom pankreasu na nivou &alpha;-&nbsp; i &beta;-ćelija. U ovoj tezi je po prvi put pokazano da tretman akrilamidom negativno utiče na broj i povr&scaron;inu &beta;-ćelija pankreasa.&nbsp; U tezi je, takođe,&nbsp; pokazan&nbsp; značajan dozno-zavisni pad u prisustvu insulina u &beta;-ćelijama&nbsp;&nbsp; pankreasa. Uprkos&nbsp; tome, kod&nbsp; akrilamidom tretiranih&nbsp; životinja&nbsp; nije konstatovana&nbsp; promena&nbsp; u&nbsp; koncentraciji serumske glukoze.&nbsp; U&nbsp; ovoj tezi je pokazano da tretman akrilamidom dovodi do&nbsp;&nbsp; statistički značajnog porasta&nbsp; u broju &alpha;-ćelija&nbsp; kod životinja koje su primale nižu dozu tretmana, dok se&nbsp; broj &alpha;-ćelija&nbsp; kod životinja koje su primale vi&scaron;u dozu tretmana&nbsp; ne razlikuje značajno od kontrole.&nbsp; Tretman akrilamidom je doveo do značajnog&nbsp; porasta u količini&nbsp;&nbsp; prisutnog glukagona&nbsp; u &alpha;-ćelijama pankreasa.<br />Tretman akrilamidom nije doveo do značajne promene u ekspresiji CAT, SOD1 i SOD2 u ćelijama Langerhansovih ostrvaca.&nbsp; Kod&nbsp; tretiranih životinja&nbsp; do&scaron;lo do značajnog dozno-zavisnog porasta&nbsp; u ekspresiji&nbsp; enzima iNOS,&nbsp; dok je ekspresija&nbsp; CYP2E1 značajno dozno-zavisno opala&nbsp; nakon tretmana. U&nbsp; tezi je pokazano da tretman akrilamidom negativno utiče na vijabilnost Rin-5F ćelija, i utvrđeno je da IC50&nbsp; koncentracija akrilamida za Rin-5F ćelije iznosi 10 mM.&nbsp; Rezultati teze pokazuju da tretman akrilamidom u IC₅₀&nbsp; koncentraciji u Rin-5F ćelijskoj liniji značajno povećava nivo malondialdehida (MDA) nakon tretmana u trajanju od 1, 12 i 24 h.&nbsp; Isti tretman&nbsp; značajno smanjuje nivo redukovanog GSH nakon tretmana od 1, 3, 6, 12 i<br />24 h, kao i nivo slobodnih&nbsp; &ndash;SH grupa nakon tretmana od 3 i 6 h. Tretman akrilamidom u IC_50&nbsp; koncentraciji signifikantno pojačava aktivnost CAT nakon tretmana od 1 h, dok tretman u trajanju od 12 h značajno smanjuje aktivnost ovog enzima. Ovaj tretman smanjuje aktivnost SOD nakon 1, 12 i 24 h, dok&nbsp; tretman u trajanju od 6 h značajno pojačava aktivnost enzima SOD.&nbsp; U tezi je, takođe, pokazan i veoma značajan porast&nbsp; u nivou prisutnih nitrita,&nbsp; koji&nbsp; je direktno proporcionalan&nbsp; sa nivoom azot-oksida i nivoom akivnosti enzima iNOS.&nbsp; Ovaj&nbsp; nalaz ukazuje na potencijalnu pojavu nitrozati vnog stresa u akrilamidom-tretiranim Rin-5F ćelijama.&nbsp; U&nbsp; tezi je po prvi put pokazano da tretman&nbsp; akrilamidom dovodi do&nbsp; značajnih&nbsp; varijacija&nbsp; u transkripciji gena za iNOS, SOD1, SOD2,&nbsp; CAT,&nbsp; CYP2E1,&nbsp; Bax i Bcl-2 u tretiranim Rin-5F ćelijama, dok isti tretman ne dovodi do&nbsp; promene nivoa&nbsp; transkripcije gena za insulin.&nbsp; Tretman akrilamidom u koncentraciji od 10<br />mM tokom rastućih vremenskih perioda dovodi do porasta u relativnoj količini iRNK<br />gena za iNOS u svim tačkama tretmana, do porasta&nbsp; nivoa&nbsp; iRNK za SOD1 i SOD2 nakon tretmana od 12 i 24 h, kao i do porasta&nbsp; količine&nbsp; iRNK za CAT nakon tretmana od 3 h.&nbsp; U&nbsp; tezi je pokazano&nbsp; i&nbsp; da akrilamid&nbsp; izaziva&nbsp; promene&nbsp; u sintezi&nbsp; iRNK&nbsp; za enzim&nbsp; CYP2E1&nbsp; koji je&nbsp; posebno značajan u kontekstu detoksikacije ove toksične supstance.&nbsp; Porast u transkripciji gena za&nbsp; CYP2E1&nbsp; je uočen&nbsp; nakon tretmana u trajanju od 0,5 i 1 h, dok je&nbsp; do smanjenja transkripcije&nbsp; do&scaron;lo&nbsp; nakon tretmana od 12&nbsp; i 24&nbsp; h.&nbsp; Tretman akrilamidom u koncentraciji od&nbsp; 10 mM tokom rastućih vremenskih perioda dovodi do porasta u relativnoj količini iRNK&nbsp; gena za Bax u svim tačkama tretmana, i do porasta u transkripciji gena za Bcl-2 nakon tretmana od 0,5, 1 i 3 h.<br />Sumirajući&nbsp; sve&nbsp; rezultate&nbsp; ove teze,&nbsp; moze se zaključiti&nbsp; da je endokrini pankreas&nbsp; jedno od&nbsp; ciljnih tkiva, na koje akrilamid ostvaruje vi&scaron;estruki negativni uticaj.</p> / <p>Acrylamide is a toxic chemical used as an important monomer for various industrial and laboratory purposes, which makes it highly present in the environment. In the last fifteen years, acrylamide has become especially interesting for wider scientific circles when it was found in staple foodstuff rich in starch, prepared at temperatures higher than 120&deg;C. The established negative health effects of acrylamide are very diverse and can be the result of the acrylamide action itself or the action of its metabolite glycidamide that occurs in vivo, when acrylamide molecule is metabolized via oxygenation of the double bond by the cytochrome P450 2E1 (CYP2E1). Acrylamide is a substance with a proven adverse effect on humans and animals, and it is classified as a possible human carcinogen. The negative effect of acrylamide on the exocrine pancreas has already been recognized, but the possible effects of acrylamide&nbsp; on endocrine pancreas are still mostly undetermined. There is a significant amount of evidence to suggest that acrylamide exerts a cytotoxic effect which manifests through the changes in level of oxidative and nitrosative stress biomarkers, as well as in the activity of antioxidant enzymes. Since, pancreas is one of the target organs for acrylamide, the main subject of doctoral thesis was to investigate the potential effect of acrylamide on the rat endocrine pancreas. The investigation was conducted on 3 experimental groups of juvenile male Wistar rats, of which one group was the control group, while two groups were treated with acrylamide at doses of 25 mg/kg bw and 50 mg/kg bw, 5 days a week, during 3 weeks. After termination of the treatment, decapitation was performed, and the complete pancreatic tissue was fixed in a 10% formalin solution for 24 h and treated according to the standard paraffin embedding procedure. Paraffin molds were cut into 5 &mu;m thick serial sections, after which they were stained with histochemical and immunohistochemical methods. Histological changes ofthe endocrine pancreas, with the emphasis on &alpha;- and &beta;-cells, were examined in three experimental groups of rats. In addition, the expression of insulin and glucagon hormone, the inducible nitric oxide synthase (iNOS) and CYP2E1 enzymes, and the expression of antioxidative enzymes catalase (CAT) and superoxide dismutases 1 and 2&nbsp; (SOD1 and SOD2) in the islets of Langerhans were also investigated. A potential change in the functionality of &beta;-cells was also examined by analyzing glucose level in the serum of rats treated with acrylamide. In pancreatic islets of Langerhans the majority of cells (&gt;80%) are &beta;-cells. Therefore, in addition to in vivo experiments, the toxicity of acrylamide was examined in vitro on rat insulinoma Rin-5F cell line.The main goal of in vitro research was to investigate the impact of increasing acrylamide concentrations on the viability of treated Rin-5F cells, and also to examine whether IC50 concentration of this substance, applied at different intervals of time (0.5, 1, 3, 6, 12 and 24 h), induce oxidative and nitrosative stress. Redox-status of Rin-5F cells treated with acrylamide was examined by analyzing oxidative and nitrosative stress biomarkers, CAT and total SOD activity, as well as changes in the expression of the CAT, SOD1, SOD2 and iNOS. In addition, the effect of the same treatment on the transcription of the insulin, CYP2E1, Bax and Bcl-2 gene was analyzed.The results of the thesis showed that acrylamide treatment does not lead to significant changes in the histological structure, diameter and number of islets of Langerhans of treated animals. Application of stereological methods indicated microstructural changes of &alpha;- and &beta;-cells ofendocrine pancreas. It has been shown for the first time that treatment with acrylamide negatively affects the number and surface area of pancreatic &beta;-cells. In addition, a significant dose-dependent decline in the amount of insulin in pancreatic &beta;-cells was also demonstrated. However, no change in serum glucose level was observed in treated animals. Acrylamide treatment led to a statistically significant increase in the number of &alpha;-cells in animals receiving a lower dose of treatment, while the number of &alpha;-cells in animals receiving a higher dose of treatment did not differ significantly from the control. Treatment with acrylamide led to a significant increase in the amount of the glucagon in &alpha;-cells. Treatment with acrylamide did not cause a significant change in the expression of CAT, SOD1 and SOD2 in islets of Langerhans. However, there was a significant dosedependent increase in the&nbsp; expression of iNOS enzyme, whereas expression of CYP2E1 significantly decreased in dose-dependent manner in treated animals. Results of the thesis showed that acrylamide exerts a negative effect on the viability of Rin-5F cell line. It has been established that the IC50 concentration of acrylamide for the Rin-5F cell line is 10 mM. The results of the thesis indicate that treatment of Rin-5F cell line with IC50 concentration of acrylamide for 1, 12, and 24 h significantly increased the level of malondialdehyde (MDA). Exposure to acrylamide for 1, 3, 6, 12 and 24 h significantly decreased the level of reduced GSH, while the level of free -SH groups was reduced after 3 and 6 h of acrylamide treatments. Treatment with IC50 concentration of acrylamide significantly enhanced CAT activity after 1 h of acrylamide exposure, while 12 h exposure significantly reduced the activity of this enzyme. Application of acrylamide reduced SOD activity after 1, 12, and 24 h exposure, while 6 h exposure significantly increased the activity of SOD enzymes. Results of the thesis also showed a very significant increase of the nitrite level, which is directly proportional to the level of nitrogen oxide (NO) and the level of the iNOS activity. This finding points to the potential occurrence of nitrosative stress in acrylamide-treated Rin-5F cells. It has been shown for the first time that acrylamide treatment leads to significant variations in transcription of iNOS, SOD1, SOD2, CAT, CYP2E1, Bax and Bcl-2 genes in treated Rin-5F cells, while the same treatment does not affect transcription of the insulin gene. Treatment with acrylamide at a concentration of 10 mM for increasing periods of time leads to an increase in the relative amount of the iNOS gene iRNA at all treatment points. Twelve and and 24 h of acrylamide exposure increased the transcription of the SOD1 and SOD2 genes. Transcription of CAT gene was increased after 3 h&nbsp; ofacrylamide exposure. Furthermore, it has been shown that acrylamide treatment leads to variations in the mRNA synthesis of CYP2E1 gene, which is particularly significant in the context of detoxification of this toxic substance. An increase in the transcription ofthe CYP2E1&nbsp; gene was observed after 0.5 and 1 h of acrylamide exposure, while the reduction of&nbsp; transcription occurred after 12 and 24 h of acrylamide exposure. The treatment with 10 mM acrylamide has led to an increase of the transcription of the Bax gene at all treatment points, and also to an increase of transcription of the Bcl-2 gene after of 0.5, 1, and 3 h of acrylamide exposure. Summarizing all the results of this thesis, it can be concluded that the endocrine pancreas&nbsp; is one of the target tissues of acrylamide, to which this substance exerts a multiple adverse effects.</p>

Introduction. The aim of the study was to introduce a new BKV PCR protocol in our centre and to verify its accuracy as well as to assess the prevalence, risk factors of BK virus replication, course of BKV infection and therapeutic approaches in simultaneous pancreas and kidney (SPK) recipients in order to design a screening protocol. Methods. The results analysed by both Affigene® and Transplantation Virology, Basel PCR protocols were compared. Thereafter 183 SPK patients were examined to assess the prevalence of BK viremia, viruria and BKVN and to identify the risk factors of BKV replication. The cases of retransplantation after a graft loss due to BKVN were retrospectively described. Results. 100 of results were analysed according to the Affigene ® and Transplantation Virology, Basel PCR protocols with the accordance of 95%, Rho = 0,946, 95% CI: 0.920 - 0.963, P<0,0001, Bland-Altman plot analyses: bias Basel PCR protocol/Affigene® BKV trender: -0,1 (mean) *±1.96 SD: -1,6 - 1,3] for both methods. Point-prevalence was assessed in 183 patients; Viruria found in 17,3 %, viremia in 3.8% of patients. High-level viruria >107 copies/mL detected in 3,7% of patiets, high-level virémia >104 in 1,6% of patients simultaneously with high-level viruria. BKVN was found in 0,5% of patients. Diabetes duration...

Type 2 diabetes is characterized by peripheral insulin resistance and insufficient insulin release from pancreatic islet β cells. However, the role and sequence of β cell dysfunction and mass loss for reduced insulin levels in type 2 diabetes pathogenesis are unclear. Here, we exploit freshly explanted pancreas specimens from metabolically phenotyped surgical patients using an in situ tissue slice technology. This approach allows assessment of β cell volume and function within pancreas samples of metabolically stratified individuals. We show that, in tissue of pre-diabetic, impaired glucose-tolerant subjects, β cell volume is unchanged, but function significantly deteriorates, exhibiting increased basal release and loss of first-phase insulin secretion. In individuals with type 2 diabetes, function within the sustained β cell volume further declines. These results indicate that dysfunction of persisting β cells is a key factor in the early development and progression of type 2 diabetes, representing a major target for diabetes prevention and therapy.

La distribution in vivo des médicaments est étudiée par des techniques quantitatives faiblement ou non résolues spatialement. Avec l'apparition des thérapies personnalisées, des études plus approfondies sont nécessaires pour connaître précisément le comportement des molécules vectorisées sous la forme de nanoparticules (NPs). Dans le cadre du programme européen Ternanomed, ce projet de recherche a pour objectif d’évaluer la capacité de deux techniques d’imagerie appliquées à l’étude de la distribution de nanomédicaments à base de squalène et de Cis platine (Cis-Pt). Ces deux techniques ont été sélectionnées pour leur apport d’informations complémentaires à l’échelle des organes et des tissus : i) l’imagerie par résonnance magnétique (IRM) pour suivre in vivo la biodistribution de NPs modèles à base de Cis-Pt et BiSqualène (BiSQ), marquées par des agents de contraste type oxyde de fer (USPIO), ii) l’imagerie de microfluorescence X, couplée au rayonnement synchrotron (SR-μXRF), qui ne nécessite pas de marquage préalable des nanomédicaments, pour le suivi tissulaire du Cis-Pt.Concernant l’approche par IRM, nous avons encapsulé avec succès nos USPIO synthétisées au sein des NPs de Cis-Pt BiSQ (210nm, polydispersité 0,1), tout en leur conférant un pouvoir contrastant à 7 tesla (r2=404 ms.mol-1 et r1=3 ms.mol-1). Ces NPs nouvellement préparées sont également traçables chez notre modèle murin Nudes. Les résultats de biodistribution montrent une arrivée rapide du contraste dans les organes épurateurs : le foie et la rate (5 minutes après l’injection). Au final, l’analyse par IRM a permis d’obtenir les données de biodistribution en temps réel des NPs à base de Cis Pt BiSQ, grâce au suivi du contraste apporté par les USPIO encapsulés. Concernant l’imagerie par SR-μXRF, nous avons démontré que cette technique est suffisamment sensible pour détecter et cartographier le Cis-Pt, vectorisé par nos NPs modèles. La distribution du Cis Pt a été quantifiée localement à partir d’une référence interne de concentration connue, le Zinc, à partir de notre méthode validée par le dosage globale du Platine par spectrométrie d’absorption atomique. Lorsque notre référence tissulaire n’est pas distribuée de façon homogène, une méthode semi-quantitative a été mise au point pour comparer la distribution à 2h, 8h et 24h, tel qu’au niveau des coupes de tumeurs PANC-1.Au final, ces travaux ont permis de démontrer, que la SR-μXRF et l’IRM sont des approches de choix pour l’étude pharmacocinétique et pharmacodynamique de nanomédicaments tels que les NPs à base de Cis-Pt. La technique de microfluorescence X contribue au caractère original et pionnier de ce travail de recherche, apportant des nouveaux résultats de détection et quantification important dans le domaine des nanomédecines. / Nowadays, the in vivo distribution of drugs is studied by non-spatial or partially spatial quantitative techniques. With the development of personalized therapies, many studies are required to know the in vivo behaviour of these innovative treatments, which target drugs, such as nanoparticles (NPs). Into the European funded program Ternanomed, the aim of this multidisciplinary research project was to evaluate two complementary imaging methods to study the distribution of squalene and Cis platinum (Cis Pt) NPs. The 2 imaging methods were selected to provide complementary data at the scale of organs and tissues: i) Magnetic resonance imaging (MRI) to monitor the in vivo biodistribution of NPs models based on Cis-Pt and BiSqualene (BiSQ), labelled with "UltraSmall Iron Oxide Particle" (USPIO) contrast agents, ii) X-ray microfluorescence imaging, coupled with synchrotron radiation (SR-μXRF) without any labelling of these nanomedicines, by following the Cis-Pt drug distribution into tissues.Regarding the MRI approach, we first successfully prepared Cis-Pt BiSQ NPs loading with USPIO (210nm, polydispersity 0,1). These NPs were given a contrast at 7 Tesla (r2 = 404 ms.mol-1 and r1 = 3 ms.mol-1). These newly prepared and characterized NPs were also trackable into our Nude murine model. The results show a rapid arrival of contrast in the liver and spleen scavengers (5 minutes after injection). Ultimately, MRI analysis yielded real-time biodistribution data for Cis-Pt BiSQ-based NPs by monitoring the contrast provided by encapsulated USPIO. Regarding the SR-μXRF imaging analysis, we demonstrated that this technique is very sensitive to detect and map the Cis-Pt distribution, the drug vectorized by our squalene NPs models. Additionally, a local quantitative analysis is feasible when a microelement present in the tissue is used as a reference, in our study the Zinc element. The distribution of Cis-Pt was quantified in the hepatic, renal and fat tissues, after 2h and 24h, with our method validated by the global Platinum microanalyse using atomic absorption spectrometry. When the tissue reference appears not homogenously distributed, a semi-quantitative analysis method is possible to compare the distribution such as into PANC-1 tumour sections.Finally, these two complementary approaches illustrate the use of SR-μXRF and lay the optimized bases of MRI to study the pharmacokinetics and pharmacodynamics of two new types of Cis-Pt/squalene NPs. The SR-μXRF technique, newly used in pharmaceutical field, had an effective contribution to these original and pioneering research studies with an original way of in vivo assessment of the distribution of drug embedded into nanomedicine system. The issue of detecting correct and measurable distribution of the drugs is extremely important, timely and relevant to improve current knowledge in the state of the art. This research study brings new data which can produce significant impact to the overall area of nanomedicine.

Indiana University-Purdue University (IUPUI) / Most pancreatic cancer patients receiving gemcitabine chemotherapy eventually develop resistance to gemcitabine. To improve survival and prognosis of pancreatic cancer patients, better understanding the mechanisms of gemcitabine resistance and discovery of new therapeutic targets are required. In this study, I investigated the molecular mechanisms of acquired gemcitabine resistance using a stepwise gemcitabine-selected pancreatic cancer cell line in comparison to the parental cell line. I found that 14-3-3&#963; is up-regulated in the drug resistant cell line due to demethylation in its first exon, and the up-regulation of 14-3-3&#963; gene expression, in turn, contributes to gemcitabine resistance. Intriguingly, I found that demethylation of the 14-3-3&#963; gene in gemcitabine resistant cells is reversibly regulated by DNMT1 and UHRF1. Furthermore, I found that 14-3-3&#963; over-expression causes gemcitabine resistance by inhibiting gemcitabine-induced apoptosis and caspase-8 activation possibly via binding to YAP1. The finding of demethylation of the 14-3-3&#963; gene in gemcitabine resistant cells led to a hypothesis that other genes may also be changed epigenetically following gemcitabine selection. By RRBS (Reduced Representation Bisulfite Sequencing) analysis, 845 genes were found to have altered methylation. One of these genes, PDGFD, was further investigated and found to have reversible demethylation at its promoter region in the drug resistant cells and contribute to gemcitabine resistance possibly via autocrine activation of the STAT3 signaling pathway. Together, these findings not only provide evidence that 14-3-3&#963; and PDGFD over-expression contribute to acquired gemcitabine resistance and that reversible epigenetic changes may play an important role in acquired gemcitabine resistance, but also demonstrate that the molecular mechanisms of acquired gemcitabine resistance in pancreatic cancer cells are complex and multifaceted.