The thesis is a study of the effects of radiation on the salivary glands in an experimental and a clinical study. Irradiation is a cornerstone in the management of head and neck cancer and is as other modalities of cancer treatment, afflicted with adverse reactions. An optimal radiotherapy regime is limited by the sensitivity of the normal tissues with regard to early and late effects. In certain cases the early effects can be so troublesome that it will cause interruption in the irradiation and questioning of the curative intention. Although DNA is the lethal target, other parts of the cell have been proposed as sensitive targets to irradiation. Different in vitro secretory models and quantitative morphological characterization and immunohistochemical evaluation of neuropeptides were performed in rat salivary glands after irradiation. The irradiation was given unilaterally or bilaterally once a day for a five-day schedule with 6 MV photons (total dose 20, 30, 35, 40, 45 Gy) or a two fractions regime in five days with a total dose of 24 or 32 Gy. The contralateral gland served as a control for unilaterally treated animals and parallel analyses were done 10 days or 180 days following the last irradiation dose. An early, dose-dependent effect of fractionated irradiation on noradrenaline-stimulated potassium fluxes (86Rb+ fluxes) was demonstrated. In contrast, the exocytotic amylase release displayed no obvious alterations, and morphologically no changes were seen. Regarding late effects (180 days) the noradrenaline-stimulated electrolyte secretion was decreased at least for the higher doses of irradiation. Amylase content and loss of acini was also dose-dependently decreased. At 10 days after bilateral irradiation there was a marked increase in the expression of the neuropeptides substance P, leu-enkephalin and bombesin in the ganglionic cells associated with the submandibular glands and in nerve fibers of the glandular parenchyme. In addition, a clinical prospective evaluation of 25 patients was performed before, during radiotherapy and 6, 12 and 18 months after the end of treatment. A great interindividual variation in the recovery was demonstrated with regard to salivary flow rate. Irradiation doses about 40-50 Gy caused generally reversible changes; sometimes salivary secretion was almost completely restored 6-18 months after the end of radiotherapy. Doses exceeding 65 Gy induced almost irreversible alterations. Even if DNA is the target for the lethal effect of irradiation, other constituents, such as the cell membrane or neuropeptide expression can be significantly affected by irradiation and cause important physiological changes. / <p>S. 1-43: sammanfattning, s. 47-164: 6 uppsatser</p> / digitalisering@umu

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21991号 / 医博第4505号 / 新制||医||1037(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 武田 俊一, 教授 坂井 義治, 教授 松田 道行 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(医科学) / 甲第19231号 / 医科博第63号 / 新制||医||科5(附属図書館) / 32230 / 京都大学大学院医学研究科医科学専攻 / (主査)教授 長船 健二, 教授 柳田 素子, 教授 斎藤 通紀 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・論文博士 / 博士(医学) / 乙第13235号 / 論医博第2175号 / 新制||医||1037(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 竹内 理, 教授 渡邊 直樹, 教授 山下 潤 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21632号 / 医博第4438号 / 新制||医||1034(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 生田 宏一, 教授 上本 伸二, 教授 竹内 理 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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Pancreatic ductal adenocarcinoma (PDAC) is the 3rd deadliest cancer in the United States with a projected 12% 5-year survival rate. Acinar cells have been proposed as a potential cell-of-origin for PDAC after undergoing acinar to ductal metaplasia (ADM). In the absence of oncogenic mutations (e.g. Kras), ADM lesions form as an adaptive response and eventually resolve to regenerate the acinar compartment, which we term as adaptive ADM. However, in the presence of oncogenic Kras mutations, the ADM lesions can transform to a pre-invasive state called pancreatic intraepithelial neoplasia (PanIN). Thus, a normally adaptive metaplastic response becomes maladaptive, which we term as oncogenic ADM. The mechanisms that drive PanIN formation in the context of injury and oncogenic mutations are poorly understood, resulting in an absence of targets to combat persistent ADM. This thesis investigates the role of FRA1 (gene name Fosl1) in acinar cell de-differentiation, PanIN transformation, and eventual PDAC tumorigenesis. Through CUT&RUN sequencing of mice undergoing recovery from caerulein-induced acute pancreatitis, we identify FRA1 as the most active transcription factor during KrasG12D mediated acute pancreatitis- mediated injury. We have elucidated a functional role of FRA1 by generating an acinar-specific Fosl1 knockout mouse expressing KrasG12D. Using a gene regulatory network and pseudotime trajectory inferred from single nuclei ATAC-seq and bulk-RNA seq, we hypothesize a regulatory model of the acinar-ADM-PanIN continuum and experimentally validate that Fosl1 knockout mice are delayed in the onset of ADM and PanIN. Furthermore, deletion of Fosl1 in an autochthonous PDAC mouse model revealed that this ADM-initiated delay eventually culminates in a significant survival advantage and a less aggressive tumor phenotype. Through investigation of upstream regulators of FRA1, we identified G-CSF as an ADM-promoting cytokine. Fosl1 depletion prevented the pro-inflammatory effects of G-CSF, indicating that the G-CSF/FRA1 signaling axis can modulate ADM. Using ex vivo acinar cultures, we also showed that G-CSF can induce FRA1 through MEK/ERK signaling. Our findings reveal that FRA1 is a mediator of acinar cell plasticity and contributes to acinar cell de-differentiation and malignant transformation. Although the majority of this thesis focuses on oncogenic ADM, we also include a chapter on the role of Prrx1 in adaptive ADM. Our comprehensive and unbiased approach identified previously the Paired-Related homebox1 (Prrx1) as the most upregulated transcription factor in the intersection of pancreatic ductal development, regeneration, and evolution of PanIN. We have demonstrated previously that Prrx1 can promote a ductal phenotype by binding the Sox9 promotor and inducing its expression during pancreatitis. In this body of work, we present a novel mechanism by which Prrx1 regulates maintenance of adaptive ADM. Using novel mouse models and ex vivo acinar culture systems, we demonstrate that Prrx1 can induce TGFβ signaling and reduce E-Cadherin expression to promote ADM. We do not know if there is any potential epistatic interaction between FRA1 and PRXX1. Overall, we reveal the rippling effects of FRA1 can have during the early stages of pre-neoplasia, and we unveil an alternative function of PRRX1 for stimulating an adaptive response to stress. This thesis presents a new understanding of how acinar cell de-differentiation occurs in the pancreas by revealing novel roles of two transcription factors, FRA1 and PRRX1, and furthers our understanding of tissue regeneration in an injured pancreas.

Pancreatic cancer is nowadays one of the neoplasms with worst prognosis, so research towards the discovery of new molecular targets for therapy and diagnosis is more than urgent. In this direction, we have deeply evaluated the role of Galectin-1 (Gal-1) - a protein that is highly overexpressed in the tumor stroma- in pancreatic tumor progression. Interestingly, we have found that Gal-1 interacts with tissue plasminogen activator (tPA) and that this interplay seems to be involved both in pancreatic tumor epithelial cells and fibroblasts migration, Erk1/2 activation and invasion, suggesting its importance in the tumor/stroma crosstalk in vitro. We have also focused on the biochemical identification of tPA/Gal-1 interaction domains. Furthermore, we have studied Gal-1 role in pancreatic tumor progression in vivo, using murine (xenografts and transgenics) and zebrafish models. We have found that Gal-1 participates in proliferation, angiogenesis, stroma formation and necrosis in Ela-1-myc pancreatic tumors, as well as in the acinar to ductal metaplasia. Importantly, these effects result in an overall significant increase in the survival of Ela-1-myc mice with reduced Gal-1 levels. We have also analyzed Gal-1 role in mouse embryonic pancreatic development, finding interesting parallelisms with tumors. Finally, the molecular mechanisms involved in Gal-1 driving tumor pancreatic progression have been addressed through a transcriptome analysis. All together, our data support Gal-1 as a new molecular target to fight against pancreatic cancer. / Avui en dia, el càncer de pàncrees representa un dels tumors amb més elevats índex de mortalitat, per la qual cosa la recerca dirigida a la identificació de molècules per teràpia i diagnosi són més que necessàries. Amb aquest objectiu, hem avaluat el paper que juga Galectina-1 (Gal-1) - una proteïna altament sobreexpressada en l’estroma tumoral- en la progressió tumoral pancreàtica. Hem trobat que Gal-1 interactua amb l’activador tissular del plasminògen (tPA), participant en la migració, l’activació de Erk1/2 i la invasió, tant en cèl4lules tumorals pancreàtiques com en fibroblasts in vitro, suggerint una importància caudal d’aquesta interacció en la comunicació entre el tumor i l’estroma. Així mateix, ens hem centrat en la identificació bioquímica dels dominis d’interacció entre Gal-1 i tPA. A més, el paper de Gal-1 en la progressió tumoral pancreàtica ha estat adreçat in vivo, utilitzant models murins (xenografts i transgènics) i el peix zebra. Així doncs hem trobat que Gal-1 participa en la proliferació, angiogènesi, formació de l’estroma i la necrosi dels tumors pancreàtics dels ratolins Ela- 1-myc, així com en la metaplasia acinar-ductal. De forma significativa, aquests efectes es tradueixen en un increment important en la supervivència dels ratolins Ela-1-myc amb nivells reduïts de Gal-1. Hem també analitzat el paper que juga Gal-1 en el desenvolupament pancreàtic embrionari murí, trobant paral4lelismes interessants amb els tumors. Finalment, hem volgut ocupar-nos dels mecanismes moleculars involucrats en els efectes produïts per Gal-1 durant la progressió tumoral pancreàtica mitjançant microarrays. Les nostres dades presenten Gal-1 com una nova diana terapèutica per lluitar contra el càncer de pàncrees.

Diss. (sammanfattning) Stockholm : Karol. inst., 2006. / Härtill 4 uppsatser.

Receptor-activated phosphatidylinositol (PtdIns) hydrolysis was examined in AR42J rat pancreatic acini. Cholecystokinin-octapeptide (CCK₈) and bombesin induced a dose-dependent accumulation of [³H] inositol monophosphate ([³H]InsP₁). Manganese (Mn²⁺), a known calcium channel blocker, did not alter basal PtdIns hydrolysis. In contrast, when added 5 minutes prior to the addition of agonists for 60 minutes, Mn²⁺ markedly inhibited secretagogue-mediated [³H]InsP1 formation. Mn²⁺ also attenuated the CCK₈-mediated increase in biologically active inositol 1,4,5-trisphosphate and inositol 1,3,4,5-tetrakisphosphate. These inhibitory effects of Mn²⁺ were mimicked by lanthanum and by EGTA. Addition of calcium to EGTA-treated cells abolished the inhibitory effects of extracellular calcium depletion. Mn²⁺, La³⁺ and EGTA exerted similar inhibitory effects on PtdIns hydrolysis in pancreatic acini. These findings suggest that receptor-activated calcium influx is required for full activation of the CCK₈-mediated signal transduction pathway that is coupled to PtdIns hydrolysis.